Investigating the role of Fwd and potential role of the Rab11-interacting protein dRip11 in Drosophila

spermatocyte cytokinesis

by

Anya Natassia Cyprys

A thesis submitted in conformity with the requirements for the degree of Master’s of Science

Graduate Department of Molecular Genetics University of Toronto

© Copyright by Anya Natassia Cyprys 2012

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Investigating the role of Fwd and potential role of the Rab11-

interacting protein dRip11 in Drosophila spermatocyte cytokinesis

Anya Natassia Cyprys

Master’s of Science

Department of Molecular Genetics University of Toronto

2012

Cytokinesis is the final separation of daughter cells after division. Membrane trafficking

increases the surface area of dividing cells and may deliver cargo needed for division. The

Drosophila PI4-kinase Fwd is required for spermatocyte cytokinesis and likely acts, in part, by

mediating Rab11-dependent trafficking to the furrow. To further understand the mechanism of

action of Fwd, I attempted to place fwd in a pathway with other cytokinesis genes encoding

Rab11, phosphatidylinositol transfer protein and a subunit of the exocyst. I also investigated a

potential role for the Rab11 interacting protein dRip11 in cytokinesis. My results suggest that

Rab11, like Fwd, is required for cell integrity during cytokinesis and that the Rab11 interacting

protein Nuf is an important candidate to investigate along with dRip11 as a relevant Fwd/Rab11

effector during this highly conserved process.

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Acknowledgments First of all, I would like to thank my supervisor, Julie Brill, for her tremendous support,

expertise and guidance during my Master’s project. Julie’s constant encouragement and scientific

curiosity propelled me forward, especially during times of frustration. Thank you for the time

and energy that you dedicated to me and my project.

Thank you to my committee members Helen McNeill and Laurence Pelletier for their

invaluable guidance and advice, as well as their time, throughout my thesis.

I would like to thank the following people for generously providing reagents: Henry

Chang, Miklόs Erdélyi, Margaret Fuller, Maurizio Gatti and Don Ready.

I would not have made it this far without the support and expertise of my lab-mates. For

help with techniques and troubleshooting, advice, and commiseration – thank you. Especially to

Gordon, Julie Tan, Phil, Lala, Lauren, Milu and Raymond: for their friendship and often strange,

but always priceless, shenanigans.

To my parents, Mary and Stan, my brother Nathan and my entire extended family: thank

you for your love and support. To Jason: for believing in me even when I didn’t and for the joy

that you bring to my life.

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Table of Contents Acknowledgments.......................................................................................................................... iii

Table of Contents........................................................................................................................... iii

List of Tables ................................................................................................................................. vi

List of Figures ............................................................................................................................... vii

Chapter 1 Introduction .................................................................................................................... 1

1.1 Membrane trafficking in cytokinesis .................................................................................. 2

1.1.1 Introduction to cytokinesis..................................................................................... 2

1.1.2 Key players in cytokinesis ..................................................................................... 2

1.1.3 Cytokinesis and membrane trafficking .................................................................. 7

1.1.4 Evidence for membrane trafficking during cytokinesis......................................... 7

1.1.5 Rab11 roles in trafficking and cytokinesis........................................................... 10

1.1.6 PIPs and PI4-kinase regulate membrane trafficking............................................ 11

1.1.7 PI4KIIIβ and PI4P in cytokinesis ........................................................................ 14

1.2 Drosophila spermatogenesis as a model to study cytokinesis ......................................... 14

1.2.1 Drosophila spermatogenesis................................................................................ 14

1.2.2 Requirements for Drosophila spermatocyte cytokinesis ..................................... 18

1.3 Rab11-family of interacting proteins (FIPs) ..................................................................... 19

1.3.1 Overview of the Rab11-family of interacting proteins ........................................ 19

1.3.2 Mammalian Class I Rab11-FIP functions............................................................ 23

1.3.3 Mammalian Class II Rab11-FIPs in cytokinesis.................................................. 25

1.3.4 Drosophila Rab11-FIPs ....................................................................................... 26

1.4 Thesis goals....................................................................................................................... 29

1.4.1 Characterizing the Fwd cytokinesis pathway in Drosophila spermatocytes ....... 29

1.4.2 Examining potential roles for dRip11 in spermatogenesis .................................. 30

v

Chapter 2 Results .......................................................................................................................... 32

2.1 Materials and Methods...................................................................................................... 33

2.1.1 Fly stocks ............................................................................................................. 33

2.1.2 Immunofluorescence and microscopy ................................................................. 33

2.1.3 Immunoblotting.................................................................................................... 34

2.2 Results............................................................................................................................... 35

2.2.1 Marker analysis in known cytokinesis mutants ................................................... 35

2.3 Examining potential roles for dRip11 in spermatogenesis ............................................... 42

2.3.1 dRip11 antibody localization ............................................................................... 42

2.3.2 Assessing dRip11 function .................................................................................. 48

Chapter 3 Discussion and Future Directions ................................................................................ 52

3.1 Drosophila cytokinesis mutants........................................................................................ 53

3.1.1 Marker analysis in known cytokinesis mutants ................................................... 53

3.1.2 Fwd in cytokinesis: open questions ..................................................................... 55

3.2 Examining dRip11 and Nuf in cytokinesis ....................................................................... 57

References..................................................................................................................................... 61

vi

List of Tables Table 1 List of fly stocks .............................................................................................................. 37

Table 2 Genes present on the duplication (Dp(1;3)DC349) that rescues lethality of dRip11KG0248 mutants ............................................................................................................... 49

vii

List of Figures Figure 1.1. Key players in cytokinesis............................................................................................ 6

Figure 1.2. The PIP metabolic pathway........................................................................................ 13

Figure 1.3. Drosophila spermatogenesis and spermatid individualization................................... 16

Figure 1.4. Drosophila male germ cell meiosis ............................................................................ 17

Figure 1.5. Schematic representation of the mammalian FIP protein family ............................... 21

Figure 1.6. Model for fwd cytokinesis pathway............................................................................ 31

Figure 2.1. PI4P localization in fun and gio mutant dividing spermatocytes ............................... 38

Figure 2.2. FwdKD localization in gio mutant dividing spermatocytes ......................................... 40

Figure 2.3. FwdKD localization in rab11 mutant dividing spermatocytes..................................... 41

Figure 2.4. dRip11 antibody localization in wild type and fwd male germ cells.......................... 44

Figure 2.5. dRip11 antibody localization in Rab11 dominant negative expressing cells ............. 46

Figure 2.6. Examining dRip11 mutant spermatocytes .................................................................. 50

1

Chapter 1

Introduction

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1.1 Membrane trafficking in cytokinesis

1.1.1 Introduction to cytokinesis Cell division is a crucial event in the life of a cell, mediating many processes, including

proliferation, tissue maintenance, embryogenesis and gamete production. During metaphase,

chromosomes align at the cell equator and, at anaphase, are pulled to opposite poles of the cell

by spindle microtubules that emanate from the centrioles (Fig. 1.1, A). In addition to spindle

microtubules, another population of interdigitating, anti-parallel microtubules is present in the

area between the separating chromosomes, forming the midzone or central spindle (Douglas and

Mishima, 2010). During telophase, a contractile ring composed of F-actin and myosin II forms at

the cell equator. The contractile ring and central spindle are likely interdependent structures. For

instance, mutations affecting the contractile ring often show defects in central spindle

morphology and vice versa (Giansanti et al., 1998; Somma et al., 2002). In addition, creating a

physical barrier between the equatorial cortex and central spindle causes cytokinesis and central

spindle defects, presumably by blocking signals sent between the two (Cao and Wang, 1996).

Cytokinesis is the final phase of division, during which the two daughter cells physically

separate. The contractile ring constricts, forming a plasma membrane (PM) indentation called the

cleavage furrow, which pinches in to separate the daughter cells. This seemingly simple process

is mediated by a number of factors.

1.1.2 Key players in cytokinesis Cytokinesis must be spatiotemporally regulated, occurring between segregated

chromosomes at the cell equator and after completion of anaphase. Rho GTPase is a key factor in

cytokinesis initiation and regulates contractile ring formation in two ways. Rho activates

formins, which nucleate unbranched actin filaments (Piekny et al., 2005). In addition, Rho

activates the Ser/Thr kinases ROCK and citron kinase (CK), which phosphorylate myosin

regulatory light chain, thereby activating myosin contractility. Depletion of RhoA causes

cytokinesis defects and disrupts actin and myosin localization (Yuce et al., 2005). Thus, Rho

activity modulates two key components of the contractile ring.

For successful cytokinesis, Rho localization and activation need to be tightly regulated.

Like most GTPases, Rho cycles between active GTP-bound and inactive GDP-bound forms. The

3

exchange of GDP for GTP is catalyzed by a guanine nucleotide exchange factor (GEF) and the

hydrolysis of GTP to GDP is aided by a GTPase activating protein (GAP). The RhoGEF ECT2

localizes to the furrow and is required for cytokinesis (Yuce et al., 2005). ECT2 interacts with

centralspindlin, a complex composed of MgcRacGAP/Cyk-4 (RacGAP) and a kinesin-6 MKLP1

(Fig. 1.1, C). It is not clear whether RacGAP acts as a GAP towards Rho or other Rho family

members, such as Rac (Canman et al., 2008; Miller and Bement, 2009). However, it is required

for cytokinesis (Yuce et al., 2005). RacGAP and ECT2 bind in a cell-cycle regulated manner,

and RacGAP may activate the GEF activity of ECT2, perhaps by relieving ECT2 autoinhibition.

The sequential action of the GAP and GEF may be required for continual flux of RhoA through

the GTPase cycle (Bement et al., 2005). Alternatively, RacGAP may not target RhoA, or may act

late to decrease RhoA activity for contractile ring disassembly.

A zone of active GTP-bound RhoA localizes to the equator in echinoderm and amphibian

embryos prior to furrowing (Bement et al., 2005). The zone of active RhoA links central spindle

MTs to contractile ring formation. Indeed, mechanically shifting the spindle demonstrates that a

cortical RhoA zone forms wherever there is a signal from the central spindle. This likely occurs

through MKLP1-dependent transport of Rho regulators such as ECT2. Further regulation occurs

by polo-like kinase 1 (Plk1), which phosphorylates RacGAP, enabling it to recruit ECT2 to the

central spindle (Petronczki et al., 2007; Wolfe et al., 2009).

Centralspindlin likely restricts the zone of active RhoA (Yuce et al., 2005).

Mislocalization of RacGAP and ECT2 away from the equator by depletion of MKLP1 did not

disrupt RhoA localization, but rather the Rho active zone is expanded, together with actin and

myosinII. Thus, while ECT2 at the central spindle restricts RhoA activity, other mechanisms

contribute to the equatorial localization of RhoA.

Another regulator of cytokinesis is the chromosomal passenger complex (CPC). CPC is

composed of Aurora B kinase and the MT binding proteins INCENP, Survivin and Borealin,

which localize and activate Aurora B (Ruchaud et al., 2007). CPC has diverse roles in mitosis,

including regulation of kinetochore attachment to MTs, spindle checkpoint, sister chromatid

cohesion and cytokinesis. CPC localizes to chromosomes in prophase, centromeres during

metaphase and to central spindle MTs in anaphase. At the central spindle, Aurora B

phosphorylates and activates MKLP1 (Guse et al., 2005) and RacGAP (Minoshima et al., 2003),

4

thus regulating furrow induction by relaying signals from the central spindle to the plasma

membrane (Fig. 1.1, C). Aurora B phosphorylation of RacGAP may induce a latent GAP activity

towards RhoA (Minoshima et al., 2003). It has recently been suggested that CPC has additional

roles in contractile ring assembly that are independent of centralspindlin and may involve

interaction with septins (Lewellyn et al., 2011). The Drosophila testis-specific CPC component,

Australin, is required not only for centralspindlin recruitment, but also anillin recruitment to the

equator (Gao et al., 2008).

Septins are GTP-binding proteins that form non-polar filaments, which arrange into

higher structure rings or gauzes (Estey et al., 2011). In budding yeast, septins form a collar at the

mother-bud neck that splits into two, with one ring located on each side of the neck area

(Dobbelaere and Barral, 2004; Lippincott et al., 2001). The rings form a diffusion barrier to

restrict proteins to the bud neck. In mammalian cells, septins bind myosin and may act as a

platform where kinases can activate myosin (Joo et al., 2007). Septins interact with F-actin likely

through anillin, and septins and F-actin may be co-dependent for spatial organization (Kinoshita

et al., 2002).

Anillin is a multi-domain scaffolding protein critical to cytokinesis that can bind myosin

II, actin, septins and RacGAP (Piekny and Maddox, 2010). Anillin may link the spindle MTs and

the contractile ring by binding RacGAP and recruiting septins to the furrow (D'Avino et al.,

2008; Gregory et al., 2008). For constriction of the ring to exert force, it must be attached to the

plasma membrane, and both septins and anillin are candidates to link the contractile ring to the

plasma membrane (Fig. 1.1, B, D).

Membrane trafficking is an important component of cytokinesis. Once furrowing

generates a thin intercellular bridge, a final separation occurs, termed abscission. Both the

ESCRT complex (endosomal sorting complex required for transport) and FIP3-positive

endosome fusion may be required to resolve abscission (Schiel and Prekeris, 2011). The ESCRT

subunits TSG101 and CHMP4 each form two rings, one on either side of the midbody. CHMP4

is also found at the membrane cut site adjacent to the midbody prior to abscission (Elia et al.,

2011; Guizetti et al., 2011). Electron tomography revealed cortical filaments that formed helices

at the intercellular bridge, that were absent in cells depleted of the ESCRT-III core subunit

CHMP2A (Guizetti et al., 2011). This suggested that the filaments may be composed of ESCRT-

5

III subunits, which could mediate membrane constriction. The order of appearance at the

midbody suggests that the centromere protein CEP55 recruits the ESCRT accessory protein,

Alix, and the ESCRT-I member, Tsg101, to the midbody. These then recruit ESCRT-III

members, including CHMP4B. Prior to abscission, CHMP4B and other ESCRT-III subunits

extend from the midbody to the future cut site and form filaments that mediate abscission. In

addition to ESCRT, FIP3-endosomes may also help mediate abscission and will be discussed in

Section 1.3.3. Membrane trafficking is important in abscission, but also in earlier stages of

cytokinesis. My thesis focuses on membrane trafficking pathways in cytokinesis.

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Figure 1.1. Key players in cytokinesis.

Schematic diagram of cell in anaphase (A) and cytokinesis (B), with boxed regions magnified below (C, D). During anaphase, chromosomes are pulled to opposite poles by kinetochore MTs (A). Central spindle MTs interdigitate at the equator. The chromosomal passenger complex (CPC) and centralspindlin are localized at central spindle MTs (C) and lead to the localization and activation of the RhoGEF ECT2. ECT2 activates Rho, which promotes contractile ring formation. During cytokinesis (B), the actomyosin ring constricts to pull in the equatorial PM. Septins and anillin are also present at the cleavage furrow, and may help anchor the contractile ring to the PM (D). Figure adapted from (Goldbach, 2011).

7

1.1.3 Cytokinesis and membrane trafficking

For cytokinesis to be successful, both contractile ring constriction and membrane

trafficking are required (Albertson et al., 2005; Montagnac et al., 2008). Trafficking may serve

several purposes. Delivery of membrane to the cleavage furrow may mediate the necessary

increase in surface area of the dividing cell. Trafficking may also deliver specific cargo

molecules to the cleavage furrow that function in division or spindle alignment (Skop et al.,

2001). Additionally, delivery of membrane may mediate abscission (Albertson et al., 2005;

Montagnac et al., 2008).

Membrane trafficking is an important process that allows regulated transport of material

between membrane-bound organelles via vesicles or tubules (Stenmark, 2009). Steps in

trafficking include budding of the vesicle, its transit to a specific site, docking at this site, and

finally fusion. These steps are regulated by various factors (Prekeris, 2003). For instance, coat

proteins, such as clathrin, mediate vesicle budding. Molecular motors transport the vesicle along

cytoskeletal components, such as actin filaments or microtubules. Vesicle docking and fusion at

the target membrane is facilitated by soluble N-ethyl-maleimide-sensitive factor attachment

protein receptors (SNAREs), such as VAMPs and syntaxins, which are present on the vesicle and

target membranes, respectively. Several pathways exist for transit throughout the cell.

Endocytosis and recycling involve uptake of material at the cell surface and subsequent recycling

back to the plasma membrane (PM), either by trafficking directly from early endosomes (EE) to

the PM (fast recycling) or by passing through the Golgi or recycling endosome (RE) on its way

to the PM (slow recycling). Secretory trafficking involves de novo generation of vesicles at the

Golgi. Endocytosis/recycling and secretion are the main routes that will be discussed. However,

other pathways exist, as does crosstalk between pathways. Many regulators of membrane

trafficking are required for cytokinesis and different trafficking pathways are involved.

1.1.4 Evidence for membrane trafficking during cytokinesis Several studies indicate a requirement for membrane trafficking during cytokinesis, either

from secretory and/or endocytosis and recycling pathways. Brefeldin A (BFA), a drug that

inhibits secretion from the Golgi, was used to demonstrate a requirement for secretory trafficking

in dividing C. elegans blastomeres (Skop et al., 2001). BFA-treated cells initiate furrow

8

ingression, but fail late in cytokinesis to produce binucleate cells. While BFA also blocks

cytokinesis in human HeLa cells (Gromley et al., 2005), it does not have the same effect in

monkey BSC1 cells (Boucrot and Kirchhausen, 2007). It is likely that the contribution of

membrane from either endocytosis/recycling or through Golgi-derived secretion differs among

cell types (Montagnac et al., 2008). Hyalin was used as a marker for Golgi-derived vesicles in

sea urchin zygotes, since it is secreted from the zygote in a SNARE-dependent manner. Both

FM1-43, a membrane probe that inserts into the outer leaflet of the PM, and hyalin were found to

be deposited at the late cleavage furrow, marking this region as the site of membrane addition

(Shuster and Burgess, 2002). Mutations in genes required for Golgi function including four way

stop (fws), which encodes the Drosophila homolog of Cog5, a protein involved in Golgi

trafficking, can lead to cytokinesis defects (Farkas et al., 2003). RNA interference (RNAi)-based

screens for cytokinesis players have identified genes involved in various aspects of membrane

trafficking (Echard et al., 2004; Eggert et al., 2004; Skop et al., 2004). Some key questions in

this field are where in the cell the membrane comes from, where it goes and when, and how these

processes are regulated and coordinated.

Experiments by Gromley et al. (2005) suggest that secretory vesicle fusion at the furrow

is critical to cytokinesis. RNAi knockdown of the exocyst, a protein complex that acts to target

vesicles to sites of fusion, and SNARE components, which are also required for the targeting and

fusion of secretory vesicles, resulted in cytokinesis defects. Depleting the exocyst subunit sec5

resulted in aberrant accumulation of v-SNARE containing vesicles at the midbody. Based on

colocalization with a luminal-GFP marker, they identified these vesicles as secretory in nature.

These experiments demonstrated that targeted fusion of secretory vesicles to the midbody is

critical for cytokinesis.

Schweitzer et al. (2005) demonstrated that endocytosis and recycling are required for

cytokinesis and are spatiotemporally regulated. Fluorescently labeled transferrin was used as a

marker for endocytosis and recycling. Transferrin localized to the poles of HeLa cells in early

division and to the cleavage furrow in late telophase. They asked whether transferrin

accumulation at the cleavage furrow was due to endocytosis within the furrow region or to

transport from the poles to the furrow. They pulsed mitotic cells with transferrin at early

cytokinesis and observed its localization at early and late timepoints. The results from pulse-

chase experiments and live cell imaging indicate that endocytosed vesicles trafficked from the

9

polar region of the cell to the furrow during cytokinesis, and that transferrin endocytosis can also

occur in the region of the furrow during late cytokinesis. To disrupt clathrin-mediated

endocytosis, they expressed dominant negative (DN) Eps15, an accessory protein found in

clathrin-coated pits that is essential for clathrin-mediated endocytosis. Expression of DN-Eps15

inhibited transferrin internalization and resulted in cytokinesis failures. These experiments

suggest that clathrin-mediated and receptor-mediated endocytosis is vital for proper cytokinesis,

and that endocytosis and recycling are regulated in a spatio-temporal manner. Endocytosis within

the cleavage furrow may act to terminate cytokinesis by removing factors that are no longer

needed. While vesicle fusion is generally thought to be required for abscission, endocytosis

within the furrow may also aid in the final resolution of the daughter cells by acting to seal off

the plasma membrane. Endocytosis at the midbody may also balance membrane delivery and

retrieval for the maintenance of proper surface area.

Boucrot and Kirchhausen (2007) proposed that cells create and store vesicles during

metaphase, at which time endocytosis continues but recycling is blocked, and only resume

recycling to the PM during late telophase. Transferrin undergoes rounds of endocytosis and

recycling. However during cell division, the rate of transferrin internalization did not decrease in

spite of decreased levels of transferrin at the cell surface. Only during anaphase and telophase

did PM levels of transferrin recover. Overexpression of dominant negative forms of the SNAREs

VAMP3 and VAMP7, which normally dock endosomes to the PM, showed decreased recovery

of both the transferrin receptor and a late endosome marker to the PM after anaphase. This

suggested that recycling is blocked during metaphase and resumes in telophase. These

experiments were done in cells that visibly round up off of their substrate during mitosis and it is

not clear if storing vesicles prior to cytokinesis is a general or cell-type specific feature.

These studies demonstrate that endocytosis and recycling as well as secretion are required

for cytokinesis in different cell types. Depending on factors such as cell type, cell size and rate of

cytokinesis, cells may meet their requirement for membrane remodeling and addition during

cytokinesis in many ways: by storing vesicles to later exocytose, by secreting new membrane

from the Golgi, by endocytosis and recycling of membrane, or by a combination of these events.

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1.1.5 Rab11 roles in trafficking and cytokinesis Rab GTPases are crucial for many steps in trafficking; they interact with coat complexes

that mediate vesicle budding, motors that move the vesicle along cytoskeletal filaments, and

SNAREs that target the vesicle to an acceptor membrane (Stenmark, 2009). Rab GTPases cycle

between GTP-bound active forms and GDP-bound inactive forms, with the exchange of GDP for

GTP catalyzed by guanine nucleotide exchange factors (GEFs). The hydrolysis of GTP to GDP

is promoted by both intrinsic GTPase activity and by GTPase-activating proteins (GAPs). The

Rab active state is generally membrane bound and can promote the recruitment of effectors to

specific membranes (Barr, 2009). Rab family members localize to distinct sites, thereby acting as

address tags for different trafficking compartments.

Rab11 localizes to both the Golgi and recycling endosome (RE) in mammalian cells

(Chen et al., 1998; Ullrich et al., 1996). In Chinese hamster ovary cells, expression of a

constitutively GDP-bound dominant negative Rab11 mutant (Rab11S25N) blocks movement of

transferrin out of Rab5-labelled early sorting endosomes (Ullrich et al., 1996). In baby hamster

kidney (BHK) cells, Rab11S25N slows the recycling of transferrin back to the PM. These

experiments were the first to suggest that Rab11 acts at the recycling endosome. The authors

speculated Rab11 may function to regulate cargo arriving at the RE from the sorting endosome

and/or traffic between the RE and Golgi.

To test whether Rab11 regulates de novo secretion out the Golgi, Chen et al. (1998)

tracked VSV-G as a marker of the secretory pathway. VSV-G is a membrane protein encoded by

the vesicular stomatitus virus and targeted to the basolateral cell surface. Expression of

Rab11S25N greatly inhibited secretion of VSV-G from the Golgi to the cell surface, as

demonstrated by immunofluorescence and by quantification of radiolabeled VSV-G protein at

the PM. The defect in VSV-G secretion was rescued by coexpressing wild-type or activated

Rab11, suggesting that the effect was specific to Rab11. Moreover, Rab11S25N did not affect

secretion of influenza hemagglutinin, an apically targeted protein, suggesting Rab11 may

regulate trafficking of a distinct subset of cargoes out of the Golgi. Rab11 regulated transport out

of the TGN, and not from the ER to Golgi, or intra-Golgi transit, as shown by following a

temperature-sensitive VSV-G mutant (Chen et al., 1998). This suggested Rab11 may directly

11

control either exit from the Golgi, perhaps by regulating vesicle budding, or flux through the RE

of cargo from both endocytic and secretory pathways.

Rab11 is required for cytokinesis during C. elegans blastomere division (Skop et al.,

2001), and in Drosophila spermatocyte cytokinesis (Giansanti et al., 2007) and embryo

cellularization, a specialized form of cytokinesis driven mainly by membrane addition (Pelissier

et al., 2003). Rab11 is required for cytokinesis in HeLa cells, as shown by the presence of

binucleate cells when overexpressing dominant negative (DN) Rab11, depleting Rab11 by

siRNA or injecting anti-Rab11 antibodies (Wilson et al., 2005). Rab11-positive vesicles are

found at the cleavage furrow in both mammalian tissue culture cells and Drosophila

spermatocytes undergoing cytokinesis (Giansanti et al., 2007; Wilson et al., 2005), where they

may mediate membrane addition to the furrow. Rab11 likely recruits effectors needed for

targeting and docking vesicles at the furrow (Section 1.3.3).

1.1.6 PIPs and PI4-kinase regulate membrane trafficking Phosphatidylinositol phosphates (PIPs) are membrane lipids that make up only a small

percent of the lipids within a cell and have key roles in membrane trafficking. The PIP precursor,

phosphatidylinositol (PI), can be phosphorylated on the three, four or five prime position of the

inositol ring to generate different PIPs. PIPs are synthesized by specific lipid kinases and

dephosphorylated by lipid phosphatases. PIP metabolism is governed by the balance of these

enzymes, which are regulated in time and space by being restricted to specific subcellular sites

(D'Angelo et al., 2008). This allows for unique distributions of the various PIPs within a cell.

PI4P is the product of PI 4-kinase (PI4K) activity on PI and is also important as the

precursor to PI(4,5)P2 (Fig. 1.2). PI4-kinases were categorized into types II and III based on

sensitivities to adenosine and wortmannin, respectively (D'Angelo et al., 2008). There are four

PI4K isoforms in mammalian cells, and two of these, PI4KIIα and PI4KIIIβ are found at the

Golgi. There are two main classes of PI4P effectors, adaptor and coat complexes, such as AP-1,

GGA proteins and EpsinR, and lipid-transfer proteins, like OSBP, CERT, FAPP1 and FAPP2. In

particular, the FAPPs bind both PI4P and Arf1 at the Golgi, where they are required for post-

Golgi trafficking (Godi et al., 2004). Another PI4P binding partner is GOLPH3. In mammalian

cells, GOLPH3 knockdown disrupts both Golgi morphology and transport out of the Golgi to the

PM (Dippold et al., 2009).

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Yeast has three PI4Ks, Stt4, Pik1 and Lsb6. While all three kinases produce PI4P, they

each have distinct, non-redundant roles. For instance, Stt4 was found to be involved in actin

cytoskeleton organization, cell wall integrity and vacuole morphology, whereas Pik1 was

required for proper secretion out of the Golgi and for trafficking to the vacuole (Audhya et al.,

2000; Walch-Solimena and Novick, 1999). Many of the genes that interact with pik1 have roles

in membrane trafficking, including the yeast homolog of Rab11 (Sciorra et al., 2005).

In addition to a role in Golgi function, Pik1 may be required for the maintenance of Golgi

structure. The temperature-sensitive mutant pik1ts showed the abnormal presence of

interconnected membrane tubules that may represent distorted Golgi, termed Berkeley bodies

(Audhya et al., 2000). Arf1, a small GTP-binding protein, is needed for the maintenance of Golgi

structure. arf1 mutants displayed decreased PI4P and PI(4,5)P2 levels similar to pik1ts mutants,

and arf1 and pik1ts double mutants displayed synthetic growth and transport defects at

permissive temperatures, indicating a genetic interaction. Therefore Pik1 and Arf1 may interact

for the maintenance of the structure and function of the Golgi in yeast. Indeed, mammalian Arf1

recruits PI4KIIIβ to Golgi membranes and both Arf1 and PI4KIIIβ are required for Golgi

integrity (Godi et al., 1999). COS-7 cells transfected with catalytically inactive PI4Kβ (D656A)

had disorganized localization of the Golgi proteins giantin and GM-130. BFA treatment

reversibly disrupts the Golgi, causing the redistribution of Golgi markers into the ER. Cells

transfected with mutant PI4Kβ did not recover as easily after BFA washout; reassembly after

drug addition was slower and the reformed Golgi morphology irregular, suggesting that PI4Kβ

could be required for the structural integrity of the Golgi in mammalian cells.

Mammalian PI4KIIIβ co-localizes with Rab11 at the Golgi and binds Rab11, with higher

affinity for the activated GTP bound form (de Graaf et al., 2004). PI4KIIIβ was found to recruit

activated Rab11 to the Golgi, whereas PI4KIIIβ localization did not depend on Rab11. To probe

the functional importance of the Rab11-PI4KIIIβ interaction, three mutants of PI4KIIIβ were

overexpressed: a catalytically inactive form, one that could not bind Rab11 and another that

sequestered Rab11 from the Golgi. While the catalytically inactive form did not inhibit VSV-G

secretion, expression of the mutated PI4KIIIβ that could not bind Rab11 or that mislocalized

Rab11 reduced VSV-G transport to the PM. Therefore the interaction of PI4KIIIβ and Rab11 is

likely required for transit out of the Golgi, independent of PI4KIIIβ catalytic activity. Thus

PI4Kβ may act as a scaffold protein to promote Rab11-dependent post-Golgi trafficking.

13

In addition to Arf1 and Rab11, another PI4KIIIβ binding partner is the Ca2+-binding

protein Neuronal calcium sensor-1 (NCS-1). Both yeast and mammalian NCS-1 (Frq1 in yeast)

bind PI4KIIIβ (Hendricks et al., 1999; Zhao et al., 2001). Mammalian NCS-1 may modulate

PI4P levels by influencing PI4KIIIβ, for instance, NCS-1 can increase PI4P levels and stimulate

trafficking through the recycling compartment (Kapp-Barnea et al., 2006). Therefore key

PI4KIIIβ binding partners, Rab11, Arf1 and NCS-1, are important modulators of secretion and

recycling.

Figure 1.2. The PIP metabolic pathway.

PI can be phosphorylated to form different PIPs. PI is phosphorylated by PI4K to generate PI4P, which can be phosphorylated to produce PI(4,5)P2 (or PIP2). PIP2 can be hydrolyzed by phospholipase C (PLC) to form diacylglycerol (DAG) and inositol triphosphate (IP3) or it can be further phosphorylated to give PIP3. All PIPs can be dephosphorylated by specific phosphatases to regenerate precursor PIPs and PI, as denoted by the arrows. PI can be moved among different cellular membranes by PI transfer proteins (PITPs), such as Giotto (Gio). Drosophila has three PI4Ks, PI4KII, PI4KIIIα and Fwd. Drosophila proteins are in blue.

14

1.1.7 PI4KIIIβ and PI4P in cytokinesis PI4KIIIβ has been implicated in cytokinesis in several organisms. PI4KIIIβ is required

for cytokinesis in Trypanosoma brucei (Rodgers et al., 2007) and in the fission yeast S. pombe

(Park et al., 2009). The Drosophila melanogaster gene four wheel drive (fwd) encodes PI4KIIIβ

and fwd mutants are male sterile due to defects in spermatocyte cytokinesis and sperm tail

maturation (Brill et al., 2000; Polevoy et al., 2009). In dividing fwd spermatocytes, the

contractile ring begins to ingress, but is unstable and later regresses, resulting in multinucleate

spermatids (Brill et al., 2000). During normal spermatocyte cytokinesis, PI4P, a secreted GFP

marker and the membrane trafficking regulator Rab11 accumulate at the midzone. fwd mutants

failed to accumulate PI4P, secreted GFP and Rab11 at the midzone, leading to cytokinesis and

spermatogenesis failures (Polevoy et al., 2009). Fwd binds Rab11, and activated Rab11 partially

rescues the fwd cytokinesis defect. Therefore, Fwd likely regulates cytokinesis, in part, by

promoting Rab11-dependent membrane addition to the ingressing furrow. While mammalian

PI4KIIIβ recruits Rab11 to the Golgi (de Graaf et al., 2004) and Rab11 trafficking is required for

cytokinesis in mammalian cells (Wilson et al., 2005), whether PI4KIIIβ or PI4P have a role in

mammalian cytokinesis is not known.

1.2 Drosophila spermatogenesis as a model to study cytokinesis

1.2.1 Drosophila spermatogenesis Drosophila spermatogenesis is an excellent model in which to study cytokinesis, given

the combination of favourable cytology and powerful genetic techniques. At the start of

Drosophila spermatogenesis, a germline stem cell at the apical tip of the testis divides to produce

a stem cell and a spermatogonial cell (Fuller, 1993) (Fig. 1.3, A). Each spermatogonial cell is

surrounded by a pair of somatically derived cyst cells. During the remainder of spermatogenesis,

the progeny of the spermatogonial cell remain enclosed by two cyst cells and together this unit is

referred to as a cyst. All germ cells within any given cyst develop in synchrony, going through

spermatocyte mitosis, growth, meiosis and finally spermatid differentiation together. The

spermatogonial cell first undergoes four rounds of mitosis, resulting in a cyst of 16 primary

spermatocytes. After the four mitotic divisions, the primary spermatocytes undergo an extensive

growth period, during which they increase 25 times in volume and transcribe many genes

15

required for spermatogenesis. The spermatocytes then undergo two rounds of meiotic

cytokinesis, generating a cyst of 64 haploid spermatids. During both the mitotic and meiotic

divisions, the developing cells undergo incomplete cytokinesis. The contractile ring constricts,

but does not completely separate the two daughter cells, which remain connected by intercellular

bridges termed ring canals throughout their development. At the end of meiosis, the

mitochondria within each spermatid aggregate to form the mitochondrial derivative, termed the

Nebenkern, which appears as a dark structure by phase-contrast microscopy (Fig. 1.4, C). This

feature allows for easy identification of cytokinesis failure. Normally, each spermatid contains

one dark circle, the mitochondrial derivative, and one light circle, the nucleus. When cytokinesis

fails, the resulting spermatids are multinucleate, as identified by the presence of more than one

nucleus per cell. These cells also contain enlarged mitochondrial derivatives due to the

aggregation and fusion of mitochondria that would have been divided among many cells (Fig.

1.4, D).

To develop into mature sperm, spermatids must undergo a dramatic elongation event

(Fuller, 1993). An axoneme grows from the basal body, which is present at the base of the

nucleus. Mitochondria elongate along the growing axoneme, extending the sperm tail length to

about 1.8 mm. After elongation occurs, the sperm undergo individualization (Fig. 1.3, B).

Investment cones composed of F-actin form around each nucleus within a cyst. The actin cones

migrate the length of the cyst in synchrony, pushing out excess cytoplasm and investing each

sperm with its own membrane.

16

Figure 1.3. Drosophila spermatogenesis and spermatid individualization.

(A) Schematic representation of Drosophila spermatogenesis. The germline stem cell (S) divides to produce a gonial cell (G), which undergoes four rounds of mitosis. The 16 spermatocytes shown here are the contents of one cyst and each testis contains many cysts. After mitosis, the cells undergo a period of growth during which they increase in size and begin expression of genes needed for further differentiation. Spermatocytes undergo two rounds of cytokinesis to produce early spermatids. Spermatids then undergo extensive membrane remodeling to elongate and develop into mature sperm. Following each division that occurs during spermatogenesis, the cells form intercellular bridges, termed ring canals, which are depicted in green. (B) Schematic representation of Drosophila spermatid individualization. Throughout spermiogenesis, the spermatids remain connected via ring canals through which the cells share cytoplasm. To fully mature, each sperm must be invested with its own membrane through a process called individualization. To the left is a cyst of elongated spermatids (a normal cyst would contain 64 spermatids; three are shown here). Actin-rich investment cones (red) form around the nuclei (blue). As individualization proceeds (right), the 64 actin cones migrate the length of the spermatids in synchrony, investing each mature sperm with its own membrane. Excess cytoplasm and waste is pushed out of the sperm and forms a cystic bulge in front of the migrating actin cones. Diagram in (A) was kindly provided by Margaret T. Fuller and modified by Julie A. Brill. Diagram in (B) was adapted from a diagram kindly provided by Tatsuhiko Noguchi and Kathryn G. Miller.

17

Figure 1.4. Drosophila male germ cell meiosis.

Phase contrast micrographs demonstrate the morphological features of male germ cells in wild-type (A-C) and fwd mutant testes (D). (A) A spermatocyte prior to meiotic cytokinesis contains a phase-light nucleus. (B) Spermatocytes undergoing the first meiotic cytokinesis are detected by the phase-dark mitochondria that line up along the spindle. Arrows point to the cleavage furrows. (C) After meiosis, each spermatid contains one phase-light nucleus (arrowhead) and one phase-dark mitochondrial derivative (arrow) of equal size. (D) When cytokinesis fails, as in fwd mutants, each spermatid contains more than one nucleus (arrowhead). The presence of four nuclei indicates failure of both rounds of meiotic cytokinesis. The mitochondrial derivative is enlarged (arrow), as the mitochondria that would have been partitioned into four cells have now accumulated in a single cell.

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1.2.2 Requirements for Drosophila spermatocyte cytokinesis

Drosophila spermatocytes are large cells (20μm) that cleave rapidly (within about 15

minutes) (Wong et al., 2005). Spermatocytes undergo two meiotic divisions in rapid succession

(about 45 minutes apart), making them highly susceptible to perturbations in trafficking

components. Several genes that encode factors involved in secretion and endocytosis/recycling

are required for spermatocyte cytokinesis, such as fwd and rab11. The COG (conserved

oligomeric Golgi) complex is involved in ER-Golgi, intra-Golgi and endosome-Golgi trafficking

(Albertson et al., 2005; Laufman et al., 2011; VanRheenen et al., 1998; VanRheenen et al., 1999;

Walter et al., 1998; Whyte and Munro, 2001). Four way stop (Fws), the homolog of the

mammalian Cog5, is required for cytokinesis (Farkas et al., 2003). The homolog of syntaxin 5, a

SNARE required for ER to Golgi transport, is also required (Xu et al., 2002). The exocyst

complex functions to target vesicles on the plasma membrane to specific sites and the exocyst

subunit mutants funnel cakes (fun) and onion rings (onr) display defects in spermatocyte

cytokinesis (Giansanti et al., 2004). Some of these genes, for example fwd and fws, are required

for spermatocyte cytokinesis, but not for mitotic cytokinesis in other Drosophila tissues (Brill et

al., 2000; Farkas et al., 2003), suggesting that this cell type has specific requirements for

membrane addition and remodeling during division and is a sensitive system in which to

examine trafficking during cytokinesis.

fwd, giotto (gio, also called vibrator) and rab11 mutants have defects in F-actin ring

constriction (Giansanti et al., 2004). gio encodes a PI transfer protein (PITP) (Gatt and Glover,

2006; Giansanti et al., 2006). PITPs regulate the availability of PI at spatially restricted sites in

the cell and thus function with PI kinases to regulate membrane trafficking (Cockcroft, 2001).

Both fwd and gio mutants fail to accumulate Rab11 at the cell poles and cleavage furrow and

fwd, gio and rab11 mutants fail to localize the Rab11 effector Nuf to the furrow (Giansanti et al.,

2007; Polevoy et al., 2009). Since fwd and rab11 mutants localize Gio properly at the poles and

furrow, gio may act upstream. These studies suggest that fwd, gio and rab11 act in the same

pathway during cytokinesis to mediate membrane accumulation at the furrow.

In addition to a requirement for Fwd, other parts of the PIP metabolic pathway are critical

to cytokinesis in dividing Drosophila spermatocytes. Either depleting PIP2 or blocking PIP2

hydrolysis causes cytokinesis to fail (Wong et al., 2005). Inhibiting PIP2 metabolism by drug

19

addition led to regression of the cleavage furrow and depletion of actin and myosin from the

contractile ring (Wong et al., 2007).

Drosophila has three PI4Ks, PI4KII, PI4KIIIα and PI4KIIIβ, also known as four wheel

drive (fwd). Neither PI4KII nor fwd are essential for viability, but the double mutant is inviable

(Burgess, J. and Brill, J.A., unpublished). Knockdown of PI4KII and fwd together results in

highly cytosolic PI4P marker distribution (Hipfner, D. R., Ho, K., Burgess, J. and Brill, J.A.,

unpublished). Fwd has a role in cytokinesis and sperm individualization whereas PI4KII

spermatocytes undergo normal cytokinesis and exhibit a late defect in spermatogenesis,

highlighting the distinct roles of these kinases (Burgess, J. and Brill, J.A., unpublished). Fwd and

PI4KII appear to direct different processes while presumably producing the same phospholipid

(PI4P) at the same organelle (the Golgi). This is likely due either to non-catalytic roles of the

kinases in binding different partner proteins, which then carry out distinct functions, or to

generation of different pools of PI4P in different domains. Complexes of kinase, effector(s) and

downstream PIP may lend specificity to directing particular subcellular events. For instance,

Rab11 and Fwd bind, they co-localize at the Golgi and activated Rab11 partially rescues the

cytokinesis defect of fwd, suggesting that interaction between Rab11 and Fwd is critical for Fwd

function in cytokinesis. Further exploration of potential Fwd binding partners is important to

expand our understanding of the mechanism of Fwd action in cytokinesis.

1.3 Rab11-family of interacting proteins (FIPs)

1.3.1 Overview of the Rab11-family of interacting proteins Rab11 interacts with different effectors that serve to specify and carry out its functions.

Some of these effectors include Myosin Vb, PI4KIIIβ, and the exocyst complex (Horgan and

McCaffrey, 2009). One important family of Rab11 effectors is the Rab11 family of interacting

proteins (Rab11-FIPs) (Fig. 1.5). At their carboxyl-termini, Rab11-FIPs (FIPs) contain a

predicted coiled-coil region with an amphipathic α-helical Rab11-binding domain (RBD), which

can bind Rab11 family members (mammalian Rab11a, Rab11b, and Rab 25). Differences in their

N-termini separate FIPs into two classes. Class I FIPs contain a phospholipid-binding C2

domain, which may mediate their ability to target Rab11-positive vesicles to the PM (Albertson

et al., 2005; Hales et al., 2002; Lindsay and McCaffrey, 2004). Class II FIPs do not contain C2

domains, and instead have calcium-binding EF-hand domains and Arf-6 binding domains. There

20

are three mammalian Class I FIPs, Rab-coupling protein (RCP), Rab11-FIP2 (FIP2) and Rip11,

and two mammalian Class II FIPs, FIP3 and FIP4. Whereas Class I FIPs have roles in recycling

of various cargoes to the PM, Class II FIPs have been specifically implicated in cytokinesis.

Rab11a only partially colocalizes with any individual FIPs, suggesting that FIPs may

share Rab11 and likely have distinct roles (Meyers and Prekeris, 2002). For instance, while BFA

caused the tubulation of Rip11-organelles, it had no affect on FIP3 localization.

Immunofluorescence and membrane fractionation demonstrated that FIPs localize to overlapping

but distinct subpopulations of endosomes. FIPs form mutually exclusive complexes with Rab11

and likely compete with each other and other Rab11 effectors for binding to Rab11. Indeed the

specificity of Rab11 function is mediated by these various ensembles of effectors, which may be

regulated spatially and temporally within the cell.

21

22

Figure 1.5. Schematic representation of the mammalian FIP protein family.

Mammalian Class I FIPs include Rip11, FIP2 and RCP, and Class II FIPs include FIP3 and FIP4. All FIPs are characterized by a highly conserved C-terminal Rab11-binding domain (RBD). Class I FIPs contain a phospholipid binding C2 domain at the N-terminus and conserve a MARK2 phosphorylation site at a serine residue. FIP2 contains three NPF (asparagines-proline-phenylalanine) repeats, which are binding motifs for EH domain-containing proteins. FIP2 also has a Myosin Vb binding region. RCP contains three PEST domains, which may promote degradation. Class II FIPs are characterized by calcium sensing EF hands, Cyk-4 binding domains and Arf6 binding domains. In addition, FIP3 has a proline rich region. Adapted from (Simon and Prekeris, 2008).

23

1.3.2 Mammalian Class I Rab11-FIP functions While all Class I FIPs contain a C2 domain and RBD, the central region of these proteins

is highly divergent and may mediate specific functions for each FIP, for instance by targeting the

FIP for posttranslational modification or for interaction with other proteins (Lindsay and

McCaffrey, 2005). In addition to Rab11, Class I FIPs can also bind and colocalize with

mammalian Rab14 (Kelly et al., 2010). As a group, the Class I FIPs are involved in numerous

trafficking pathways to the PM.

One approach to investigating Class I FIP function has been the overexpression of FIP

truncations lacking the C2 domain. These truncations contain the RBD, which can sequester

Rab11 into a collapsed membrane compartment and strongly inhibit Rab11-dependent trafficking

(Meyers and Prekeris, 2002). While this approach disrupts Rab11 trafficking, it may not reveal

unique functions of the different FIPs, but rather general defects. For instance, overexpression of

Rip11, RCP or FIP2 truncation mutants lacking the C2 domain all inhibits transferrin receptor

(TfR) recycling from REs to the PM (Lindsay and McCaffrey, 2002; Peden et al., 2004).

However, siRNA knockdown of RCP has no effect on transferrin recycling, but rather decreases

total levels of TfR, likely by missorting TfR to lysosomes for degradation. Therefore, RCP may

have a role in sorting TfR away from lysosomal degradation and towards the recycling pathway

(Peden et al., 2004). RCP has also been implicated in retrograde transport from EE/RE to the

TGN, likely through an interaction with Golgin-97 (Jing et al., 2010). Again, this role is unique,

as knockdown of Rab11a/b or RCP, but not Rip11, inhibited TGN transport of several

molecules.

Overexpression of FIP2 truncations has implicated FIP2 in IgA apical recycling and

transcytosis in MDCK cells and transferrin recycling in HeLa cells (Hales et al., 2002; Lindsay

and McCaffrey, 2002). A FIP2 mutant, FIP2SARG, contains two point mutations

(S229A/R413G) that affect residues in the central portion of the protein, between the C2 domain

and RBD. FIP2SARG can still interact with Rab11a or the MyoVb tail by yeast two-hybrid

analysis (Ducharme et al., 2007). Like the FIP2 truncation (FIP2ΔC), expression of FIP2SARG

inhibits basolateral to apical transcytosis in MDCK cells. However unlike FIP2SARG, FIP2ΔC

affects the morphology of the early endosome compartment. This suggests FIP2 may act at

multiple points in endosomal trafficking pathways. FIP2 carries out many of its functions

24

through interaction with Myosin Vb. A ternary complex of Rab11, FIP2 and MyoVb is important

for the transport and recycling of many proteins, including AMPA receptors (Wang et al., 2008),

aquaporin-2 (Nedvetsky et al., 2007), and Niemann-Pick C1-like protein 1 (Chu et al., 2009).

In addition to its functions in recycling to the PM, FIP2 also has roles in early endosome

(EE) trafficking and endocytosis. FIP2 contains three NPF motifs that can bind the EH-domain

containing Reps1, a substrate for the EGF receptor. FIP2 overexpression inhibited the

endocytosis of the EGF receptor through interaction with Reps1, Rab11 and the α-adaptin

subunit of the coat adaptor protein AP-2 (Cullis et al., 2002). Many markers of the EE

compartment, such as EEA-1, epsin 4 and Rab5b became mislocalized when expressing FIP2ΔC,

suggesting FIP2 may affect early endosomes (Ducharme et al., 2011). The C-terminal coiled-coil

of FIP2 can homo-oligomerize (Cullis et al., 2002) and by yeast two-hybrid assays the C and N

termini can bind to themselves and to each other (Lindsay and McCaffrey, 2002). Moreover,

full-length FIP2 can bind full-length RCP (Cullis et al., 2002). Hence, expression of a truncated

protein may disrupt trafficking by sopping up either itself, its effectors, including Rab11, or other

FIPs. It is important to verify results obtained using FIP2 truncations using other methods such

as RNAi, to ensure that the defects observed are not simply due to a general disruption of the

recycling system, but are specific to FIP2.

Lastly, FIP2 also has a role in establishment of polarity in MDCK cells (Ducharme et al.,

2006). FIP2 contains a serine residue (Ser227) that is phosphorylated by MARK2/EMK1/Par-

1Bα (MARK2), a member of the partitioning-defect (PAR) family first characterized in C.

elegans. While FIP2(S227A) is no longer phosphorylated by MARK2, it still binds MyoVb and

Rab11 and its overexpression does not affect the transcytosis or apical recycling of polymeric

IgA, which is trafficked through Rab11a vesicles (Ducharme et al., 2006). This suggests

MARK2 phosphorylation regulates FIP2 functions distinct from recycling. Cell polarity is

disrupted in MDCK cells by exposure to calcium-free media; once calcium is added back, the

cells reestablish tight and adherens junctions and cell polarity. Cells expressing FIP2S227A did

not reestablish proper adherens junctions after readdition of calcium, suggesting that FIP2 may

have roles in cell polarization, possibly through affecting trafficking pathways other than

Rab11a-mediated recycling.

25

1.3.3 Mammalian Class II Rab11-FIPs in cytokinesis Class II FIPs, FIP3 and FIP4, have been implicated in mammalian cytokinesis and

abscission. Class I FIPs do not localize to the cleavage furrow in HeLa cells and RNAi against

RCP or Rip11 has no effect on cytokinesis (Wilson et al., 2005). In contrast, FIP3 is enriched at

the furrow along with Rab11, and RNAi against Rab11 or FIP3 causes a late defect in

cytokinesis. Expression of a FIP3 mutant (FIP3 1737E) defective in Rab11 binding increases the

number of binucleate cells, demonstrating that the interaction between Rab11 and FIP3 is

required for cytokinesis. However, FIP3 1737E still localizes to the midbody, likely through an

interaction with Arf6. Arf6 is a GTPase present at the midbody and expression of a GDP-bound

form of Arf6 decreases recruitment of FIP3, FIP4 and Rab11 to the midbody (Fielding et al.,

2005). Arf6 has been shown to interact with the exocyst complex and pull down experiments

demonstrate that FIP3/4 can interact with the Exo70p subunit of the exocyst in a Rab11-

independent manner. Depletion of Exo70p results in cytokinesis failure and decreased FIP3 and

Rab11 localization to the midbody. These experiments suggest Rab11 may serve to recruit FIP3

to recycling endosomes, which then traffic to the midbody. In this model, FIP3 interacts with

Arf6 for the tethering of FIP3-Rab11 vesicles to the midbody. Through an interaction between

FIP3, Arf 6 and the exocyst, these vesicles dock and later fuse to the plasma membrane,

mediating late cytokinesis and abscission. It is not clear whether FIP3 and FIP4 have identical

functions in this process or if they have tissue or cell type specific roles (Wilson et al., 2005).

In addition to binding to Arf6 and the exocyst at the furrow, FIP3 binds the

centralspindlin component Cyk-4/MgcRacGAP/RacGAP1 (henceforth referred to as Cyk-4)

(Simon et al., 2008). Cyk-4 is a GAP that targets Rho GTPases, although it is not clear whether it

affects RhoA or Rac (Canman et al., 2008; Miller and Bement, 2009). The centralspindlin

complex is composed of Cyk-4 and the kinesin-6 motor MKLP. Centralspindlin is critical for

formation of the central spindle and recruits effectors such as the RhoGEF ECT2 to help regulate

RhoA, which mediates contractile ring formation (Douglas and Mishima, 2010). Knockdown of

MKLP disrupts FIP3 midbody localization, suggesting that the central spindle localizes FIP3

(Simon et al., 2008). ECT2 accumulates at the furrow in early division, and once its levels at the

furrow decrease, FIP3 endosomes then accumulate there in late telophase. The removal of ECT2

may allow FIP3 to interact with Cyk-4. Indeed, overexpression of a portion of ECT2 that binds

Cyk-4 inhibits FIP3 recruitment to the midbody, perhaps by blocking FIP3 access to Cyk-4.

26

However, although both the timing of their localization and biochemical binding experiments

demonstrate that ECT2 and FIP3 form mutually exclusive complexes with Cyk-4, knock-down

of ECT2 does not alter the timing of FIP3 recruitment to the midzone, suggesting that multiple

binding partners act to fine-tune FIP3 localization.

Using fluorescence recovery after photobleaching (FRAP), it was demonstrated that

FIP3-positive endosomes move from the centrosome to the furrow (Simon et al., 2008). Since

nocodazole inhibits FIP3 movement and FIP3 localizes along microtubules, FIP3-endosomes

may travel along microtubules to reach the furrow. In this model, ECT2 binds Cyk-4 at the

midbody during anaphase and early telophase, where it activates RhoA, allowing actomyosin

ring formation. At this time, FIP3-containing vesicles can move in and out of the midzone, but

do not accumulate there, as ECT2 binding to Cyk-4 prevents this. Once ECT2 dissociates from

Cyk-4 in late telophase, FIP3-vesicles bind Cyk-4 and accumulate in the midbody. The tethering

of these vesicles is enhanced by the exocyst complex. Recently it has also been suggested that

FIP3-positive endosomes may be responsible for a secondary ingression event, characterized by

further thinning of the intracellular bridge to 100-200nm wide, that promotes abscission (Schiel

et al., 2011). RNAi of FIP3 or VAMP8 resulted in a reduction of plasma membrane dynamics at

the midbody and inhibition of the secondary ingression event, as visualized by time-lapse

microscopy and correlative high-resolution 3-D tomography. Thus FIP3 plays an important role

in cytokinesis and abscission, and its localization to the furrow is ensured by interactions with

many proteins, including Rab11, Arf6, Cyk-4 and the exocyst.

1.3.4 Drosophila Rab11-FIPs Drosophila has one FIP from each class; dRip11 is the Class I FIP and Nuclear fall-out

(Nuf) is the Class II FIP. dRip11 aligns with the human Class I FIPs, Rip11, Rab11-FIP2 and

RCP. Based on ClustalW alignments to the three human Class I FIPs, dRip11 is most similar to

FIP2 (Shaye et al., 2008). dRip11 contains a C2 domain, but does not have the NPF domains that

link FIP2 to the EH-domain containing Reps1 (Cullis et al., 2002). FIP2 contains a serine residue

(Ser227) that is phosphorylated by the Par family kinase MARK2 in MDCK cells (Ducharme et

al., 2006). FIP2 is phosphorylated on the first serine residue of SMSxL, a noncanonical MARK2

phosphorylation site. RCP, Rip11 and FIP1 also conserve this site, and RCP and FIP1 are

phosphorylated by MARK2 in vitro. dRip11 conserves this residue at S262, but whether it is

27

phosphorylated by Drosophila PAR-1 is unknown. Similar to FIP2, dRip11 interacts with Rab11

and MyoV for trafficking (Li et al., 2007). Like RCP, dRip11 contains two PEST domains that

may promote its degradation.

dRip11 interacts with Rab11 for the trafficking of rhodopsin in the fly eye (Li et al.,

2007). Vesicles bearing dRip11, Rab11 and the light-sensing pigment rhodopsin are trafficked to

the base of the rhabdomere, where they are delivered to the growing membrane. This trafficking

depends on Myosin V and occurs along actin filaments. Li et al. (2007) found that in dRip11

mutant photoreceptor cells, MyoV, Rab11 and rhodopsin no longer traffic to the base of the

rhabdomere and actin localization becomes more diffuse. In addition, the cytoplasm of dRip11

mutant photoreceptor cells is abnormally filled with vesicles. dRip11 also plays a role in tracheal

cell intercalation. During tracheal development, epithelial cells intercalate to extend the dorsal

branches from the dorsal trunk, the tube that connects the trachea to the outside. The

transcription factor Spalt (Sal) inhibits intercalation in the dorsal trunk, likely by mediating the

accumulation of Rab11 and dRip11 in the dorsal trunk (Shaye et al., 2008). Overexpressing

dRip11 leads to increased Rab11 accumulation in the dorsal trunk and a concomitant 56% of

embryos show dorsal branch intercalation defects. Expression of a DN-dRip11 truncation leads

to dorsal trunk intercalation defects in 20% of embryos and dRip11 mutant embryos also show

abnormal dorsal trunk intercalation. This suggests dRip11 may modulate Rab11 accumulation in

the dorsal trunk, where Rab11 impacts the recycling of adherens junction components, thereby

affecting intercalation through the remodeling of cell-cell junctions.

The Drosophila Class II Rab11-FIP Nuf is the Class II Rab11-FIP in Drosophila. It was

identified by and named after its embryonic ‘nuclear fallout’ phenotype. Cellularizing nuf mutant

embryos have defects in furrow invagination and the nuclei do not align along the cortex, but

rather ‘fall’ inside the embryo (Sullivan et al., 1993). While Nuf shares sequence homology with

mammalian Class II FIPs, Nuf does not contain an EF-hand domain and cannot bind Arf6

(Wilson et al., 2005). Therefore, there may be differences in Drosophila Nuf function compared

to that of the mammalian Class II FIPs. Nuf is required for embryo cellularization, which is an

unconventional form of cytokinesis. During Drosophila development, nuclei undergo 13 mitotic

divisions within a syncytial embryo (Mazumdar and Mazumdar, 2002; Strickland and Burgess,

2004). Cellularization occurs once the nuclei enter interphase of cycle 14. Plasma membrane

invaginates to form furrows between the nuclei, separating each nucleus completely, and creating

28

a polarized epithelial sheet that surrounds the now cellularized embryo. Drosophila

cellularization shares many features with conventional cytokinesis. The invaginating membrane

front, called the furrow canal, contains several contractile ring components, including F-actin,

myosin II, anillin, septins and other actin-binding proteins (Lecuit, 2004). Despite the presence

of actin and myosin, it is thought that the primary force driving invagination of the furrow canal

is vesicle trafficking. Embryos injected with DN-Rab11 have defects in furrow invagination and

show a ‘nuclear fallout’ phenotype (Pelissier et al., 2003). Nuf localizes near the centrosomes in

prophase and is cytoplasmic during other phases of division. nuf mutants have gaps in their

metaphase furrows so that by cellularization, furrows are incomplete and encompass multiple

nuclei (Rothwell et al., 1998). It is hypothesized that Nuf may act at the centrosome to initiate

the transport of actin or actin-remodeler-bearing vesicles to the furrow, since both membrane-

containing puncta and actin fail to localize to the furrow in nuf mutant embryos (Cao et al., 2008;

Rothwell et al., 1998; Rothwell et al., 1999). Injection of a constitutively active mammalian

RhoA(Q63L) into nuf embryos rescued the furrow defects at the site of injection. Hence, Nuf

likely acts by stabilizing actin at the furrows, possibly acting upstream of RhoA-mediated actin

polymerization (Cao et al., 2008).

There are several outstanding questions regarding the roles of Nuf and dRip11. While

Nuf is required for cellularization, its role and potential binding partners in conventional

cytokinesis are not known. It is also not known whether dRip11 may play a role in Drosophila

cytokinesis. Although Rab11 is required for the successful completion of spermatocyte

cytokinesis, neither dRip11 nor Nuf, both Rab11 effectors, have been examined for functions in

Drosophila spermatocyte cytokinesis. While Class II, and not Class I, FIPs are involved in

mammalian cell cytokinesis, it is not clear whether this distinction will hold in Drosophila,

especially considering differences in Nuf binding domains as compared to mammalian Class II

FIPs. Hence, both Nuf and dRip11 are excellent candidates to be investigated for potential roles

in spermatocyte cytokinesis.

29

1.4 Thesis goals

1.4.1 Characterizing the Fwd cytokinesis pathway in Drosophila spermatocytes

Fwd likely regulates Drosophila spermatocyte cytokinesis, in part, by regulating Rab11-

mediated trafficking to the cleavage furrow (Polevoy et al., 2009). To further understand how

fwd acts, my goal was to place fwd in a pathway with other genes known to be required for

Drosophila spermatocyte cytokinesis. funnel cakes (fun) and giotto (gio) were among male-

sterile mutants that, like fwd, gave defects in F-actin ring constriction during Drosophila

cytokinesis (Giansanti et al., 2004). Also, like fwd, gio mutants fail to properly localize Rab11 to

the midzone (Giansanti et al., 2007). These genes were thus chosen as candidates to be involved

in a Fwd cytokinesis pathway.

I hypothesized that Gio is the PITP responsible for transferring PI to membranes that are

accessible to Fwd, that the exocyst subunit Fun may target PI4P vesicles to the furrow, and that

Rab11 may act to reciprocally regulate Fwd (Fig. 1.6). Gio may be required for PI4P localization

since local enrichment of PI near the Golgi may allow Fwd to generate PI4P during cytokinesis.

Mutations in funnel cakes (fun), which encodes the Sec8 subunit of the exocyst component, lead

to cytokinesis defects (Giansanti et al., 2004). In yeast, disrupting the exocyst results in the

accumulation of vesicles at the mother-bud neck (Salminen and Novick, 1989). Mutants for Sec8

accumulate vesicles near the site of division and cannot complete separation (Wang et al., 2002).

Exo70p, a component of the mammalian exocyst complex, is required for cytokinesis and Rab11

localization to the furrow (Fielding et al., 2005). If the exocyst directs PI4P-containing vesicles

to the cleavage furrow for fusion, I would expect to see aberrant accumulations of PI4P in fun

mutants. Since Fwd localizes Rab11 to the Golgi (Polevoy et al., 2009), I tested whether the

reciprocal is true by examining Fwd localization in rab11 mutants. I tested whether gio mutants

mislocalize PI4P or Fwd and whether fun mutants mislocalize PI4P during cytokinesis. These

experiments will place fwd in a cytokinesis pathway with these genes.

30

1.4.2 Examining potential roles for dRip11 in spermatogenesis

To further understand the mechanism by which Fwd regulates cytokinesis, we

collaborated with the lab of Anne-Claude Gingras to perform affinity purification coupled to

mass spectrometry (AP-MS) on the human homolog of Fwd. One potential interactor that was

identified was Rab11-FIP2 (Kean, M. and Gingras, A.-C., personal communication). FIP2 was

identified in biological duplicates of AP-MS using FLAG-tagged PI4KIIIβ. As a validation of

the AP-MS, known interactors were identified, including 14-3-3 proteins, which may positively

regulate PI4KIIIβ by protecting the kinase from deactivating dephosphorylation, and Rab11 (de

Graaf et al., 2004; Hausser et al., 2006; Polevoy et al., 2009). The list of potential interactors

excluded proteins identified in eight FLAG alone samples. The list also excluded proteins that

are commonly identified, i.e. proteins with > 30% occurrence in an internal database containing

information from > 700 independent AP-MS experiments. This served to increase the probability

of identifying specific interactors. FIP2 had an averaged spectral count of 3.5, which was above

the cut-off of 2. Based on these results, we hypothesized that Fwd and dRip11, the Drosophila

homolog of FIP2, may interact in the fly. Since dRip11 and Fwd have been characterized in

different Rab11-dependent trafficking processes, I investigated whether dRip11 has a function

during spermatogenesis. I performed immunostaining and genetic analysis to determine its

localization in the testis and whether it has a role in cytokinesis.

31

Figure 1.6. Model for fwd cytokinesis pathway.

Diagrams of a portion of a spermatocyte at a stage prior to (A) or during (B) cytokinesis, highlighting steps of the model to be tested. The model: (1) Prior to cytokinesis, Gio may deliver PI to membranes accessible by Fwd. (2) Fwd is situated at the Golgi, where it generates PI4P and recruits Rab11. During cytokinesis (B), the Golgi disperses. (3) PI4P and Rab11 accumulate at the cleavage furrow, likely on vesicles, which may be targeted to the furrow by the exocyst subunit Fun (4). N (nucleus), ER (endoplasmic reticulum), TGN (trans-Golgi network). Adapted from a figure by Ronit Wilk.

32

Chapter 2

Results

Experiments examining localization of Nuf in wild type and Rab11 dominant negative lines were

performed by Lauren Del Bel.

33

2.1 Materials and Methods

2.1.1 Fly stocks Flies were raised on standard cornmeal molasses agar at 25ºC. FM7i, Act-GFP/C(X) and

the duplication Dp(1;3)DC349, which contains a portion of the X chromosome duplicated on the

third chromosome, were obtained from the Bloomington Stock Center (Bloomington, Indiana).

The ethyl methanesulfonate (EMS)-induced rab1193bi stock was kindly provided by M. Erdélyi

(Hungarian Academy of Sciences, Szeged, Hungary; Jankovics et al., 2001). The rab1193bi

hypomorphic allele has an arginine to tryptophan missense mutation at amino acid 104

(Giansanti et al., 2007). funz3-1010 and gioz3-3934 alleles were graciously provided by M. Gatti and

M. Fuller (Università “La Sapienza”, Rome, Italy and Stanford University School of Medicine,

Stanford, California; Giansanti et al., 2004). The EMS-induced fwd3 allele has a stop codon at

amino acid 310, resulting in a truncated protein (Brill et al., 2000). fwd3 is a genetically null

allele and was analyzed over the deficiency Df(3L)7C, which removes the entire fwd coding

region (Brill et al., 2000). The dRip11KG02485 mutant was provided by D. Ready (Purdue

University, West Lafayette, IN; Li et al., 2007). Wild-type Rab11-GFP and two dominant

negative Rab11-GFP (N124I) stocks were provided by H. Chang (Purdue University, West

Lafayette, IN). YFP-PH-FAPP and GFP-FwdKD constructs are previously published (Polevoy et

al., 2009; Wei et al., 2008).

2.1.2 Immunofluorescence and microscopy Testis preparations were from 0-2 day-old males, except where otherwise noted. YFP-PH-

FAPP and fluorescent Fwd constructs were examined in live squashed testis preparations. Testes

were dissected in testis isolation buffer (Casal et al., 1990) containing 8.3μg/ml Hoechst 33342

(Sigma-Aldrich) for DNA staining, cut with a tungsten needle on poly-lysine coated slides, and

squashed with a coverslip. For immunostaining, squashed testis preparations were frozen in

liquid nitrogen. The coverslips were removed, and samples were chilled in 95% ethanol for at

least 10 minutes, fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) for 7

minutes at room temperature, permeabilized twice for 15 minutes each in PBS containing 0.3%

Triton X-100 and 0.3% sodium deoxycholate, washed for 10 minutes in PBS with 0.1% Triton

X-100 (PBT), blocked for 30 minutes in PBT with 3% bovine serum albumin (PBTB) at room

temperature, and incubated at 4ºC overnight with primary antibody. Primary antibodies used

34

included rabbit anti-dRip11 antibody (1:1000) from D. Ready (Purdue University, West

Lafayette, IN; Li et al., 2007), rabbit anti-Nuf antibody (1:500) from B. Riggs and W. Sullivan

(University of California, Santa Cruz, CA; Riggs et al., 2003) and mouse anti-GFP antibody

(1:1000; Molecular Probes). Samples were washed 3 times for 5 minutes each and once for 15

minutes in PBTB, incubated with Alexa fluorochrome-conjugated secondary antibodies (1:1000;

Molecular Probes) and/or rhodamine-phalloidin (1U/ml; Molecular Probes) in PBTB for 1 hour

at room temperature and washed for 15 minutes in PBT. Samples were then incubated in 1 μg/ml

DAPI (Sigma-Aldrich) in PBT for 10 minutes and washed twice for 15 minutes each in PBT.

Samples were mounted in 9:1 glycerol/PBS containing 100 mg/ml p-phenylenediamine, sealed

with nail polish and examined within 1-2 days. Testes preparations were observed with a 40x and

63x phase-contrast objectives on an upright Zeiss Axioplan 2 epifluorescence microscope

equipped with an Axiocam camera using Axiovision software (Carl Zeiss). Images were

imported into Adobe Photoshop and adjusted for brightness and contrast. Control and

experimental samples were imaged in identical conditions and adjusted using identical

manipulations.

To collect dRip11 mutant larvae, flies from the stock dRip11 KG02485/FM7,GFP were allowed

to lay eggs on fruit juice agar plates for 1-3 hours. Larvae were sorted the next day using a Leica

fluorescence stereoscope. GFP-negative larvae were mutant for dRip11 (dRip11 KG02485/Y). 30-40

larvae were collected per vial. The larvae were allowed to develop until 3rd instar or pharate adult

stages, and then dissected and examined for defects or immunostained as described above.

2.1.3 Immunoblotting Samples were prepared using approximately 30 pairs of testes, 10-20 adult fly heads, or

11 larvae per genotype. Embryos were collected overnight on fruit juice agar plates. Samples

were ground with a pestle and boiled for 10 minutes in SDS sample loading buffer. The

supernatant was isolated after centrifugation. Proteins were separated in a 10% SDS-

polyacrylamide gel and transferred to a nitrocellulose membrane (Amersham Hybond-ECL GE

Healthcare, Piscataway, NJ) using a wet transfer apparatus (Hoefer Blot Module, Holliston,

MA). Blots were probed with anti-dRip11 antibody (1:10,000; Don Ready) and anti-alpha-

tubulin antibody (1:250; Amersham, Piscataway, NJ). HRP-conjugated anti-rabbit or anti-mouse

35

secondary antibodies (Amersham; Piscataway, NJ) were used at 1:10,000 and visualized by

chemiluminescence (Amersham ECL Western Blotting Detection Reagents, Piscataway, NJ).

2.2 Results

2.2.1 Marker analysis in known cytokinesis mutants To further understand how Fwd acts during cytokinesis, my goal was to place Fwd in a

genetic pathway in relation to other genes known to affect cytokinesis. I generated several fly

stocks (Table 1) that contained both the mutation of interest and the marker of interest. To

examine localization of PI4P, I used a fusion protein containing the pleckstrin homology (PH)

domain of four-phosphate-adaptor protein (FAPP) fused to yellow fluorescent protein (YFP)

(Dowler et al., 2000). FAPP is a PI4P-binding protein that also recognizes Arf1 GTPase at the

Golgi (Godi et al., 2004). To examine localization of Fwd, I used a catalytically inactive form of

Fwd fused to GFP (FwdKD-GFP) (Polevoy et al., 2009).

While it has been reported that monomeric red fluorescent protein (mRFP)-PH-FAPP

accumulates at the equator in dividing spermatocytes (Polevoy et al., 2009), I found that the

YFP-marked version did not always appear to mark the furrow, was very dim, and bleached

quickly. These caveats meant that it was not possible to clearly distinguish between PI4P

localization in wild-type versus mutant cells since there was variability in localization even in

wild type, where at times the marker would localize to the furrow and at other times would

appear more diffuse. I examined animals with only one copy of the YFP-PH-FAPP marker. I

found that two copies occasionally caused cytokinesis defects, as seen by the presence of

multinucleate spermatids. This likely occurs because of titration of PI4P by the marker, again

confirming the importance of PI4P in cytokinesis. With these caveats in mind, I will describe

some of the images.

giotto (gio) mutants exhibit defects in cytokinesis (Gatt and Glover, 2006; Giansanti et

al., 2006). gio encodes a PI transfer protein. One hypothesis is that gio may transfer PI to

membranes accessible to Fwd, where Fwd could then generate the PI4P needed for cytokinesis.

If this were the case, gio mutants might show defects in PI4P localization. In control cells (YFP-

PH-FAPP; gio/TM6B), localization of the FAPP marker is variable: at times it is punctate at the

poles of dividing cells, while at other times it is more diffuse (Fig. 2.1, E). Although flies

36

dissected may differ in having either one or two copies of the YFP-PH-FAPP transgene, control

cells for fun mutants (YFP-PH-FAPP/CyO; fun/TM6B), which all had one copy of the PI4P

marker, also showed variability for this marker, suggesting that it is not a copy number issue. As

a further control, these experiments could be repeated using a stock I generated with FAPP

maintained over a balancer chromosome to allow tracking of transgene number (YFP-PH-

FAPP/CyO; gio/TM6B). Whether the signal appears more punctate or diffuse may depend on

specific stages of division, i.e., early and late anaphase, or early and late telophase. gio mutant

telophases had puncta at the poles of the dividing cell, and occasionally at the midzone (Fig. 2.1,

F), which in one instance appeared to accumulate aberrantly at the furrow in a non-punctate

localization (Fig. 2.1, G). Further numbers would be needed to verify whether this non-punctate

accumulation is representative. It is possible that subtle differences in localization are not easily

detected by fluorescence microscopy or without the aid of a contrasting second marker, for

instance, a Golgi marker or a marker to highlight the secretory pathway.

funnel cakes (fun) encodes the Sec8 subunit of the exocyst complex (Fabian et al., 2010).

fun is required for cytokinesis, and the mutants exhibit cytokinesis defects, presumably due to

defects in targeted membrane delivery (Giansanti et al., 2004). If the exocyst is required for

targeted fusion of PI4P-containing organelles at the furrow, then fun mutants may aberrantly

accumulate PI4P-containing organelles either at the furrow or on their way to the furrow. My

preliminary data suggest that this is not the case; as in wild type, PI4P in fun mutants was present

in puncta at the poles of the dividing cells and occasionally near the furrow, and, importantly, the

signal did not appear to aberrantly accumulate in dividing cells (Fig. 2.1, A-D). Further

experiments would be useful to ensure that it is not a timing issue. For instance, very late

telophase cells may show a phenotype, whereas early telophase may not. In the future,

localization of Rab11 or a secretory marker could be examined in fun mutants. This would

demonstrate whether fun affects PI4P and Rab11 localization similarly. The mutant onion rings

(onr), which encodes the Exo84 subunit of the exocyst, gives cytokinesis defects and these

experiments could be repeated in this mutant to verify results.

37

Table 1. List of fly stocks. Fly stocks generated to analyze the localization of PI4P, using YFP-

PH-FAPP, and Fwd, using GFP-FwdKD, in cytokinesis mutants. Stock with an asterisk (*) was

generated by Gordon Polevoy.

Experiment Genotype

PI4P in fun w ; P{w+, YFP-PH-FAPP} / CyO ; fun z3-1010 / TM6B

PI4P in gio w ; P{w+, YFP-PH-FAPP} / CyO ; gio z3-3934 / TM6B *

FwdKD in rab11 w, P{w+, GFP-FwdKD} ; ; rab1193bi / TM6B

FwdKD in gio w, P{w+, GFP-FwdKD} ; CyO / +; gio z3-3934 / TM6B

38

Figure 2.1. PI4P localization in fun and gio mutant dividing spermatocytes.

Phase contrast micrographs (left) and corresponding fluorescence micrographs (right) of dividing spermatocytes expressing YFP-PH-FAPP to mark PI4P. (A, A’, B, B’, E, E’) Wild-type (fun/TM6B and gio/TM6B) dividing cells contain YFP-PH-FAPP puncta at the poles (arrows) and occasionally near the furrow (arrowhead). (C, C’, D, D’) fun mutant dividing cells (fun/fun) contain YFP-PH-FAPP puncta at the poles (arrows). (F, F’, G, G’) gio (gio/gio) mutant dividing cells have YFP-PH-FAPP puncta at the poles (arrows) and occasionally near the furrow (arrowhead). This localization is at times less punctate (G’) at the furrow (arrowhead). Scale bar, 20 μm.

39

To investigate whether gio localizes Fwd, I examined GFP-FwdKD in gio mutants.

Similar to results using the FAPP marker, GFP-FwdKD in gio mutants gave inconclusive results,

with the marker displaying variability in terms of punctate versus diffuse localization in wild-

type cells. In gio mutants, GFP-FwdKD was at times found at the poles of dividing cells, as in

control cells (Fig. 2.2, C-D). gio is an essential gene, and because the mutant is not a null allele,

it is possible that potential defects in the localization of PI4P and Fwd are overcome by some

residual protein function. Alternatively, while providing the substrate for Fwd, Gio may not be

required to localize Fwd.

Since fwd mutants fail to properly localize Rab11 (Polevoy et al., 2009), I wanted to

determine whether rab1193bi mutants fail to localize Fwd. Interestingly, the GFP-FwdKD signal

appeared to be largely extracellular in the rab1193bi mutants, compared to only rarely in wild

type. This is similar to what had been observed previously for GFP-FwdKD in fwd mutants (Wei,

H.-C. and Brill, J. A., unpublished; see Discussion). However, GFP-FwdKD still localized to

puncta at the poles of the dividing cells and occasionally near the furrow (Fig. 2.3, C-D). Since

the allele of rab11 used is a weaker allele, this experiment could be repeated using a more

penetrant rab11 mutation, for instance the trans-heterozygous rab11E(To)11/rab1193bi, which gave

42% multinucleated spermatids (Giansanti et al., 2007).

40

Figure 2.2. FwdKD localization in gio mutant dividing spermatocytes.

Phase contrast micrographs (A-D) and corresponding fluorescence micrographs in grayscale (A’-D’) or merged (A’’- D’’) of dividing spermatocytes expressing a GFP tagged, catalytically inactive form of Fwd (GFP- FwdKD). (A-B’’) Dividing wild-type cells (gio/TM6B) contain GFP- FwdKD puncta at the poles (arrows). (C-D’’) gio mutant dividing cells (gio/gio) also have GFP- FwdKD puncta at the poles (arrows). Merged images show GFP-FwdKD in green and DNA in magenta (A’’- D’’). Scale bar, 20 μm.

41

Figure 2.3. FwdKD localization in rab11 mutant dividing spermatocytes.

Phase contrast micrographs (A-D) and corresponding fluorescent micrographs of grayscale images (A’-D’) or merged images (A’’-D’’) of dividing spermatocytes expressing a GFP tagged, catalytically inactive form of Fwd (GFP- FwdKD). (A-B’’) GFP-FwdKD localizes to puncta at the poles of dividing wild-type spermatocytes (arrows) and occasionally near the furrow (arrowhead). (C-D’’) GFP-FwdKD localizes to puncta at the poles of dividing rab11 mutant spermatocytes (arrows) and occasionally near the furrow (arrowhead). Note that the GFP-FwdKD signal appears more outside of the cell in rab11 mutants (C’, D’). Merged images show GFP-FwdKD in green and DNA in magenta (A’’-D’’). Scale bar, 20 μm.

42

2.3 Examining potential roles for dRip11 in spermatogenesis

2.3.1 dRip11 antibody localization To assess dRip11 localization at different stages of sperm development, I used anti-

dRip11 antibody generously provided by Don Ready (Li et al., 2007). In dividing spermatocytes,

the dRip11 antibody colocalized with F-actin at the cleavage furrow and is also present in puncta

at the poles of the cell (Fig. 2.4, A). This localization is similar in fwd dividing cells (Fig. 2.4, B).

At the onset of spermatid individualization, investment cones composed of F-actin form at the

base of the elongated nuclei. At early stages, these appeared to lack any dRip11 antibody

staining. However, as the cones migrated away from the nuclei, puncta of dRip11 antibody

staining were localized adjacent to the leading edge of the actin cones (Fig. 2.4, C-D). These

puncta remained associated with the migrating actin cones during individualization (Fig. 2.4, E).

fwd mutants exhibit defects in individualization, making it is difficult to assess whether the actin

cones have migrated away from nuclei, since the nuclei and cones are both scattered. However,

in many instances, actin cones appeared to have dRip11 antibody-positive puncta at the leading

edge (Fig. 2.4, F-H).

Since dRip11 is a Rab11 effector, I examined its distribution relative to wild-type Rab11

(GFP-Rab11-WT) and a dominant-negative Rab11 mutant (GFP-Rab11-DN). In non-dividing

spermatocytes, the dRip11 antibody localized in the nucleus and in small puncta within the

cytoplasm (Fig. 2.5, A). These puncta did not appear to colocalize with GFP-Rab11-WT. dRip11

antibody localization was similar in GFP-Rab11-DN expressing non-dividing and dividing

spermatocytes (Fig. 2.5, B). Although GFP-Rab11-DN spermatocytes fail cytokinesis, they do

form an actin ring at the equator, suggesting that a functional contractile ring is formed (Fig. 2.5,

G, H). In addition to the nuclear and punctate distribution of dRip11 in wild-type spermatocytes,

a dot of dRip11 antibody staining can be seen adjacent to the nucleus of spermatids (Fig. 2.5, E).

GFP-Rab11-DN spermatids are multinucleate, and appear to have multiple dots of dRip11

antibody staining next to each nucleus, rather than just one (Fig. 2.5, F). Further experiments are

needed to determine whether this staining localizes to a particular structure.

Although dRip11 antibody localization appears normal in dividing GFP-Rab11-DN

spermatocytes (Fig. 2.5, C, D), preliminary evidence from Lauren Del Bel suggests that Nuf

43

localization may be perturbed. Lauren found that in wild-type cells, Nuf localizes tightly to the

cleavage furrow (Fig. 2.5, I). In GFP-Rab11-DN cells, Nuf-positive puncta are often found in a

line perpendicular to the cleavage furrow axis (Fig. 2.5, J). Although the images shown for Nuf

are from two different GFP-Rab11-DN lines (line 6 for Nuf and line 1 for dRip11), my

preliminary results suggest dRip11 localization is similar in both GFP-Rab11-DN lines.

Interestingly, a similar phenotype had been observed in fwd mutants, where Nuf accumulates in a

line perpendicular to the cleavage furrow axis (Wei, H.-C. and Brill, J.A., unpublished).

To understand whether dRip11 antibody localization pointed towards a role for dRip11 at

these structures, I next examined dRip11 mutant male germ cells.

44

45

Figure 2.4. dRip11 antibody localization in wild type and fwd male germ cells.

Fluorescence micrographs of dRip11 antibody staining in wild-type (A, C-E) and fwd mutant (B, F-H) male germ cells. dRip11 antibody colocalizes with F-actin at the cleavage furrow (arrows) and is present in puncta at the poles (arrowheads) in both wild-type (A) and fwd mutant (B) spermatocytes. In wild type, dRip11 antibody does not stain spermatids early in individualization, when the actin cones have not yet begun to migrate (C). Puncta of dRip11 antibody staining are found adjacent to the leading edge of the actin cones as they start to migrate away from the nuclei (D) and remain in adjacent to the actin cones as the cones migrate the length of the sperm tails (E). In fwd mutant sperm, it is difficult to assess whether the actin cones have begun to migrate away from the nuclei, since both the nuclei and actin cones are out of register. Actin cones are found with (G) and without (F) dRip11 antibody stained puncta near the leading edge. Actin cones that have clearly migrated away from the nuclei exhibit dRip11 antibody staining near the leading edge (H). Merged images show dRip11 in green, F-actin in red and nuclei in blue. Scale bar, 20 μm.

46

47

Figure 2.5. dRip11 antibody localization in Rab11 dominant negative expressing cells.

Fluorescence micrographs of dRip11 antibody staining in wild-type (GFP-WT-Rab11-GFP; A, C, E) and Rab11 dominant negative (Rab11-DN-GFP, line 1; B, D, E) male germ cells. In wild-type spermatocytes, GFP-WT-Rab11 localizes to puncta throughout the cytoplasm (A) and dRip11 antibody stains the nucleus and small puncta in the cytoplasm (A’). In Rab11-DN spermatocytes, GFP-DN-Rab11 is diffuse (B) and dRip11 antibody stains the nucleus and small puncta in the cytoplasm (B’). Wild-type dividing cells show dRip11 antibody staining at the cleavage furrow (C’; arrow). GFP-Rab11-DN cells also show dRip11 antibody staining at the cleavage furrow (D’; arrow). (E, F) Phase contrast micrographs of wild-type and GFP-Rab11-DN spermatids. Wild-type spermatids have one dRip11 antibody-positive punctum next to each nucleus (E’), whereas GFP-Rab11-DN spermatids have multiple dRip11 antibody-positive punta next to the nuclei (F’). In the merged images for A-F, Rab11 is in green, dRip11 is red, and DNA is blue. GFP-Rab11-DN cells stain for actin at the contractile ring (H’; arrows point to cleavage furrow) as do wild-type cells (G’). In the merged images (G’’, H’’), Rab11 is in green, actin is red and DNA is blue. Images in I-J were taken by Lauren Del Bel. I and J show signals from anti-GFP antibody. Nuf accumulates at the cleavage furrow in wild-type cells (I’). GFP-Rab11-DN cells (line 6) accumulate a line of Nuf staining (arrows point parallel to cleavage furrow) perpendicular to the cleavage furrow. Scale bar, 20 μm.

48

2.3.2 Assessing dRip11 function dRip11 is encoded by an essential gene on the X chromosome (Li et al., 2007). Therefore

any males that are hemizygous for dRip11 do not survive to adulthood. To confirm that the

lethality is due to loss of dRip11, I identified a fly line that contains a portion of the X

chromosome duplicated onto chromosome III. The duplication (Dp(1;3)DC349) spans six genes,

including dRip11 (Table 2). The lethal P-element, dRip11KG02485, is contained within this region

and likely affects dRip11 transcription, as it is inserted 83 base pairs upstream of the dRip11 start

site. When I introduced the duplication into the dRip11 mutant stock, it rescued lethality, and

mutant dRip11 male progeny containing the duplication were viable

(dRip11KG02485/Y;;Dp(1;3)DC349). This confirmed that the P-element insertion dRip11KG02485

affects at least one of six genes present on the duplication.

Since dRip11 is on the X chromosome and adult male mutants are lethal, I used a system

that allowed me to identify dRip11 mutant male larvae before they died. I generated a fly stock

containing a loss of function mutant dRip11 allele (dRip11KG02485) balanced over a GFP-marked

balancer chromosome (FM7-GFP). This allowed for the selection of dRip11 mutant male larvae

(dRip11/Y), as they were GFP-negative. The dRip11 mutant larvae lived to 3rd instar, although

their body length remained that of first instar larvae. Based on the size and number of mouth

hooks, they were classified as third instars. Occasionally, larvae developed to pupal stages and,

very rarely, there were a few adult escapers.

Immunofluorescence experiments on larval testes showed that the dRip11 antibody

localized to the nucleus and to ring canals in wild-type (w1118/Y) larvae (Fig. 2.6, A).

Surprisingly, this localization was the same in dRip11 mutant (dRip11KG02485/Y) larvae (Fig. 2.6,

B). Immunofluorescence on pupal testes revealed that the dRip11 antibody stained puncta near

the leading edge of actin cones, just as in wild type (Fig. 2.6, C, D). This suggested that either

the dRip11 antibody recognizes other epitopes or that the dRip11KG02485 allele may not reduce

dRip11 protein levels (see Discussion). To investigate these possibilities, I performed

immunoblotting on whole larval extracts. The dRip11 antibody bound to two polypeptides

present in wild-type larval lysates that migrated at approximately 225 kDa and between 102 kDa

and 150 kDa (Fig. 2.6, F). In dRip11 larval lysates, the polypeptide at 225 kDa was reduced in

abundance, and the polypeptide between 102 kDa and 150 kDa was barely visible, even at high

exposures. Levels of α-tubulin were similar in both wild type and dRip11. A separate

49

immunoblot experiment using adult head and testis lysates revealed a single polypeptide between

102 kDa and 150 kDa. These immunoblots need to be repeated. It is possible that there is an

additional protein product from the dRip11 locus expressed in larvae, accounting for the larger

polypeptide. In any case, the decrease in signal in dRip11 mutant larvae suggests that

dRip11KG02485 could be a loss-of-function allele of dRip11. By phase-contrast microscopy, two

out of 60 spermatids were multinucleate and had abnormal sized nuclei. The sample size was too

small to determine whether this is a statistically significant defect. Further numbers are needed to

determine whether dRip11KG0248 mutant spermatids have cytokinesis defects.

Table 2. Genes present on the duplication (Dp(1;3)DC349) that rescues lethality of dRip11KG0248

mutants.

Gene name Product Function References

dRip11 Class I Rab11 FIP Rhodopsin trafficking

in fly eye; tracheal

cell intercalation

(Li et al., 2007; Shaye

et al., 2008)

wengen (wgn) Tumor necrosis factor

(TNF) receptor

Interacts with ligand

Eiger for TNF

signalling

(Kanda et al., 2002)

CG15040 Unknown - -

CG43289 Unknown - -

CG6540 Unknown - -

CG6617 Unknown - -

50

51

Figure 2.6. Examining dRip11 mutant spermatocytes.

Fluorescence micrographs of wild-type (A, C) and dRip11 (B, D) cells. dRip11 antibody stains the nucleus and ring canals (arrows) in both wild-type (A) and dRip11 mutant (B) spermatocytes from larval testes. dRip11 antibody stains puncta near the leading edge of the actin cones in both wild-type (C) and dRip11 mutant (D) sperm from pharate adult testes. (E) Phase contrast micrograph demonstrating the absence of a gross cytokinesis defect in dRip11 mutant spermatids, as assayed by the presence of one nucleus and a normal-sized mitochondrial derivative per cell. (F) Immunoblot of wild-type (lane 1) or dRip11 (lane 2) larval lysates probed with anti-dRip11 antibody. Polypeptides at ~225 kDa and between 102 kDa and 150 kDa were reduced in the dRip11 mutant lysate (top panel). α-tubulin was used as a loading control (~50 kDa; bottom panel). (G) Immunoblot of wild-type embryos (lane 1), adult heads (lane 2) and testes (lane 3), and fwd testes (lane 4), probed with dRip11 antibody reveals a polypeptide between 102 kDa and 150 kDa in each sample. Embryo lysates gave additional bands (~76 kDa, ~52 kDa). Scale bar, 20 μm.

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Chapter 3

Discussion and Future Directions

53

3.1 Drosophila cytokinesis mutants

3.1.1 Marker analysis in known cytokinesis mutants Since gio, fun and rab11 are likely part of the same pathway as fwd, I tested whether

these genes affect the distribution of the PI4P marker YFP-PH-FAPP or GFP-FwdKD in dividing

spermatocytes. To begin to test whether these genes regulate Fwd localization, I examined GFP-

FwdKD, a catalytically inactive form of Fwd, in rab11 mutants. While I found GFP-FwdKD-

positive puncta at the poles in rab11 mutants, the GFP-FwdKD signal appeared more frequently

extracellular compared to wild type. Since GFP-FwdKD also appears extracellular in fwd mutants,

one speculation is that the plasma membrane of fwd and rab11 mutants may be compromised

due to defects in trafficking of components needed for membrane stability or for control of

osmotic pressure, rendering the cell more fragile. This may explain why the fluorescent signal is

found outside of the cell, since perhaps the cell’s contents are being released during preparation

of samples for microscopy. Indeed, a previous student in the lab found that fwd cells are more

difficult to prepare for live imaging due to their fragility (Wong, R. and Brill, J.A., unpublished).

In support of this, knockdown of Rab11 in C. elegans embryos reportedly causes the embryos to

be more sensitive to pressure and osmotic strength (Zhang et al., 2008).

In cells expressing a dominant negative Rab11, preliminary data indicate that Nuf puncta

localize along a line parallel to the spindle, rather than accumulating at the cleavage furrow.

Interestingly, this Nuf phenotype also occurs in fwd mutants. Thus, both fwd and rab11 may

regulate Nuf trafficking to the furrow, as disruption of either leads to defects in Nuf targeting.

These similarities again suggest the involvement of fwd and rab11 in the same cytokinesis

pathway and raise the possibility that Nuf plays a role in spermatocyte cytokinesis.

To visualize PI4P, I used YFP fused to the PH domain of FAPP. The YFP-PH-FAPP

marker showed variable localization in otherwise wild-type cells that were homozygous for

mutations of interest. Thus, either the marker is not optimal for PI4P visualization or the

mutations are not fully penetrant and have some residual function that allows for proper PI4P

localization. Alternatively, the genes may not affect PI4P distribution. Using a fluorescent tag

that is brighter and does not bleach quickly, such as mRFP-PH-FAPP, would be beneficial for

future experiments. Alternatively, a different PI4P marker could be used, like the Golgi localized

PI4P-binding protein GOLPH3. To validate GOLPH3 as a fluorescent PI4P marker, it could be

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examined in fwd mutants to see whether its signal decreases. GOLPH3 could be co-expressed

with mRFP-PH-FAPP to compare the extent of co-localization. In addition, GOLPH3

localization could be examined in cells expressing catalytically inactive Fwd to see whether its

localization is more diffuse.

Besides exploring a different PI4P marker or brighter fluorescent tag, visualizing a

second marker in tandem with a PI4P marker may make analysis simpler by providing a point of

comparison. For instance, a marker of the secretory pathway, a Golgi marker or Rab11 could be

expressed together with a PI4P marker to differentiate subtle differences in localization between

wild type and mutant. Moreover, the stage of division may have an impact on PI4P localization

and could be controlled for in future experiments by using markers to distinguish between early

and late telophase. Centrioles migrate during cytokinesis and using a centriole marker, such as

Unc-GFP (Baker et al., 2004) or GFP-PACT (Basto et al., 2006), or observing β-tubulin-GFP to

mark astral microtubules (Giansanti et al., 2004) would relay the timing of cytokinesis. In early

and mid-telophase of meiosis I, each spindle pole has one centrosome from which astral

microtubules emanate. As telophase proceeds, the sister centrioles within each centrosome

separate and form two distinct arrays of astral microtubules. In late telophase, the two arrays

migrate to opposite sides of the nucleus before the second meiotic division. Thus, following the

degree of centriole or astral microtubule separation can serve as a marker for telophase

progression.

To aid in interpretation of the fluorescence imaging results, the number of dividing cells

that contain puncta at the poles and equator could be quantified, as could the average number of

puncta within a cell. The signal intensity could also be measured in a specific area, such as the

cleavage furrow, and compared between wild-type and mutant cells. Measuring signal intensity

of puncta compared to the surrounding cytoplasm could be useful in determining whether the

signal is more diffuse in mutant cells. These measurements can be repeated for a number of cells

to determine whether any differences are of statistical significance.

It should be noted that the mutant alleles of rab11, gio and fun used in these experiments

were not null alleles. Therefore it is possible that the presence of some residual protein function

may be sufficient to localize Fwd or PI4P but not enough for successful cytokinesis, depending

on the nature of the mutant protein expressed. Alternatively, YFP-PH-FAPP localization may not

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appear perturbed because of redundant or parallel pathways that target PI4P to the furrow.

Perhaps PI4P-containing vesicles have multiple interactors that can lead to successful targeting

and fusion at the furrow. Thus, mutation of one gene may compromise the pathway so that

cytokinesis fails, but some of the players may reach their proper destinations due to redundant

localization tags.

To examine Fwd function in the future, I would troubleshoot using multiple markers as

discussed above in order to examine subtle defects in PI4P and Fwd localization. This can be

piloted using already characterized fwd mutants to confirm whether defects are interpretable.

Immuno-electron microscopy (immuno-EM) is another powerful tool to examine organelle

morphology and accumulation of PI4P in mutants. For example, if exocyst mutants have an

increase in number of vesicles or membrane structures near the furrow, these organelles could be

examined for PI4P or Rab11 immuno-gold labelling. This would demonstrate which organelles

PI4P and Rab11 label at the furrow and whether their localization is affected in the mutants.

Looking in the area of the cleavage furrow would require correlative light and transmission EM,

but would allow a thorough characterization of the defects in the mutants. To analyze EM data,

organelle morphology could be examined for defects by quantifying the surface area and density

of membrane compartments. Additionally, the number of gold particles could be quantified per

compartment or per area to determine whether there is a decrease in labeling within a specific

compartment, or a difference in distribution among compartments. The experiments proposed

above would strengthen our understanding of the fwd cytokinesis pathway.

3.1.2 Fwd in cytokinesis: open questions An assumption made in these experiments is that PI4P is present at the furrow on Rab11-

vesicles. However, the nature of the PI4P-containing organelle is not known. Giansanti et al.

(2007) observed the movement of Rab11 puncta by time-lapse microscopy and found that in

mid/late telophase, 88% of Rab11 puncta traveled from the poles of the cell to the equator. This

suggests that Rab11 is present on post-Golgi vesicles. Imaging of PI4P has not revealed directed

movement of PI4P to the furrow (Polevoy et al., 2009). Higher resolution microscopy or brighter

PI4P markers may allow the visualization of PI4P vesicle movement, although the possibility

remains that PI4P may not accumulate on vesicles, but some other organelle. For instance, PI4P

may accumulate within ER at the cleavage furrow or at ER exit sites that contact the PM at the

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furrow. In Drosophila spermatocytes, an ER-derived structure called the spindle envelope

surrounds the spindle. Rab11 is found along the spindle envelope and in similar regions as an ER

marker (Giansanti et al., 2007). Rab11 also partially colocalizes with an ER marker in C. elegans

embryos and knockdown of Rab11 gives defects in ER morphology during metaphase (Zhang et

al., 2008). Thus rab11 or fwd may affect ER morphology. It is not clear whether Drosophila

spermatocytes contain REs. In prophase, Rab11 does not accumulate in a pericentriolar location,

but is found associated with Golgi throughout the spermatocyte cytoplasm (Giansanti et al.,

2007). Dividing spermatocytes exhibit Rab11 and an ER marker concentrated in pericentriolar

regions and these do not appear as distinct REs. Thus Drosophila male germ cells may not have

a canonical pericentriolar RE. Immuno-EM would allow us to discern the nature of the organelle

that PI4P and Rab11 decorate, using antibodies against recycling endosomes, secretory

endosomes, Golgi and ER.

In addition to the nature of the organelle at the furrow, another unknown is the primary

defect in fwd mutants. In fwd mutant spermatocytes, Golgi markers appeared either more diffuse

or in smaller puncta by immunofluorescence (Polevoy et al., 2009). In the future, these defects

can be studied at the ultrastructural level by EM. In Drosophila spermatocyte metaphase I, Golgi

stacks break down into smaller structures, the nature of which is not known (Giansanti et al.,

2007). In 96% of wild-type telophases, puncta stained for the Golgi protein Lava lamp (Lva)

were found near the poles and excluded from the region of the cleavage furrow. However, 48%

of rab11 mutant telophases accumulated Lva-positive puncta at the furrow. This phenotype also

occurs in gio and fwd mutants (Giansanti et al., 2006; Polevoy et al., 2009). These Lva-positive

puncta may be post-Golgi vesicles that fail to fuse with the furrow membrane in these mutants

(Giansanti et al., 2007). However, this seems somewhat unlikely, since Lva is a golgin and

would not be expected to localize to post-Golgi vesicles. Thus, a second possibility is that

accumulation of Lva at the equator in these mutants represents disorganization of Golgi, rather

than a failure of vesicle fusion. PI4Kβ has been implicated in Golgi organization in yeast and

mammals (see Section 1.4). If Fwd is required for Golgi structure or function in Drosophila

spermatocytes, fwd may affect cytokinesis via a defect in Golgi morphology, which could be

analyzed by EM. If Golgi structure appears normal, the defect in fwd mutants may be more

specific, for instance, in localizing Rab11 and mediating trafficking through Rab11 vesicles.

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Fwd and Rab11 may impact cytokinesis through effects on the cytoskeleton. 31% of fwd

mutant telophases show disrupted actin organization near the contractile ring, with actin

accumulating near the ring and not fully colocalizing with myosin II (Brill et al., 2000). In rab11

mutants, a subset of telophase cells displayed actin rings that were incompletely constricted,

broken or abnormally thick (Giansanti et al., 2007). The degree of central spindle disruption

correlated with the degree of actin ring defect, highlighting the interdependence of these

structures. rab11 mutant telophases that divided normally had normal central spindles, whereas

rab11 mutant telophases that failed division had disrupted central spindles that fell apart as the

furrow regressed. Thus, the stability of central spindle microtubules requires interaction with the

contractile ring (Giansanti et al., 2007; Giansanti et al., 2004), further complicating the search for

a primary defect. Rab11 may also impact the cytoskeleton through an effect on spindle

orientation (Zhang et al., 2008). Knockdown of Rab11 in C. elegans embryos led to improper

spindle positioning and violent rocking movements of the spindle, as well as shorter metaphase

astral microtubules that do not reach the cortex. This raises the question of whether activated

Rab11 improves the outcome of fwd cytokinesis by coordinating favourable spindle orientation.

To test this, spindle orientation could be examined in wild type, fwd and fwd expressing Rab11-

GTP. This would indicate whether the fwd cytokinesis defect is due in part to defects in spindle

orientation and whether the role of Rab11 in this pathway is mediated in part through its effects

on the spindle.

3.2 Examining dRip11 and Nuf in cytokinesis dRip11 has been characterized in the eye as an adaptor that links Rab11-containing

vesicles bearing rhodopsin to MyoV motor for trafficking (Li et al., 2007). In the tracheal

system, dRip11 mediates the accumulation of Rab11 (Shaye et al., 2008). The Gingras lab found

that human PI4KIIIβ associates with FIP2 by mass spectrometry. This prompted us to consider

whether dRip11 may interact with Rab11 for trafficking in spermatocytes. My initial localization

of dRip11 antibody to the contractile ring appeared promising, but I subsequently found that the

dRip11 antibody signal did not decrease by immunofluorescence in dRip11 KG02485 mutant larval

testes. My preliminary immunoblot analysis suggested that the dRip11 KG02485 mutant may be a

loss of function mutation, since the polypeptides appeared reduced in the mutant lane. However,

the size of the bands did not correspond to predicted weights. The two predicted dRip11

transcripts would yield different polypeptides of 91.9 kDa and 44.6 kDa (Flybase.org). Li et al.

58

(2007) report a single polypeptide at ~100 kDa from fly head lysates. When I performed

immunoblot analysis of adult head and testis lysates, I also obtained a single polypeptide

between 102 kDa and 150 kDa. Embryo lysates gave additional polypeptides, at approximately

76 kDa and 52 kDa. When I performed an immunoblot against whole larval lysates, I obtained a

polypeptide between 102 kDa and 150 kDa and also an unexpected polypeptide at approximately

225 kDa. An immunoblot using lysates from both larvae and adult testes could be performed to

compare these samples in the same experiment. Although I observed a general reduction in

dRip11 antibody signal in dRip11KG02485 larval lysates, it is possible that the mutant may not

affect dRip11 levels in the testis. dRip11KG02485 mutation is a P-element insertion 83 base pairs

upstream of the start site. There may be a testis transcript that employs an alternate start site that

the P-element insertion does not affect. To rule out this possibility, reverse-transcriptase coupled

PCR could be performed on testes to examine which transcripts are expressed in this tissue.

While the antibody may be showing dRip11 levels by Western blot, it could be cross-

reacting to other epitopes in the testis and therefore not revealing dRip11 localization. In the fly

eye, dRip11 KG02485 mutant photoreceptors show a decrease in dRip11 antibody staining (Li et al.,

2007). There was a minor signal still detectable, suggesting that the antibody might recognize a

non-specific epitope or that there may be some protein perdurance in these clones. It is possible

that there is tissue dependent background with the antibody, where there is little background in

the eye but more in the testis. In any case, the antibody needs further validation. One approach

would be to first incubate the antibody with GST-dRip11 to deplete the antibody and then use the

antibody for immunofluorescence or immunoblotting. If the antibody is specific, this should

greatly decrease its signal. Alternatively, a dRip11 fluorescent marker could be generated to

examine dRip11 localization compared to the antibody. I attempted to clone dRip11, but

encountered major difficulties. If this could be done, then a fluorescently tagged dRip11 would

be useful for localization studies.

To examine dRip11 function in the testis, I generated a fly stock containing dRip11KG02485

allele and a fluorescent balancer. This allowed me to identify dRip11 mutant larvae by the

absence of GFP. dRip11 mutant larvae did not show highly penetrant cytokinesis defects by

phase-contrast microscopy, although further numbers would needed to verify this. It is possible

that the P-element insertion may be hypomorphic, as it does not contain a stop codon and is

upstream of the start site. This may lead to low level of functional protein that could rescue any

59

potential defects. Considering that the mutant allele needs further validation, it is important to

follow-up these results with different approaches. RNAi could be used to deplete both dRip11

transcripts. Additionally, another allele of dRip11 (dRip11G0003) has been reported to be protein

null, based on immunostaining with anti-dRip11 antibody in mutant embryos (Shaye et al.,

2008). If this allele represents a stronger loss of function, it should be investigated for effects on

dRip11 levels by Western blot and on dRip11 antibody signal. While mutant larvae could be

generated for these experiments, mutant clones could also be used. dRip11 is an essential gene

on the X chromosome. dRip11G0003 mutant clones could be generated by using the FLP-FRT

system to create clones of cells that lack a rescue transgene and are therefore mutant for dRip11.

Duplication Dp(1;3)DC349, which I found rescues dRip11 lethaliy, can be used as a rescue

transgene, since it duplicates a portion of the X chromosome on the third chromosome. The

duplication could be crossed onto an appropriate FRT chromosome for use in making mosaic

flies. Since I had hypothesized that dRip11 may act to anchor Rab11 vesicles to the furrow, if the

cells lacking dRip11 show strong cytokinesis defects, Rab11, PI4P and actin could be examined

in dRip11G0003 telophase cells.

The experiments outlined above will enable further investigation of a potential role for

dRip11 during cytokinesis. While this hypothesis needs further testing, it is plausible that Nuf

may be the relevant Rab11 effector during spermatocyte cytokinesis. Nuf is required for

cellularization, yet its role in spermatocyte cytokinesis is unknown. Nuf antibody localizes to the

cleavage furrow and both Nuf and Rab11 have been reported to be missing from the furrow in

fwd, rab11 and gio mutants (Giansanti et al., 2007; Polevoy et al., 2009). Expression of activated

Rab11 rescues Nuf localization in fwd mutants. These studies suggest that a cytokinesis pathway

comprising fwd, rab11 and gio is critical for the localization of Rab11 and its effector Nuf to the

furrow, where they may mediate vesicle docking or fusion. nuf is on the third chromosome. To

examine its functions, mutant clones can be made by FLP-FRT mediated recombination. These

can be examined by phase contrast microscopy for cytokinesis defects. Mammalian studies

indicate that Class II FIPs and not Class I FIPs are involved in cytokinesis. However, since

spermatocytes are particularly sensitive to changes in membrane trafficking during cytokinesis, it

is possible that both may play a role in Drosophila cytokinesis or may have roles somewhat

different from their mammalian counterparts. For instance, as in mammals, Drosophila Arf6 is

required for cytokinesis. Nonetheless, whereas Drosophila Arf6 localizes with Rab11 at the

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furrow, it does not control Rab11 recruitment to the furrow, as it does in mammalian cells (Dyer

et al., 2007; Fielding et al., 2005). One reason for this discrepancy may be that Drosophila Arf6

does not bind Nuf, whereas mammalian Arf6 binds FIP3/4. The experiments proposed above

will help establish which FIPs are involved in Drosophila spermatocyte cytokinesis.

Although FIP2 came out of the mass spectrometry screen as a potential interactor of

PI4KIIIβ, this interaction is not necessarily direct or biologically relevant. Moreover, even if it is

biologically relevant, the interaction may be important for a process other than cytokinesis. To

determine whether dRip11 and Fwd interact, yeast two-hybrid or co-immunoprecipitation with

bacterially-expressed and purified proteins could be performed. Gordon Polevoy in our lab

performed yeast two-hybrid analysis of Fwd and dRip11 and did not detect an interaction. It is

possible that Rab11 may serve as a linker between the two proteins and this experiment could be

repeated in cells expressing activated Rab11. If an interaction is detected between Fwd and

dRip11, but no role for dRip11 can be found in the testis, another avenue to explore is whether

Fwd may function alongside dRip11 in the fly eye (Li et al., 2007). To determine if Fwd

functions in the eye, fwd mutant photoreceptors could be examined from either fwd mutant flies

or by generating fwd mutant clones. Photoreceptor cells could be immunostained for rhodopsin,

dRip11, Rab11 or MyoV to determine whether these target properly to the base of the

rhabdomere. Disrupted targeting may indicate a role for Fwd in Rab11 and dRip11-mediated

trafficking in photoreceptor cells. Since Rab11/FIP2/MyoV-trafficking is also critical in

mammalian systems (Chu et al., 2009; Hales et al., 2002), this would suggest that Fwd/PI4KIIIβ

regulation of this triple complex may be conserved.

In conclusion, while it is likely that Fwd and Rab11 act together to mediate Drosophila

spermatocyte cytokinesis, their exact mechanism of action is unknown. Further exploration of

roles for dRip11 and Nuf, together with ultrastructural analysis of known cytokinesis mutants,

will allow a more detailed picture of this important process.

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