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Understanding the Role of the Actin Cytoskeleton in Vesicle Trafficking to Restrict Growth of Drosophila Epithelia
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I. Introduction
The ability of eukaryotic cells to assume a variety of shapes and to carry out
coordinated and directed movements depends on a complex network of protein
filaments that extends throughout the cytoplasm. The extent and pattern of actin
filament polymerization drives the protrusive force and shape of migrating cells, as
well as the cortical tension necessary for maintaining adhesion contacts between cells
and between the cell and its substrate.
1. Actin dynamics and its regulation by actin binding proteins
Actin exists in a cell in two main forms: the monomeric globular actin (G‐actin),
and the polymeric filamentous actin (F‐actin). Under physiological conditions, ATP
bound G‐actin incorporates into growing filaments at a fast‐growing barbed (+) end,
undergoing thereafter a slow hydrolysis into ADP‐actin, as actin monomers are shifted
along the filament towards the slow‐growing pointed (‐) end 1. Actin dynamics and
structure are controlled by a large variety of Actin Binding Proteins (ABPs), including
actin nucleators, depolymerisation factors, actin‐bundling proteins and actin‐
crosslinking proteins. Furthermore, some ABPs link filaments to the plasma membrane
or organelles within the cell, while others use actin filaments as tracks upon which to
move vesicles and organelles 2‐4.
1.1 Actin filament nucleation
The nucleation of actin filaments is an energetically unfavourable event, being
promoted by certain ABPs, such as the Arp2/3 complex and formins. For instance, the
Arp2/3 complex can generate a stable trimer with G‐actin along the side of an actin
filament, producing a new filament branch 5 (Fig.1).
Extensive nucleation occurs mainly at the leading edge of motile cells, where
the meshwork of filaments acts as a platform against which actin polymerization can
push 6, 7 (Fig.x). The morphology of the membrane protrusion varies according to the
growth extent and bundling of the filaments, ranging from filopodia, that arise from
long and bundled filaments, to lamellipodia, that rely on a highly branched network of
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short actin filaments. In general, actin polymerization induced by the Rho‐like GTPases
Cdc42 and Rac, respectively promotes the formation of filopodia and lamellipodia,
downstream of the Arp2/3 complex 5.
Fig.1 – Actin dynamics is controlled by a large array of ABPs. The actin nucleators, such as the Arp2/3
complex, promote de novo actin filament nucleation and branching. Profilin sequesters G‐actin
monomers, translocating them to sites of active filament assembly and promoting prolimerization.
ADF/cofilin factors severe the actin filaments and promote dissociation of the actin monomers from the
pointed (‐) end. Capping proteins (CPs) restrict the access to the barbed (+) end, forming a protein cap
that impedes further addition and loss of actin monomers.
1.2 Actin filament depolymerization and sequestration of actin monomers
Actin depolymerization factors also regulate the extent and spatial pattern of
actin polymerization. For instance, members of the ADF/Cofilin family are known to
dramatically accelerate actin filament turnover in vitro 8, which occurs mainly through
filament severing and monomer dissociation from the pointed end 9 (Fig.1). Similar to
the Arp2/3 complex, ADF/Cofilin seems to be regulated by the activity of Rho‐like
GTPases. For instance, Rho and Cdc42 respectively induce the formation of actin stress
fibers and filopodia 10, 11.
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Besides ADF/Cofilin, other ABPs, like the Cyclase Associated Proteins (CAPs),
regulate the availability of actin monomers by sequestering them and prevent their
incorporation into filaments 2, 12. On the other hand, Profilins enhance exchange of
ADP for ATP on sequestered actin monomers, thereby promoting actin filament
polymerization 13. A generally devised model of actin filament ‘tread‐milling’ assumes
that filament turnover is driven by actin monomer dissociation through ADF/Cofilin,
whereas at sites of rapid actin assembly, the pool of ADP‐actin monomers undergoes a
rapid nucleotide exchange driven by Profilin, to promote new incorporation into the
actin filaments 14 (Fig.1).
1.3 Actin filament termination
Actin filament termination occurs mainly through the activity of filament end‐
binding proteins, such as proteins of the Gelsolin superfamily and Capping proteins
(CPs). For instance, gelsolin is able to dissolve actin gels in vitro 15, but it is still
controversial whether it does so through a severing or a capping activity over the
actin filaments 16, 17. On the other hand, CPs form a highly conserved αβ heterodimer
(CP) that binds to the barbed end of actin filaments, thereby forming a protein cap
that arrests actin polymerization by locally preventing the further addition and loss of
actin monomers 18 (Fig.1). Functional CP, along with the Arp2/3 complex, support the
assembly of a dominantly branched network of small actin filaments, giving rise to the
lamellipodia of migrating cells 19(Fig.2). When capping of actin filaments by CP is
inhibited, long actin filaments can become bundled, giving rise to filopodia 20.(Fig.2)
Intriguingly, besides having a critical role in the termination of filaments, CP has been
suggested to mediate actin filament attachment to the plasma membrane 21, 22.
Although much is now understood about how actin dynamics is regulated,
most work addressing this issue has been developed in cultured cell lines. Few studies
have addressed the importance of cytoskeletal dynamics in the regulation of tissue
homeostasis and morphogenesis in the context of an intact metazoan organism 23.
Throughout this thesis I will concentrate on the role of actin dynamics during
epithelial development of the Drosophila wing imaginal disc.
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Fig.2 – Functional CPs and the Arp2/3 complex support the assembly of a dominantly branched
network of small actin filaments, giving rise to lamelipodia. (b) When the capping of actin filaments is
inhibited, actin filaments can become bundled, giving rise to filopodia. Some molecular components
have been identified that could inhibit or counteract the capping activity: the Ena/VASP family of
proteins seems to exhibit anticapping and antibranching activities and PIP2, CARMIL and
Melanotrophin/V‐1 have been proven to bind and block the C‐terminal capping portion of CP, in vitro 24‐27.
2. Epithelial cell‐cell adhesion and polarity
Simple epithelia generally consist of a laterally coherent layer of cells that have
a distinct apico‐basal polarity, having a apical surface that borders a lumen, a lateral
surface that adheres to neighbouring cells, and a basal surface that adheres to the
extracellular matrix (ECM) or basal lamina 28. A great variety of epithelia line the walls
of cavities and channels or, in the case of the epidermis, serve as the outside covering
of the body, pertaining a variety of functions, such as the selective absorption of
Understanding the Role of the Actin Cytoskeleton in Vesicle Trafficking to Restrict Growth of Drosophila Epithelia
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solutes, the glandular production of hormones, digestive enzymes and termo‐
regulatory fluids, and serving as a physical and immunological barrier against pathogen
attack.
Epithelial cells have a polarized actin system, with a band of filaments
concentrated at the apical zonula adherens (ZA), long actin filaments around the
basolateral cortex and stress fibres localized at the basal surface. The apical actin
network supports cell‐cell adhesion through the adherens junctions (AJ), whereas cell‐
matrix adhesion is supported by stress fibre anchoring to hemiadherens junctions 29.
Cell‐cell adhesion is mainly accomplished by cadherin homophilic interaction
between cells at the AJ 30. In turn, the cytoplasmic domain of cadherins is able to
interact with proteins of the armadillo repeat family, such as Armadillo/β‐catenin
(Arm) and p120‐catenin, thereby regulating actin dynamics at adhesive contacts 31.
For instance, the cadherin‐β‐catenin complex is thought to trap α‐catenin, which
would otherwise compete with the Arp2/3 complex in binding to the actin filaments 32, 33. Although by no means clarified, the apical ‘actin belt’ seems to be of great
importance for the establishment and maintenance of cell‐cell adhesion, therefore
assuring proper epithelium development.
Besides the cadherin complexes, other macromolecular complexes are required
for the establishment of cell‐cell adhesion. For instance, in the subapical region (SAR)
of epithelial cells, the Bazooka (Baz/Par3)/Par6/aPKC complex is required for
establishing the AJ, mainly through the recruitment of apical polarity determinants and
the Rho‐like GTPases Cdc42 and Rac 34‐39. In Drosophila epithelial cells, the mature
Par6‐aPKC complex drives the apical recruitment of the transmembrane protein
Crumbs (Crb) and its cytozolic associates, Stardust (Sdt) and Discs Lost/DPatJ (Dlt),
whose activity is required to further recruit and concentrate sparsely distributed DE‐
cadherin into the AJ 40, 41 (Fig.3). Thus, mutants for components of the Crb polarity
complex fail to develop a normal AJ, with DE‐cadherin being diffusely distributed and
with apical and basolateral membrane markers overlapping each other 42, 43.
Functional regions of Crb residing in its cytoplasmic tail assure the correct
biogenesis of the AJ and the establishment of apical‐basal polarity 41, 44. In particular,
the cytosolic tail of Crb interacts with moesin, an ERM protein that tethers the
complex to the actin cytoskeleton 45. Interestingly, in Drosophila, expression of
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cytoplasmic truncated versions of Crb leads to loss of proper DE‐cad distribution and
consequent multilayering of simple epithelia 44.This phenotype seems to be linked to
the ability of the Crb complex to repress the activity of Discs‐large (Dlg), Scribble (Scrib)
and Lethal giant larvae (Lgl) 46, 47(Fig.3). These proteins form the septate junction (SJ) in
Drosophila, being the functional equivalent to the vertebrates’ tight junction, although
being localized below the AJ. Importantly, the SJ constitutes the paracellular barrier
and restricts the localization of apical polarity determinants, such as Crb (Fig.3). When
mutated, the components of the SJ cause epithelial cells of Drosophila to aberrantly
distribute Crb and DE‐Cadherin. As a result, epithelial cells become unpolarized, giving
rise to a multilayered tissue that overproliferates and acquires invasive properties 48, 49
(Fig.3).
Fig. 3 – Epithelial polarity complexes are located in stratified regions along the apico‐basal axis of cells.
In Drosophila, the subapical is located above the zonulae adherens and the septate junction is located
below. The Crb/Sdt/Dlt complex is located in the SAR and, along with the Baz/Par6/aPKC complex, is
responsible for maintaining the integrity of the AJ, counteracting the basolateral Lgl/Scrib/Dlg septate
complex. In Drosophila’s ovarian follicle epithelium, mutant cells for components of the septate junction
overproliferate and stream into the germ line cyst, where they penetrate between nurse cell
membranes 28, 48 (as shown in the series below).
Understanding the Role of the Actin Cytoskeleton in Vesicle Trafficking to Restrict Growth of Drosophila Epithelia
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2.1 Vesicle trafficking and the control of epithelial cell polarity and proliferation
Epithelial cell polarity relies upon polarized transport of proteins and lipids to
the apical, basolateral and junctional domains in a process called exocytosis. In this
case, recently synthesized proteins are released from the trans‐Golgi network (TGN) to
apical or basolateral membrane domains, following specific guiding signals 50. In
addition, many membrane‐linked receptors are recruited into endocytic vesicles being
either recycled back to the plasma membrane or targeted to lysosomal degradation 51,
52 (Fig.4).
Early endosomes, budding from the plasma membrane, are characterized by
their enrichment in certain phosphoinositides and for the presence of Rab5 GTPases.
These endosomes mediate recycling exocytosis to the apical or basolateral membrane
or endocytosis to a degradative lysosomal route. The degradative route is
characterized by a series of maturation steps, leading to progressive endosomal
acidification and to the formation of multivesicular bodies (MVBs). Ultimately, when
the endosome reaches its maturity, Rab7 GTPases mediate its fusion with a lysosome,
where proteolytic degradation occurs 53(Fig.4).
Importantly, the actin cytoskeleton seems to be essential for the sorting of
vesicle cargo to the basolateral membrane, through several sorts of myosin motors 54,
55. For instance, in MDCK cells, the use of actin polymerization inhibitors compromises
the sorting of proteins to the basolateral membrane domains, forcing them to take
apical recycling routes 56. On the other hand, the microtubule network seems to be
essential for transporting vesicle cargo to the apical epithelial surface, essentially
through dynein and kinesin (‐) end‐directed motors 57. A generally devised model is
that, according to the peripheral location of actin filaments in many cell types, the
actin cytoskeleton essentially mediates vesicle trafficking events close to the
membrane, whereas the microtubule network drives ‘long‐range’ transport of vesicles
across the cell cortex 54, 57. Accordingly, for endocytosis to proceed, actin filaments are
known to increase the uptake of membrane‐linked receptors into vesicle‐coated pits,
whereas the microtubule network is required to assure deep‐cortical endocytic
transport and maintain the distribution of the late endocytic compartments 58‐60.
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Fig. 4 – Overview of vesicle trafficking in epithelial cells. The trans‐Golgi network (TGN) releases
recently synthesized proteins to apical or basolateral membrane domains and the recycling endosome,
which mediates recycling exocytosis to the apical or basolateral membrane, through apical recycling
routes. The endocytic pathway can lead to recycling back to the plasma membrane or to lysosomal
degradation, and each route is characterized by endosome compartments that possess typical
regulatory proteins (in red and purple). The endosome markers labelled in purple have been shown to
suppress tumour formation in Drosophila, as indicated by the tumoral phenotypes of the respective
mutant imaginal discs 61 (as shown in the picture to the left).
When endocytosis is impaired, accumulation of proteins occurs at the level of
the plasma membrane or intermediate endosomal compartments. Interestingly,
several tumour suppressor genes (TSGs) have been characterized in Drosophila that
are key components of the endossomal system, being required for endosome
maturation 62.(Fig.4) For instance, genes required for late endosome maturation, such
as Vps23 (erupted, tsg101) and Vps25 have been shown to be neoplastic tumor
suppressors in Drosophila 63‐65. Disrupting the function of these genes seems to
prevent the degradation of membrane signalling receptors, leading to transduction of
an excessive mitogenic or stress signal 66. Interestingly, mutant cells for avalanche, a
synthaxin required for early endossome sorting (Fig.4B), lead to the neoplastic
development of several Drosophila epithelia, which correlates to vesicular
WT
Rab5
avl
vps25
vps23
Understanding the Role of the Actin Cytoskeleton in Vesicle Trafficking to Restrict Growth of Drosophila Epithelia
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accumulation of the polarity determinant Crb 67. However, until now, no evidence has
clarified how endocytosis of this polarity determinant contributes to the regulation of
epithelial growth.
2.2 The development of epithelial neoplasms
During aberrant epithelium development, cells often loose adhesion between
each other, forming epithelial cysts that eventually extrude to the basal lamina.
Frequently, these abnormal tissue configurations disrupt the normal growth of the
tissue, giving rise to morphogenetic distortions and overgrowths typical of epithelial
tumors or neoplasms 68‐70 (Fig.5).
Neoplastic development is a multistep process, which is thought to involve
cooperation between several mutations, as well as between the tumor and its
microenvironment 71. Indeed, in a chimeric tissue environment, abnormal epithelial
cells, that would potentially drive neoplastic transformation, might undergo apoptosis
triggered by their tissue surroundings, a phenomenon designated as cell competition 72, 73. For instance, when a clone of mutant cells for the tumour suppressor scrib arises
among normal tissue, the surrounding wild‐type cells actively eliminate the
unpolarized mutant cells, triggering their apoptosis 74. In this case, a synergistic
interaction with the Ras or Notch oncogenes is required for the formation of scrib
mutant invasive tumors 74, 75. In this sense, multiple alterations, either arising within
the tumor or in the surrounding normal tissue, seem to be required for promoting
unrestrained tumor growth and lethality 76.
In Drosophila, tumoral epithelia have been classified as hyperplastic whenever
they show increased proliferation, although epithelial architecture is maintained, or
neoplastic if tissue overproliferation occurs concurrently with disruption of epithelial
structure, as cells become unpolarized. Neoplastic epithelia are also characterized by
their inability to terminally differentiate, with cells frequently acquiring migratory and
matrix degradation potential, allowing them to cross the basal lamina and invade
adjacent tissues 62, 69 (Fig.5).
During tumour development, loss of cell‐cell adhesion is usually correlated with
disruption of the apical actin network, while metastatic invasion is associated with
enhanced protrusive activity of the basal actin stress fibers 77. In particular, many ABPs
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have been shown to be misregulated during the metastatic process, including the
Arp2/3 complex components, CPs and members of the ADF/cofilin family 78, 79.
Therefore, actin dynamics at adhesive contacts seems to be crucial for assuring proper
epithelial architecture and tissue homeostasis.
Fig.5‐ Monoclonal tumors are thought to arise by the loss of heterozygozity, leading to overexpression
of a certain oncogene or to a decreased expression of a certain tumour suppressor gene. This event is
then thought to trigger complex rearrangements of the cellular architecture, leading to an epithelial‐to‐
mesenchymal transition. Loss of proliferation control and the acquisition of resistance to cell death are
thought to be triggered by this transition. In Drosophila, tumour suppressor pathways have been found
that link cell polarity to proliferation control. In the depicted example, a series of neoplastic epithelia
arise from overexpression of the Crb polarity complex (as show in the A‐C series, compared to WT).
3. Actin dynamics and the control of cell proliferation
The use of drugs to manipulate actin dynamics has been widely used to
investigate links between actin and stress signalling. For instance, several studies
using mammalian cell lines in culture have shown that, depending on the cell type,
either depolymerisation or stabilization of F‐actin is accompanied by the induction of
an apoptotic stress 80. Interestingly, one striking difference between cell types is the
ratio of G‐actin to F‐actin, this being tightly regulated by the activity of several ABPs.
In this sense, several studies have suggested a role of ABPs in regulating cell survival
WT A B C
Understanding the Role of the Actin Cytoskeleton in Vesicle Trafficking to Restrict Growth of Drosophila Epithelia
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and death 80. For instance, gelsolin activity seems to prevent the induction of
apoptosis and ADF/cofilin has been shown to induce cytochrome c leakage and trigger
apoptosis 81‐83. However, very few studies have actually revealed the significance of
ABPs’s function during the homeostatic processes of cell growth and death within
tissues. For instance, recent studies in Drosophila, concerning the role of CP in wing
tissue morphogenesis, may provide us some insight into this question.
3.1 CP prevents cell extrusion within restricted regions of Drosophila epithelia
As expected, in vivo studies showed that CP prevents excessive actin filament
polymerization within Drosophila epithelia, confirming its already known role in actin
filament termination. However, CP doesn’t have the same developmental function in
all epithelia. For instance, loss of either capping protein α (cpa) or β (cpb) subunits in
the wing imaginal discs of Drosophila gives rise to different cellular outcomes along the
proximal‐to‐distal axis 84. The wing imaginal disc of Drosophila can be subdivided into
three fate‐defined regions: the notum, in the most proximal part, then the hinge, and
finally the blade, in the most distal part. In the blade, CP mutant cells lose polarity,
extrude basally and undergo cell death, while mutant cells that develop in the
remainder of the wing disc, sustain polarity and survive 84.(Fig.6) This behaviour is
correlated to a specific actin filament network present in mutant cells: while in the
wing blade epithelium, excessive actin filaments are observed throughout the entire
cell cortex, in the remainder of the wing disc, actin filaments accumulate mainly at the
cell’s apical surface 84.
3.2 CP prevents neoplastic development of Drosophila epithelia
Recently, it was found that cpa mutant cells in the follicle epithelium of
Drosophila are prone to become neoplastic, lacking polarity and cell‐cell adhesion
(Janody, unpublished; Fig.6). Furthermore, RNA interference (RNAi) leading to CP
depletion in the presumptive blade region of the wing imaginal disc, resulted in tissue
overproliferation, possibly due to the outward resistance of depleted cells to cell
death 85 (Fig.6). Interestingly, it was found that like mutations in genes associated with
the endocytic pathway, including the syntaxin avalanche (avl) and Rab5 86, loss of CP
results in punctated Crb accumulation (Janody, unpublished; Fig.6). Overall, the
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similarities between endocytosis defective phenotypes and the ones observed for CP
mutant cells suggest that the actin dynamics machinery is responsible for maintaining
the appropriate network of actin filaments to support vesicle trafficking within
epithelial cells. Interestingly, the Dictyostelium CARMIL protein was found to link CP
and the Arp2/3 complex to Myosin I 87, whose activity seems to involve the transport
of Golgi‐derived vesicles 88. In this sense, CPs might directly be involved in vesicle and
organelle movement or have an indirect role in regulating trafficking events, trough
their effect on actin dynamics.
Fig.6 – CP mutations have different cellular outcomes depending on the tissue context. cpa mutant
clones developed in the hinge or notum are maintained within the wing epithelium, while clones
developed in the wing blade undergo extrusion and apoptosis, as indicated by anti‐activated Caspase3
(C3) staining84 (A). CP mutant clones induced in the ovarian follicle epithelium form a multilayered
epithelium in the anterior and posterior poles of the egg chamber (Janody, unpublished)(B). Extruding
cpa mutant cells (green) accumulate Crb in punctated structures (Janody, unpublished) (C‐C’).
Depletion of CP by RNAi leads to neoplastic overgrowth of the wing epithelium (D‐D’).
B
Arm GFP Dlg/C3
Phal GFP Dapi Crb
Crb GFP
Phal Phal
WT sd>UAScpbRNAiC10
cpa107E clones
cpa69E clones
A
C
C’
D E
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4. Patterning of the Drosophila wing imaginal disc
Highly conserved mechanisms in epithelial development, such as the
establishment and maintenance of epithelial cell polarity, have been intensively
studied in Drosophila, paving the way for the discovery of homologous mechanisms in
vertebrates. In particular, the imaginal discs and the ovarian follicle epithelium of
Drosophila constitute excellent models for the study of epithelial differentiation in
postembryonic development.
The Drosophila egg hatches into a larva that has no resemblance to the adult
fly, since it possesses no external evidence of motile appendages. However, the
primordial adult appendages are already present in the larvae, deriving from
invaginating epidermal cell clusters that become segregated from the larval tissues.
These structures are called the imaginal discs, since they are going to give rise to most
of the adult insect body, also known as the imago (Fig.7). The imaginal discs follow a
developmental program distinct from that of their larval ‘host’, proliferating from 20‐
50 cells to a final size of 20000‐50000 cells 89. In particular, imaginal discs have been
preferred for the analysis of the combinatorial effects of many conserved signalling
molecules in the specification of positional values for differentiation and growth
(Fig.8). Because of its well characterized developmental circuitry and ease of dissection
I will focus on the wing imaginal disc as an epithelial model, and therefore I proceed to
briefly explain its major patterning events, as known to date.
The majority of the wing disc consists of a columnar epithelium, which will give
rise to most adult cuticular structures, being covered by a thin layer of squamous
epithelium called the peripodial membrane. The luminal face gives rise to the external
surface of the adult structures, following eversion of the disc during metamorphosis.
Specifically, the outmost region of the wing disc gives rise to the thorax structures (the
notum and the pleura), the next ‘ring’ will make the wing hinge, and the central pouch
of the disc will make the wing blade 89 (Fig.7).
Very early in larval development, an invaginating cell cluster that constitutes
the haltere and wing common precursor, inherits the apposed stripe pattern of
engrailed (en) and wingless (wg) expression from the anterior‐posterior (A‐P) boundary
of an embryonic parasegment.
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Fig.7 – Imaginal disc that will give rise to the adult appendages are present within the larvae. Fate
determination along the proximal to distal axis is believed to result from complex interactions between
the anterior‐posterior and dorso‐ventral morphogen‐mediated signalling, leading to the specification of
the wing blade, the wing hinge and the notum, that will give rise to the adult hemithorax.
In particular, the wing disc further derives from the dorsal part of the common
precursor, therefore inheriting a portion of the En stripe that specifies the posterior
compartment of the wing 90 (Fig.8). Although still elusive, distinct adhesive properties
between the posterior and anterior cells of the wing disc seem to prevent their
intermingling, therefore defining a clear compartment boundary, termed the A‐P
boundary. In the posterior compartment En seems to promote hedgehog (hg)
expression 91, 92, which acts as a short‐range morphogen to specify the expression of
decapentaplegic (dpp) in a narrow stripe of cells adjacent to the A‐P boundary 93
(Fig.8). Dpp constitutes a member of the TGF‐β family, promoting patterning and
differentiation of the wing beyond the central domain through a concentration‐
dependent mechanism, inducing the expression of target genes, such as spalt (sal),
optomotor‐blind (omb) and vestigial (vg) 94‐99. Besides giving genetic positional values
along the medial‐to‐lateral wing, the Dpp activity gradient seems to indirectly promote
wing growth 100, being counterbalanced by the brinker transcriptional repressor, which
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is expressed in a complementary lateral‐to‐medial fashion, being itself repressed by
Dpp activity 101, 102.
The important contribution of Dpp signaling to the proximal‐distal patterning of
the wing disc is further completed by intersection of signals coming from the dorsal‐
ventral (D‐V) boundary of the wing disc. During the early stages of larval development
the D‐V boundary is established by the antagonist relationship between dorsal
Epidermal Growth Factor Receptor (EGF‐R) signaling and ventral Wingless (Wg)
signaling, resulting in the expression of apterous (ap) in the dorsal region of the disc 90,
103 (Fig.8). Ap further specifies the activation of Notch at the D‐V boundary, through a
complex regulation of its ligands Serrate and Delta 104‐106. Notch is then assumed to
directly activate the transcription of genes at the D‐V boundary, including wg and cut,
which themselves further restrict the expression of the Notch ligands, fine‐tuning
Notch activity to a narrow stripe 107.
At the D‐V boundary Notch and Wg signaling synergistically specify the
development of the sensory cell precursors (SOPs) that will give rise to the margin
bristles. Furthermore, in the presumptive wing blade, the selector genes scalloped and
vestigial are transcriptionally activated by the combinatorial action of Notch, Wg and
Dpp signaling 108‐110 (Fig.8), providing the organizer functions of the D‐V boundary,
such as promoting growth along the proximal‐distal axis 111‐113, and specifying a
gradient of cell‐affinities that differentiates the presumptive blade from the hinge 114.
The combinatorial action of vestigial together with two of its target genes ‐ nubbin and
rotund ‐ seems to be required for the induction of a ring of Wg expression in the distal
hinge 115 (Fig.8). This is required for the proximal expression of the transcription factor
homothorax (hth), which together with teashirt (tea) is required for proper hinge
development 116, 117 (Fig.8). Furthermore, Homothorax provides an autoregulatory loop
that maintains the ring of wg expression essential for the normal growth of the hinge.
On the other hand, the iroquois gene complex (iro‐C), induced through dorsal EGF‐R
signaling and restricted to the notum‐hinge boundary by Dpp signalling 103, 118, 119
(Fig.8), is responsible for patterning the proximal and intermediary hinge structures,
providing cell‐affinity differences between the notum and hinge cells 120, 121. Most
proximally, pannier (pnr) and U‐shaped are critical for specifying notum dorsocentral
bristles through activation of wg expression in an anterior‐posterior stripe 122 (Fig.8).
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Fig.8 – Wing imaginal disc patterning. The domain of expression of some patterning molecules is
depicted from the most primordial stages of wing development (left) to the most advanced 3rd instar
imaginal disc (right).
In the end, this complex circuitry of signalling molecules seems to drive the
establishment and maintenance of a region specific cell architecture, possibly
providing cues for the modification of adhesive properties and cytoskeletal
architecture. This seems to occur mainly through the regulation of cell asymmetries
both in the apico‐basal axis and in the plane of the tissue, to which contribute the
amazingly complex array of adhesion molecules, polarity determinants and
cytoskeletal genes.
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II. Aims of the Project
The actin cytoskeleton is essential to support polarized vesicle transport within
epithelia and for endocytosis to take place. Most studies that have addressed these
issues used epithelial cell lines in culture. However, very few have analysed how actin
itself or specific ABPs are implicated to promote vesicle trafficking in vivo, in part,
because for most components of the actin cytoskeleton, their role within a tissue
remains obscure. In this project I aimed to: analyse whether the tumoral behaviour of
CP depleted cells results from Crb accumulation due to impaired endocytosis (Task 1);
analyse whether accumulation of other membrane surface receptors is evident in CP
mutant or depleted cells due to endocytic trafficking defects (Task 2); determine
whether loss of CP affect only a subset of vesicle trafficking events (Task 3) and finally
elucidate whether CP acts directly on vesicle trafficking or indirectly through
maintenance of a particular actin filament network (Task 4).
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III. Materials and Methods
1. Fly strains and genetics
1.1 Fly husbandry
All fly stocks were raised at 25oC, according to standard conditions 123. Crosses
were cultured in small vials containing a yeast‐glucose‐agar medium.
1.2 Fly Strains: mutant alleles and transgenic lines available for this study
The mutant alleles cpa107E and cpbM143 used during the course of this work are
likely null alleles, leading to a truncated protein lacking the actin‐binding domain,
since both have been shown to include stop codons in the beginning of the sequence
coding for the N‐terminal domain 124, 125. The majority of fly stocks used in this work
were available at the IGC, and some were kindly supplied by the labs of Ginés Morata
(CBMSO, Madrid) and António Jacinto (IMM, Lisbon). The Gal4 drivers and UAS‐
transgenes used in this study are reported in Table 1.
1.3 Fly Stocks generated
In order to robustly overexpress actin in wing imaginal discs, a recombinant line
was produced between the UAS‐act5C::GFP and UAS‐act42A::GFP 126, 127 transgenes,
both located on the second chromosome. Since the P‐elements used to insert both
transgenes carry the white (w+) marker, giving rise to an orange eye colour in a w‐
background, recombinant flies were selected for having a stronger, red eye colour. As
expected, driving the recombinant line with nub‐Gal4 gave rise to a enhanced wing
phenotype compared to flies overexpressing either UAS‐act5C::GFP or UAS‐
act42A::GFP alone (Sup. Fig.1).
To analyse Cpa colocalization with endosomes, the following transgenic lines w‐
; UAS‐Rab5::GFP/CyO, MKRS/ TM6β, w‐; UAS‐Rab7::GFP/CyO; MKRS/ TM6β were
crossed to w; sp/CyO; UAS‐HA::cpa/ TM2.
In order to evaluate a possible genetic interaction between CP and aPKC, two
independent P‐element insertions carrying the cpb RNAi constructs (w; UAS‐
cpbRNAiC10; MKRS/ TM6β and w; UAS‐cpbRNAiD4; MKRS/TM6β) were crossed with w;
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sp/CyO; UAS‐aPKCCAXXDN/TM6β, carrying a dominant negative version of aPKC to obtain
the line w; UAS‐cpbRNAiC10; UAS‐ DaPKCCAXXDN / TM6β. In addition, to induce clones of
cells mutant for either cpa or cpb that expressed the aPKCCAXXDN dominant negative
form, w; FRT42D, cpa107E/ CyO or w; FRT40A, cpbM143/ CyO flies were crossed to w; sp/
CyO; UAS‐aPKCCAXXDN/TM6 yielding w; FTR42D, cpa107E; UAS‐aPKCCAXXDN and w; FRT40A,
cpbM143; UAS‐aPKCCAXXDN, respectively.
Table 1 – Gal4 drivers and UAS constructs used during the course of this work.
Gal‐4 Drivers Spatio‐temporal domain of activity in the wing disc
engrailed‐Gal4 (en‐Gal4) 128
From end of embryogenesis to end of larval stage in the
posterior compartment (Fig.2)
nubbin‐Gal4 (nub‐Gal4) 129
From 2nd larval instar (48h) to end of larval stage in the
prospective blade and proximal hinge (Fig.2)
scalloped‐Gal4 (sd‐Gal4) 130
From 1st larval instar (24h) in the whole disc and restricted
in the blade and proximal hinge at 3rd instar larvae.
T155‐Gal4 131 From 1st larval instar (24h) in a patchy pattern
UAS‐transgene Purpose of use
UAS‐cpbRNAiC10 85
To promote knockdown of cpb expression
UAS‐act5C::GFP 127
To overexpress actin 5C tagged to GFP
UAS‐act42A::GFP 126
To overexpress actin 42A tagged to GFP
UAS‐crbstrong (minigen32.12C)42
To overexpress the entire crb coding region
UAS‐crbweak (minigen30.11d)42
To overexpress the entire crb coding region
UAS‐myc::crbintra 42
To overexpress the intracellular domain of crb
UAS‐rab5::GFP 133
As an early endosome GFP marker
UAS‐rab7::GFP 133
As a late endosome GFP marker
UAS‐HA‐cpa 84
To overexpress cpa tagged to an HA epitope
UAS‐aPKCCAXXDN 134
To express a ‘kinase‐dead’ dominant negative form of aPKC (DN) that is targeted to the membrane (CAAX)
Gaspar P. Master Thesis
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1.4 Genetic tools
1.4.1 The UAS‐Gal4 system
Expression of UAS constructs was performed at 25oC, unless mentioned
otherwise, using the Gal4 system. The Gal4 protein is a yeast transcription factor that
activates GAL genes required for glucose metabolism. The Gal4 gene has been widely
cloned and used to genetically manipulate expression of genes in foreign organisms
such as Drosophila. Promoter sequences containing a consensus binding site for
activating Gal factors, termed UAS sequences, have been introduced in a number of
eukaryotic expression vectors, allowing for the production of transgenic constructs
that are conditionally expressed upon the availability of Gal4 135. Thus, in Drosophila, a
parental stock expressing Gal4 can be crossed to another carrying a UAS‐insert,
therefore driving expression of the later in the F1 progeny (Fig.1). Furthermore, several
well characterized enhancer sequences have been ‘trapped’ by the Gal4 coding gene
to yield spatio‐temporal specific drivers of gene expression 128.
1.4.2 Generation of clones by mitotic recombination
The FLP/FRT system was used to generate mitotic clones. The Flipase (FLP)
recombinase belongs to the family of λ integrases from Saccharomyces cerevisiae. The
members of this family are known to direct site‐specific recombination of two DNA
strands, which in the case of the FLP occurs through the recognition of Flipase
Recombination Target (FRT) sites. The FLP/FRT technique has been particularly
interesting for generating mosaics to study recessive mutations that would otherwise
cause embryonic lethality if present in the entire organism 136.
Several p‐element insertion stocks have been generated in Drosophila, introducing FRT
sites close to the centromere of each chromosome as well as a transgene encoding FLP
driven by a heat‐shock inducible promoter (hsp70). The fly stocks constaining FLP and
FRT sites, used during the course of this work to generate somatic clones, are indicated
in Table2. In this sense, mitotic recombination between FRT sites in homologues
chromatids occurs following a 1 hour treatment at 37oC of individuals at the stage of
interest. In this work, heat‐shock was performed at 1st and 2nd larval instar. As a result,
in a heterozygous setup, recombination between nonsiter chromatids results in the
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generation of a daughter cell homozygous for the mutation distal to the FRT site.
Subsequent cell divisions of this homozygous mutant cell give rise to a mutant clone
that can be analyzed phenotypically. Conversely, from the same mitotic recombination
event, a wild‐type homozygous sibling cell is generated, giving rise to the twin spot of
the mutant clone. Usually, the wild‐type FRT chromatid contains a cell marker, such as
GFP under the control of a ubiquitous promoter. This allows for the generation of
negatively marked mutant clones (Fig.2).
Fig.1 – The Gal4 system in Drosophila. One of the parental stocks carries the Gal4 gene in close
proximity to a known enhancer of gene expression. This stock can be crossed to another containing a
UAS‐transgene, and the resulting progeny will express this transgene in the pattern of expression of the
Gal4 factor, which is specified by its associated enhancer region. A nubbin‐Gal4 line drives UAS‐GFP
expression in the presumptive wing pouch and distal hinge of the wing disc. The engrailed‐Gal4 line
drives UAS‐GFP expression in the posterior compartment of the wing disc.
en>UAScpbRNAiC10 nub>UAScpbRNAiC10
Phal Phal
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On the other hand, mosaic analysis with a repressible cell marker (MARCM) was
used to positively mark mutant cpa and cpb clones, as well as to generate constitutive
expression of UAS‐transgenes in marked clones 137. The MARCM technique relies on
the combined use of Gal4 together with its repressor Gal80, arranged in a manner such
that FLP‐mediated mitotic recombination causes the genetic loss of Gal80 and the
concomitant derepression of Gal4. Such a derepression can be set to happen in a
mutant clone in a way that allows for the expression of a certain UAS‐marker, like UAS‐
GFP or another UAS‐transgene (Fig.2).
Table 2 – FLP/FRT system stocks used during the course of this work.
FLP/FRT stock Purpose of use
y‐,w‐,hsFLP122, UAS‐GFP; FRT42, tub‐Gal80;
tub‐Gal4/ TM6β 137
To generate cpa mutant clones, positively marked by GFP and expressing another UAS‐transgene.
y‐,w‐,hsFLP122, UAS‐GFP; FRT40, tub‐Gal80;
tub‐Gal4/ TM6β 137
To generate cpb mutant clones, positively marked by GFP and expressing another UAS‐transgene.
w‐; FRT42, tub‐GFP; T155‐Gal4, UAS‐FLP 138
To generate cpa mutant clones, negatively marked by GFP
2. Immunohistochemistry
Third instar larvae were dissected in 0,1M phosphate buffer (pH 7,2) and fixed in 4%
formaldehyde in PEM (0,1M PIPES pH 7,0; 2mM MgSO4; 1mM EGTA) for 30 minutes on
ice. As a standard protocol, except for anti‐Crb and anti‐Notch staining, larvae were
then washed and permeabilized in PBS 0,2% Triton X‐100 (PBT) for 15 minutes and
incubated with the primary antibodies diluted in PBT, supplemented with 10% donkey
serum, overnight at 4oC. The larvae were then washed 3 times, for 10 minutes each, in
PBT and incubated in the dark with secondary antibody diluted in PBT 10% donkey
serum, for 2 hours at 4oC.
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Fig.2 ‐ Mitotic recombination induced by the FLP recombinase during the G2 stage of the cell cycle
results in the loss of heterozygosity at the locus coding for a mutation (black star) and a ubiquitously
expressed cell marker (green rectangle) (A). As a consequence one of the daughter cells will become the
precursor of the mutant clone, whereas the other will become the precursor of the complementary
‘wild‐type’ population (twin spot) that is marked by the presence of the cell marker, such as GFP (A). In a
MARCM set up, Gal80 represses the expression of a UAS‐insert (green rectangle), such as UAS‐GFP, but
loss of heterozygosity at the Gal80 containing locus, as induced by FLP mediated mitotic recombination,
results in expression of the UAS‐construct in the homozygous mutant clone (B).
GFP GFP
A B
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For anti‐Crb and anti‐Notch staining, fixed larvae were previously incubated for
1 hour in a blocking solution of PBT 10% bovine serum albumin (BBT) and then
submitted to a quick dehydration‐rehydration series of 30%, 50% and 70% methanol in
BBT. This was found to greatly enhance the signal detection for membrane‐associated
proteins. The primary antibodies used were: mouse anti‐Arm, (N27A1, 1:10;
Developmental Studies Hybridoma Bank (DSHB)); rabbit anti‐activated Caspase3 (1:50;
Cell Signalling Techonology); mouse anti‐Crb (Cq4, 1:10; DSHB); mouse anti‐Notch
extracellular domain (C458.2H, 1:50; DSHB); goat anti‐Fat (1:1 cocktail of dR‐18 and
dC‐16, 1:20; Santa Cruz); rabbit anti‐Homothorax (1:500, a gift from Adi Salzberg);
mouse anti‐Cut (2B10, 1:5; DSHB); rabbit anti‐Rab5 (1:5000, a gift from Akira
Nakamura). The secondary antibodies used were donkey anti‐mouse and anti‐rabbit
conjugated to TRITC or Cy5, (1:200; Jackson Immunoresearch). Imaginal discs were
mounted in Glycerol 90%.
3. Phalloidin Staining
A phalloidin conjugated dye was used to stain F‐actin. Third instar larvae were
dissected and fixed as described in the previous section. Before mounting, tissues were
incubated with TRITC conjugated phalloidin, (1:200; Sigma), in PBT for 5‐8 minutes and
rinsed 3 times 10 minutes, before mounting in Glycerol 90%.
4. Image Acquisition and Analysis
Fluorescent images were obtained on a Zeiss LSM 510 Meta and Leica SP5
confocal microscope. Image files were processed using the Bitplane Imaris software®
to select standard z‐projections and optical cross‐sections through the epithelium. In
order to evaluate colocalization between fluorophores, binary colocalization masks
were obtained with the NIH Image J software®, using a cut off point of 50% relative to
maximum pixel intensity. Using this same software, confocal images destined for
colocalization analysis were previously submitted to standard medium filtering.
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IV. Results 1. CP mutant and depleted cells accumulate the Notch receptor and the Crumbs polarity determinant in punctuated strucures
Lethal alleles of cpa were isolated in a previous mosaic genetic screen to
identify genes required for Drosophila eye differentiation 124. Given that capping of
actin filaments relies on a functional CP αβ‐heterodimer 21, 139, whose stability depends
upon the association of each subunit with one another 140, 141, loss of one subunit or
mutations preventing the association of both are assumed to effectively cause a loss of
capping activity. This is consistent with the accumulation of actin filaments observed in
either cpa or cpb mutant clones or in tissues depleted of cpa and cpb by RNA
interference, as well as with the fact that loss of either cpa or cpb gives rise to identical
phenotypes 84.
Interestingly, cpa107E mutant cells, undergoing extrusion in the wing blade
epithelium, mislocalize Arm and DE‐Cad to basolateral positions (data not shown),
whereas they accumulate the Crb polarity determinant in punctated structures
resembling vesicles (Fig.1). As the cpa and cpb extrusion phenotype had been shown
to be region specific, with mutant clones mainly extruding in the blade region of the
wing disc 84, I decided to evaluate whether Crb accumulation in cpa107E mutant clones
was also specific to the prospective blade, possibly accounting for the region specificity
of the cell extrusion phenotype. In order to observe cpa107E mutant clones within the
blade epithelium, I used a mosaic FLP/FRT system that uses an imaginal tissue‐specific
Gal4 driver (T155‐Gal4) to direct expression of the FLP enzyme, thus catalyzing a low
frequency of mitotic recombination during all stages of larval development 138.
Therefore, cpa107E mutant clones, marked by the absence of GFP, are produced in all
larval stages, which allows for the recovery of late induced mutant clones across the
whole wing disc of 3rd instar larvae. As expected, mutant clones induced in the
prospective blade region showed basal accumulations of Crb in the form of punctated
structures (Fig. 1). Interestingly, I observed similar accumulations in clones induced in
the presumptive hinge and notum (Fig.1), ruling out the possibility for a region specific
accumulation of Crb that correlated to the blade specific extrusion of cpa and cpb
mutant clones.
Gaspar P. Master Thesis
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Fig.1 –cpa mutant clones induced in the wing imaginal disc accumulate Crb in the form of punctated
structures. cpa mutant clones are marked by the absence of nuclear GFP and Crb staining appears in red
(A); punctated Crb accumulations are seen throughout the tissue, matching GFP negatively marked
clones (A’‐AA’).
Distinct from what is observed in the clonal context, when broad regions of the
wing disc are depleted of cpb by RNAi, cells seem to undergo an epithelium to
mesenchymal‐like transition and overproliferate 85 . As expected, depletion of cpb by
RNAi using the eng‐Gal4 driver, leads to Crb accumulation. This not only occurs in
discrete punctuated structures resembling vesicles, but also at the level of apical peri‐
membrane clusters (Fig.2A‐A’’’). The same phenotype was observed, by depleting cpb,
using the sd‐Gal4 driver (Sup. Fig.2).
Accumulation of Crb at the cell membrane or in punctuated structures did not
resemble basolateral mislocalization of DE‐cadherin and Arm, that had previously been
observed in extruding cpa and cpb mutant clones 84. They were rather similar to
accumulations of membrane‐linked receptors observed in imaginal mutant tissue for
genes implicated in endocytic trafficking 67. Interestingly, similar to avl and rab5
mutant tissue, cpb depletion by RNAi, using en‐Gal4, also induces apical punctated
accumulation of the Notch extracellular domain, as indicated by anti‐Notchextra staining
(Fig.2B‐B’’’).
Crb GFP Crb
Crb GFP
A A’
A’’
cpa107E clones
cpa107E clones
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Fig.2 – CP depleted cells accumulate the Notch receptor and Crb in the wing imaginal disc. cpb depleted cells are marked by the presence of GFP (green) and either Notch or
Crb appears in red/gray (A‐A’’’’, anti‐Notch; B‐B’’’’, anti‐Crb); Notch and Crb become accumulated in the posterior compartment as shown by anti‐Notch and anti‐Crb (gray),
respectively (A’’‐A’’’, B’’‐B’’’). Cross‐section views through the disc indicate that accumulation occurs at the level of intracellular punctated structures, and at the level of
apical peri‐membrane clusters (A’’’’‐ arrows; B’’’’ ‐ arrowhead). A – anterior, P – posterior
A P
A’’’’
A P
A’
A’’ A’’’
Crb
Crb GFP
Crb
Crb
Crb
A en>UAScpbRNAiC10
Notch
B B’
B’’ B’’’
B’’’’
Notch GFP
Notch GFP
Notch
Notch
en>UAScpbRNAiC10
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Several membrane receptors and adhesion molecules, such as Notch, Fat,
EGFR, and E‐Cad have been shown to undergo endocytosis in wing disc cells through a
process that depends on the ERM proteins Merlin and Expanded 142. Therefore, in
order to determine whether other membrane associated proteins were accumulated
due to CP depletion, I analyzed the expression and subcellular localization of the
adhesion‐related proto‐cadherin Fat. Indeed, cpb depleted cells by RNAi accumulate
Fat, as indicated by anti‐Fat staining, although this seems to occur in a difuse manner
along the entire apical cell surface (Fig.3).
Fig.3 – CP depleted cells accumulate Fat in the wing imaginal disc. cpb depleted cells are marked by the
presence of GFP and Fat appears in red/gray (A‐A’’); Fat becomes apically accumulated in the posterior
compartment, as shown by anti‐Fat (gray) (A’‐A’’). A – anterior, P – posterior
Taken together, the data present here suggests that CP promotes endocytic
vesicle trafficking of Crb, Notch and Fat, or that it is able to restrict their transcriptional
upregulation. In order to test this hypothesis, I analyzed the expression levels of cut
and m8‐lacZ as a read‐out for Notch activity, since Notch upregulation induces
misexpression of these genes beyond the DV boundary 143, 144. Instead, expression of
cut and m8‐lacZ at the DV boundary, as indicated by anti‐Cut and anti‐β‐Gal staining,
A en>UAScpbRNAiC10
Fat GFP
A’
Fat
A P
A’’
Fat
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respectively, is highly reduced in posterior wing cells depleted of cpb, indicating that
Notch signalling is disrupted (Fig.4). This suggests that Notch accumulation in CP
depleted cells does not result from overexpression of Notch, but that loss of CP
prevents Notch activation. It is therefore likely that accumulation of Notch, and
possibly of Crb and Fat, in cpb depleted cells, results from defects in endosomal
trafficking.
Fig. 4 – CP depleted cells show reduced expression of Notch target genes. cpb depleted cells are marked
by the presence of GFP (A) or by increased phalloidin staining (B); Cut and β‐Gal appear in blue/gray (A‐
A’‐ anti‐Cut; B‐B’ – anti‐β‐Gal); cut expression at the DV boundary is reduced in the posterior
compartment, as shown by anti‐Cut (gray) (A’); m8‐lacZ reporter expression at the DV boundary is
reduced in the posterior compartment, as shown by anti‐β‐Gal (gray) (B’).
Phall Cut GFP
Cut
A’
Phall β‐Gal (m8‐lacZ)
B
β‐Gal (m8‐lacZ)
B’
A en>UAScpbRNAiC10
en>UAScpbRNAiC10
Gaspar P. Master Thesis
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2. Overexpression of the two cytoplasmic actin genes act5C and act42A can give rise
to hinge overgrowth and Crumbs accumulation.
Decreased expression of CP causes actin filament accumulation 84, 145. In this
sense, the putative role of CP in vesicle trafficking could be indirect, such that the actin
filament network formed in CP mutant cells would disrupt endocytic vesicle trafficking.
In order to understand whether actin filament accumulation, by itself, prevents
membrane and cytoplasmic accumulation of Notch and Crb, I’ve used the en‐Gal4 and
nub‐Gal4 drivers to overexpress the ubiquitous actin genes act5C and act42A, as their
overexpression promotes accumulation of actin filaments in the wing disc epithelium 146(Vinhas and Janody, unpublished). Interestingly, similarly to cpb depletion by RNAi,
overexpression of act5C, using en‐Gal4, leads to an expansion of the prospective distal
hinge, associated to accumulation of the apical polarity determinant Crb, as revealed
by anti‐Crb staining (Fig.5A). This accumulation occurs mainly in punctuated structures
at the apical cell surface (Sup. Fig.3). Furthermore, a relatively slight accumulation of
the Notch receptor also occurs in act5C overexpressing cells, as revealed by staining of
anti‐Notchextra (Fig.5B). Overall, this suggests that Crb and Notch accumulation,
following loss of CP, is caused by a global excess of F‐actin within the cell.
Surprisingly, overexpressing a recombinant version of UAS‐act5C::GFP and UAS‐
act42A::GFP, under the control of the nub‐Gal4 driver, comparatively enhanced the
hinge outgrowth observed in wing imaginal discs where each actin gene was
overexpressed individually (Fig.6). This suggests that hinge‐specific overgrowth
observed in CP depleted discs is attributable to actin filament accumulation, pointing
to a role of the cortical actin network in regulating growth processes in this region.
3. CP colocalizes with the early endosome marker Rab5
Punctuated accumulation of Notch, Crb and Fat in CP depleted tissue suggests
that CP might have a role in vesicle trafficking. In support of this, CP contains
hydrophobic segments that are likely to interact with membrane lipids 147. Therefore,
it is conceivable that CP could be in close contact with endocytic vesicles, which would
suggest a more direct role of CP in the regulation of endosome dynamics.
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Fig.5 – Act5C overexpression leads to accumulation of Notch and Crb in the wing imaginal disc. cells
overexpressing act5C are marked by the presence of GFP and either Notch or Crb appear in red/gray (A‐
A’, anti‐Notch; B‐B’, anti‐Crb); Notch and Crb are accumulated at the apical cell membrane of posterior
depleted cells, as shown by anti‐Notch and anti‐Crb, respectively (A’, B’).
Fig. 6 – Actin overexpression induces hinge overgrowth in the wing imaginal disc. Actin overexpressing
cells are marked by the presence of GFP; phalloidin staining of F‐actin (red) allows for visualization of
cell architecture (A‐C); hinge outgrowths are observed in wing discs overexpressing either act42A or
act5C (A,B, ‐ arrows), although they look smaller than the ones observed in wing discs overexpressing
both act42A and act5C (C ‐ arrows). Note: expression of UAS‐constructs was performed at 22oC .
B
Phal Act5C‐GFP
C
Act42A‐GFP, Phal Act5C‐GFP
A
Phal Act42A‐GFP
nub>UASact42A::GFP nub>UASact5C::GFP nub>UASact5C::GFP UASact42A::GFP
B’
en>UASact5C::GFP A
Act5C‐GFP Notch
B en>UASact5C::GFP
Act5C‐GFP Crb
Notch
A’
Crb
B’
Gaspar P. Master Thesis
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Interestingly, I found that an HA‐tagged form of cpa (HA‐cpa), which can fully
rescue the cpa and cpb mutant phenotype 84, apically localizes to punctated structures
resembling vesicles, as indicated by anti‐HA staining (Fig. 7A). Indeed, the punctated
pattern of early and late endosomes, labelled by Rab5‐GFP and Rab7‐GFP, respectively,
strongly resembled in size and position the pattern outlined by staining of HA‐Cpa
(Fig.7A‐C). Taking this into account, I’ve qualitatively analysed colocalization between
early or late endosomes marked by the presence of Rab5‐GFP and Rab7‐GFP,
respectively, and HA‐Cpa. Colocalization masks, above 50% of pixel intensity, revealed
a punctated pattern of colocalization between Rab5‐GFP marked early endosomes and
HA‐Cpa (Fig.7D‐D’’). However, a similar pattern of colocalization was not verified
between Rab7‐GFP marked late endosomes and HA‐Cpa (Fig.7E‐E’’). Furthermore,
optical cross sections through the wing imaginal discs expressing rab5::GFP or
rab7::GFP, and HA::cpa, also suggest colocalization of early endosomes with the apical
punctated pattern of HA‐Cpa (Fig.7D’’’), whereas late endosomes appear to be
localized more basally (Fig.7E’’’).
This suggests that CP directly associates with early endosomes, possibly being
relevant to the regulation of early endosome dynamics. Indeed, in cpb depleted cells
by RNAi, using the en‐Gal4 driver, I’ve observed an increased abundance of Rab5‐
positive puncta, as indicated by anti‐Rab5 staining (Fig.8). This further suggests that
early endosomes become accumulated in CP depleted cells, being either unable to
recycle back to the plasma membrane or to traffic to the lysosome.
4. crb overexpression in the wing imaginal disc gives rise to different cellular
outcomes depending on the tissue genetic context
Vesicular accumulation of Crb has been coupled to neoplastic tumor
development of avl mutant tissue 67. Interestingly, CP depletion by RNAi in the distal
wing hinge induces hyperplastic tissue overgrowth, while in the wing blade epithelium,
most cells seem to delaminate and possibly overproliferate (Janody, unpublished).
Moreover, similar to cpa and cpb mutant clones, avl mutant clones are extruded from
the wing blade epithelium 67.
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Fig.7 – Cpa partially colocalizes with early endosomes. Clones overexpressing HA‐cpa (A), rab5::GFP (B)
and rab7::GFP (C) reveal apical vesicular structures (green) that are similar in size and shape (Arm (red)
outlines the apical cell membrane) (A‐C). Early and late endosomes (green) are marked by the presence
of Rab5‐GFP (D) and Rab7‐GFP (E), respectively; anti‐HA (red) reveals a punctated pattern of HA‐Cpa
localization (D’, E’); colocalization masks above 50% of pixel intensity (white) reveal partial colocalization
of HA‐Cpa (red) with Rab5‐GFP (green) (D’’), but not with Rab7‐GFP (green) (E’’); confocal cross sections
trough the wing imaginal disc show that HA‐Cpa partially colocalizes with Rab5‐GFP marked early
endosomes (green) (D’’’), but not with Rab7‐GFP marked late endosomes (green) (E’’’).
A
B
C
Arm Rab5‐GFP
Arm HA‐Cpa
Arm Rab7‐GFP
D nub>UAScpbRNAiC10; UAS‐rab5::GFP
D’
D’’
D’’’
HA
Rab5‐GFP
E nub>UAScpbRNAiC10; UAS‐rab7::GFP
E’
E’’
E’’’
HA
Rab7‐GFP
Gaspar P. Master Thesis
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Fig.8 ‐ CP depleted cells accumulate Rab5‐positive endosomes in the wing imaginal disc. cpb depleted
cells are marked by the presence of GFP and Rab5 appears in red/gray (A‐A’’). Rab5‐positive puncta
apically accumulate in the posterior compartment, as shown by anti‐Rab5 (gray) (A’‐A’’). A – anterior, P
– posterior
In order to test whether an excessive Crb signalling activity could justify
extrusion of cpa and cpb mutant clones, I’ve induced clones overexpressing crb in the
wing imaginal disc, assuming that if Crb accumulation promotes extrusion of CP
mutant cells, groups of cells overexpressing crb should behave in a similar manner. To
induce clonal overexpression of crb, I’ve made use of the MARCM system. Accordingly,
in a clonal context, crbintra overexpression leads to cell death, as revealed by the
expression of activated Caspase 3 in GFP‐positive cells (Fig.9A‐A’’,B‐B’’). However,
dying cells are mostly observed in the presumptive wing blade epithelium, defined by
the folding formed between the presumptive blade and hinge regions and revealed
with anti‐Arm staining, which marks the apical cell membrane (Fig. 9A,A’’,B,B’’).
Optical cross‐sections through the wing disc showed that crb overexpressing cells
extrude basally in presumptive blade, whereas some cells can often be recovered in
the hinge and notum regions (Fig. 9B’’’). This clonal behaviour is reminiscent to the cpa
and cpb extrusion phenotype, suggesting that extrusion of CP mutant cells could be
linked to Crb accumulation.
A’’
Rab5 GFP Rab5
A en>UAScpbRNAiC10 A’
A P
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Fig.9 – crb overexpressing clones extrude mainly in the presumptive wing blade. crbintra overexpressing
clones are marked by the presence of GFP (green) (A‐B’’’); caspase3 positive cells (blue) are present
mainly in the wing blade (A’’, B’’), although some can also be observed in the wing hinge (B’’‐ arrow);
many clones are still maintained in the wing hinge (B’’, B’’’ – arrows). The dashed line is limiting the
boundary between the presumptive wing blade and hinge. b – blade, h ‐ hinge
To confirm that homotypical Crb overexpression can lead to a tumoral
phenotype, I overexpressed crb using the nub‐Gal4 driver. Under these conditions, crb
overexpression does indeed induce the formation of epithelial cysts and numerours
foldings (Fig.10). Larvae overexpressing crb (crbweak) develop wing discs that look
mostly hyperplastic, where the epithelial sheet looks overexpanded, particularly in the
presumptive hinge (Fig. 10A). On the other hand, overexpression of a similar but
stronger contruct (crbstrong), leads to the development of neoplastic wing discs that are
mainly comprised of round cells (Fig.10B).
A A’ A’’
B B’ B’’
B’’’
Arm GFP C3
Arm GFP C3
Arm GFP C3
C3
C3
b h
b h
GFP
GFP
UAScrb intra clones
UAScrb intra clones
Gaspar P. Master Thesis
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Fig.10 – crb overexpression in a broad tissue context leads to tumoral development of the wing imaginal
disc. Cell architecture is visualized with phalloidin staining of F‐actin in red/gray (A‐D). Epithelial
transformation in the range of neoplastic (A,C) to hyperplastic (B) is observed in crb overexpressing
discs, as compared to wild‐type (D); crbweak overexpressing discs, reveal an expansion of the distal hinge
(B‐ arrow); crbintra overexpression gives rise to neoplastic phenotypes, revealing epithelial cysts that form
preferentially in the distal hinge (C‐ arrow); confocal cross section reveals mutlilayering of the tissue (C’)
as compared to wild‐type (D’).
Furthermore, larvae overexpressing the intracellular domain of crb (crbintra)
show severe neoplastic phenotypes, leading to the formation of epithelial cysts
(Fig.10C). Optical cross sections through the wing disc epithelium show that the tissue
architecture is dramatically altered, producing an intricate plane of folding (Fig.10C’).
Phal
D WT
Phal
C nub>UAS‐crbintra
D’PhalPhal
C’
A nub>UAS‐crbweak
Phal Phal
B nub>UAS‐crbstrong
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5. crb overexpression in the wing imaginal disc induces hinge specific overgrowth
Surprisingly, I realised that the prospective wing blade, in proximity to the DV
compartment boundary, is less affected by crbintra overexpression driven with nub‐
Gal4, when compared to the surrounding outsized hinge tissue (Fig.10C; Fig.11). Cells
neighbouring the DV boundary have a normal shape, maintaining the proper tissue
organization, while the presumptive hinge cells assume a round shape, which is
concomitant with a variable loss of cell polarity manifest in folding of the tissue
(Fig.10C‐C’; Fig.11A’). This phenotype was confirmed using the rn‐Gal4 driver to
overexpress crbintra (data not shown). Thus, similar to CP loss, the crb overexpression
outcome seems to depend on tissue identity. In order to confirm this, I stained crb
overexpressing wing discs driven by nub‐Gal4, for Homothorax (Hh), whose normal
pattern of expression is restricted to the hinge and notum regions, and for Wg, which
is normally expressed in a narrow stripe of cells along the DV boundary and in two
concentric rings in the wing hinge. I did find that Hth is expressed in severely
unpolarized tissue (Fig.11B’’’), indicating that these cells have derived from a hinge‐
defined cell lineage or that they have acquired hinge genetic fate. Expansion of Wg
expression domain was also detected in the prospective hinge of crb overexpressing
discs, but not at the DV boundary (Fig.11A’’,B’’). Curiously, overgrowth observed in CP
depleted tissues also seemed to arise mainly from the hinge region (introduction
Fig.6E), suggesting that this fate defined epithelial region is more susceptible to loss of
proliferation control.
Cell death is usually suppressed in many tumoral processes, although a small pool
of dying cells is always reported, possibly as footprint of cell competition 73. Wing discs
overexpressing cbrintra show only a few cells stained positively for the activated form
of Caspase3, meaning that the cell death program is only mildly activated throughout
the tissue, at least during this stage of development (Fig.11A’’’). The same was verified
for cpb depleted wing discs by RNAi, where the majority of the overgrown tissue does
not express the activated form of Caspase3 148.
Gaspar P. Master Thesis
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Fig. 11 – crbintra overexpression induces overgrowth of the presumptive wing hinge. Cell architecture is visualized with phalloidin staining of F‐actin (red) (A‐A’, B‐B’);
Caspase3‐positive cells (blue) are few and sparse, arising mainly between severely unpolarized tissue (A, A’’’); Wg domain of expression (green) is broadened and
disorganized in the presumptive hinge (A,A’’,B, B’’), although it looks unaffected at the DV boundary (A’’) or absent if the presumptive wing blade has suffered a great
reduction (B’’), as compared to wild‐type (C); Hth domain of expression (blue) is broadened towards the central prospective blade (B,B’’’), as compared to wild‐type (D).
A’’’
C3
C WT
Wg
Hth
D WT
A
Phal Wg C3
B
Phal Wg Hth
A’
Phal
B’’
Wg
B’’’
Hth
B’
Phal
A’’
Wg
nub>UAS‐crbintra
nub>UAS‐crbweak
Gaspar P. Master Thesis
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6. CP genetically interacts with aPKC to define cell shape and prevent cell extrusion
A ‘kinase‐dead’ dominant negative form of a‐PKC (aPKCCAXXDN) has been shown
to reduce the activity of overexpressed crb and could partially suppress the polarity
and proliferation phenotypes of imaginal discs depleted of avl 67. Actually, when I
drove crbintra and aPKCCAXXDN in the wing imaginal disc, using the nub‐Gal4 driver, I
could rescue pupal lethality associated to crbintra overexpression (Sup. Fig.4). If the CP
phenotype is a consequence of Crb accumulation, I expected that expressing
aPKCCAXXDN in CP depleted cells should prevent cells from losing polarity and
overproliferate. Surprisingly, clones mutant for cpa107E or cpbM143 and expressing
aPKCCAXXDN, using the MARCM system, not only undergo extrusion and apoptosis in the
prospective wing blade, but also in the hinge and notum epithelia, as revealed by
staining of activated Caspase 3 in GFP positive cells. (Fig.12B). A cross section through
the wing blade epithelium showed that GFP‐positive cells undergo extrusion in all
regions of the wing disc (Fig.12B’). On the contrary, clones of cells expressing
aPKCCAXXDN alone are perfectly maintained within the epithelium and show no evidence
of polarity disruption, as indicated by normal apical localization of Arm (Fig.12A‐A’’).
Since expression of aPKCCAXXDN has a dominant negative effect, this suggests that CP is
absolutely required to maintain aPKC deficient cells within the epithelium tissue.
Consistent with an enhancement of the CP depletion phenotype, driving both
aPKCCAXXDN and cpb depletion using nub‐Gal4, results in a severely enhanced adult wing
phenotype, when compared to the phenotype of wings depleted of cpb alone (Fig.
13D, D’, D’’). The former show partial or total loss of vein differentiation, hair
misorientation, disorganization of the margin sensory bristles and an overall necrotic
and blistered appearance (Fig.13C, C’, C’’), while expression of aPKCCAXXDN alone has no
visible effect on wing morphogenesis (Fig.13B, B’, B’’). Moreover, cpb depletion by
RNAi along with expression of aPKCCAXXDN, using the en‐Gal4 driver, seems to induce
cell roundening and progressive loss of the columnar epithelial shape, which is evident
by the fact that cells in the posterior wing compartment look shorter along their apical‐
basal axis than anterior cells serving as an internal control (Fig.14C’,D’). This is further
associated to a significant reduction of the posterior wing compartment, suggesting a
reduction in cell size (Fig.14C,D). Cell size reduction is also evident in cpb depleted
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wings expressing aPKCCAXXDN, using nub‐Gal4, since wing hairs are much reduced when
compared to the hairs of wings depleted of cpb alone (Fig.12C’’, D’’).
All together, these results suggest that CP and aPKC work in parallel to prevent
epithelial cell extrusion and to promote and maintain the columnar cell architecture
typical of wing disc epithelial cells.
Fig. 12 – CP genetically interacts with aPKC to prevent loss of epithelial cell polarity and extrusion.
Clones expressing aPKCCAXXDN are marked by the presence of GFP (green) (A‐A’); cpbM143 mutant clones
expressing DN‐aPKC are marked by the presence of GFP (green) (B‐B’); anti‐Arm staining (red/gray)
outlines the apical cell membrane (A‐B’’); clones expressing aPKCCAXXDN don’t show any polarity or
adhesion defects, as indicated by anti‐Arm staining (A‐A’’); cpb mutant cells expressing aPKCCAXXDN
extrude in all regions of the wing disc epithelium, including the wing hinge and notum (B‐B’‐ arrows),
and many undergo apoptosis, as indicated by anti‐activated Caspase 3 staining (blue) (B‐B’).
Arm GFP C3 B’
B’’
Arm
Arm GFP C3
B
Arm GFP C3 A’
Arm GFP C3
A
A’’
Arm
UAS aPKCCAXXDN clones
UAS aPKCCAXXDN , cpbM143clones
Gaspar P. Master Thesis
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Fig. 13 – CP genetically interacts with aPKC during wing morphogenesis. cpb depleted wings (C) are
smaller than wild‐type (A); expressing aPKCCAXXDN (B‐B’’) has no effect on wing morphogenesis, as
compared to wild‐type (C‐C’’); wings depleted of cpb and expressing aPKCCAXXDN are reduced and have
loss of vein differentiation (D), as compared to wings depleted of cpb alone (C); cpb depleted wings
show an increased density and disorganization of wing margin bristles (C’), which becomes enhanced by
expressing aPKCCAXXDN at the same time (D’), as compared to wild‐type (C’); wing hairs look misoriented
in cpb depleted wings (C’’), becoming densely distributed in cpb depleted wings expressing aPKCCAXXDN
(D’’), as compared to wild‐type (A’’).
B’’
A’ B’ C’ D’
A’’
C’’ D’’
A WT C nub>UAS‐RNAicpbC10 B nub>UAS‐aPKCCAAXDN D nub>UAS‐RNAicpbC10; UAS‐aPKCCAAXDN
Understanding the Role of the Actin Cytoskeleton in Vesicle Trafficking to Restrict Growth of Drosophila Epithelia
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Fig.14 – CP genetically interacts with aPKC to maintain the epithelial columnar cell shape. No visible
enhancement of cpb depletion phenotype (A) was observed by expressing aPKCCAXXDN (B), in the nubbin
domain of expression; the posterior wing compartment is much reduced (D) and cells seem to become
more cuboidal than their anterior counterparts (D’), when aPKCCAXXDN is expressed in cpb depleted
tissue, as compared to wings depleted of cpb alone (C‐C’).
nub>UAS‐cpbRNAiC10 nub>UAS‐cpbRNAiC10 UAS‐aPKCCAAXDN
PhalPhal
Phal
A’
B’
A B
Phal
Gaspar P. Master Thesis
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VI. Discussion
1. The role of CP in endocytosis
Endocytosis and polarized exocytosis seems to be crucial for the establishment
and maintenance of epithelial cell polarity. I found that loss of CP in the Drosophila
wing imaginal disc leads to accumulation of the membrane associated proteins, Crb
Notch, and Fat (results Fig.1‐3). In particular, Notch accumulation is unlikely to be due
to transcriptional upregulation, since Notch strongly accumulates at the cell surface of
CP depleted cells but remains inactive, as suggested by downregulation of Notch target
genes (results Fig.4). Alternatively, accumulation of Crb and Notch is suggestive of
endocytic trafficking defects, since apical Notch and Crb levels have been shown to be
regulated by endocytic lysosomal degradation 67, 149.
In fact, my results suggest that CP is likely to regulate early endosome
dynamics. Foremost, I found that an HA‐tagged form of Cpa co‐localized with the early
endosomal marker Rab5‐GFP, but not with the late endosomal marker Rab7‐GFP
(results Fig.7). Additionally, I found that Notch signalling is downregulated following
loss of CP. Interestingly, Notch is endocytosed upon ligand binding and is transferred
to late endosomes where activation can proceed through cleavage by the λ‐Secretase
complex 149‐151. Accordingly, Notch accumulation in late endosomes leads to its
ectopic activation, such as in vps25 mutant tissue 63, 64, 152. However, when lysosomal
targeting is disrupted, Notch accumulates at the cell surface but is unable to signal,
such as in avl and Rab5 mutant tissue 68. In this sense, it is likely that Notch
accumulation in CP depleted cells is due to an early endocytic fusion defect, blocking
progression to late endosomes were activation of Notch takes place. Accordingly,
accumulation of Crb and Fat in CP depleted cells could also be due to an endocytic
defect.
Growing evidence indicates that the actin cytoskeleton plays a fundamental
role in endocytic trafficking 59, 60, 153. In this sense, the role of CP in early endosome
dynamics could be mediated through its well‐known function in promoting actin
filament termination. Indeed, I found that actin filament accumulation could lead to
defects in vesicle trafficking, since actin overexpression (act5C and act42A) leads to
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Notch and Crb accumulation, as was observed in CP depleted cells (results Fig.5, Sup.
Fig.3). As a fact, the apical actin web in epithelial cells seems to be crutial for the
transport of vesicles, since cytocalasin treatment, which depolymerised actin filaments
in this region, inhibits endocytosis 54. Furthermore, the actin filament cross‐linking
protein Spectrin, which localizes to the apical actin web, was shown to promote
endocytosis in enterocytes of the Drosophila gut epithelium 154. In this sense, actin
filament accumulation, due to CP depletion or actin overexpression, could lead to
filament bundling and disruption of the cross‐linked network of the apical actin web,
therefore preventing endocytosis.
In detail, strong evidence suggests that actin polymerization at sites of vesicle
assembly occurs by recruitment and activation of the Arp2/3 complex 155‐158.
Furthermore, the adaptor protein CD2AP, which regulates Arp2/3 activity at the level
of early endosomes, is able to bind CPs, in vitro 159. Thus, the combined activity of
Arp2/3 and CPs could produce a specific actin structure that is required to assist in the
early steps of vesicle formation (Fig.1A‐B). Additionally, a structural scaffold, such as
the apical actin web, might be needed to stabilize the local membrane composition of
RabGTPase effectors, arranging them in a precise architectural layout to allow for
directed vesicle fusion 160. Therefore, apart from regulating early steps of vesicle
formation, CPs could generate an actin framework required to stabilize oligomeric
protein complexes that promote directed docking and fusion events towards the
degradative or recycling pathways (Fig.1C).
To summarize, I propose that short actin filaments, terminated by the activity
of CPs, are required for the initial steps of vesicle formation and possibly for providing
a structural support for directed vesicle fusion events during endocytosis (Fig.1).
2. Endocytosis defects might contribute to growth of CP mutant tissue
Interestingly, CP depleted cells seem to overproliferate, and this could be
related to defects in internalization and lysosomal degradation of membrane
receptors. In fact, blocking the endocytic pathway induces overproliferation and can
lead to the formation of neoplastic tumors 62, 66. Curiously, neoplastic development
and overproliferation of avl or rab5 mutant cells was shown to be coupled to Crb
Gaspar P. Master Thesis
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vesicular accumulation, leading to loss of cell polarity as well to overproliferation 67.
Indeed, in a homotypical environment, crb overexpressing cells, as well as CP depleted
cells, are able to survive or even overproliferate in the distal hinge region (results
Fig.10). Therefore, Crb accumulation could at least be partially responsible for the
overgrowth of CP depleted tissue.
Fig.1 – Actin filament networks generated by the barbed end capping activity of CP could contribute to
the assembly and pinch‐off of endocytic vesicles (A‐B), as well as to oligomerize protein complexes in
vesicle membrane domains (C), to specify docking towards endocytic recycling or degradation.
In addition, my results revealed that CP depleted cells also accumulate the Fat
tumour suppressor gene (results Fig.3). There are at least two signalling branches
downstream of Fat, one concerning the establishment of planar cell polarity (PCP) and
requiring the transcriptional corepressor Atrophin 161, and another restricting cell
proliferation through the Hippo pathway 162‐164. The fact that CP depletion gives rise to
hinge overproliferation and to misorientation of the wing hairs indicates that Fat
signalling is blocked at an early stage, before giving rise to the PCP and Hippo signalling
branches (Fig.2).
A
B
C
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The conserved Hippo signalling pathway has been proposed to regulate contact
inhibition of growth 165, 166, being mediated by the kinase complexes Hippo/Salvador
and Warts/Mats and culminating in the phosphorylation and consequent inactivation
of the transcriptional co‐factor Yorkie (Yki) 163, 167‐169. By inactivating Yki, the Hippo
signalling cascade prevents transcription of growth promoting genes, such as cyclin E
and wg, as well as anti‐apoptotic genes, such as diap1 170, 171. Furthermore, it prevents
transcription of upstream components of the pathway, such as expanded (ex), in a
negative‐feedback loop 172 Indeed, disruption of Hippo signalling seems to occur in CP
mutant or depleted cells, since these upregulate ex and wg (Janody and García‐
Fernandez, unpublished).
Interestingly, mutant clones for components of the Hippo signalling cascade
show defects in endosomal trafficking, and this has been correlated to disruption of
the Notch signalling pathway 173, 174. Furthermore, Merlin (Mer) and Expanded (Ex),
two upstream members of the Hippo pathway, have been shown to colocalize with
vesicle compartments and regulate endocytosis of numerous transmembrane proteins,
including Notch and Fat 142. However, whether they do so independently of Yki
transcriptional regulation is still a matter of debate 142, 175. My results show an
extensive colocalization of CP with early endocytic compartments (results Fig.7),
suggesting that CP has a direct role in regulating vesicle trafficking rather than an
indirect role through its effect on Hippo signalling. In this sense, CPs could cooperate
with Ex and Mer to regulate endocytosis of Notch and Fat, independently of Hippo
signalling (Fig.2). However, it still needs to be addressed whether CP plays a role in
Hippo signalling through its putative influence on endocytosis, or if it does so
independently of vesicle trafficking (Fig.2).
Despite the reported similarities, CP mutant or depleted cells show additional
phenotypes that differ from those of mutants for components of the Hippo pathway.
For instance, mer and ex mutant cells shown apical accumulation of E‐cadherin 142, 175,
whereas CP mutant cells essentially mislocalize it to basolateral positions. Additionally,
the hyperplastic overgrowth of tissue mutant for components of the Hippo pathway
does not match the polarity disruptive overgrowth of CP depleted tissue. Thus, besides
potentially promoting Hippo signalling, CPs could independently regulate the stability
of cell‐cell adhesion and polarity.
Gaspar P. Master Thesis
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Fig.2 – CP and the ERM proteins Mer and Ex, could cooperate to promote steady‐state clearance of
membrane receptors. This could eventually contribute to the regulation of signalling pathways, such as
the Fat/Hippo signalling pathway.
3. crb overexpression leads to cell competition in a heterotypical tissue environment
Upon CP depletion, the accumulation of Crb occurs everywhere along the wing
disc epithelium. Although crb overexpression might be far from mimicking punctated
Crb accumulation in CP mutant cells, clones overexpressing this polarity determinant
extrude mainly in the presumptive wing blade, as do CP mutant clones (results Fig.9),
suggests that cells do not respond identically to Crb levels along the proximal‐distal
axis. On the other hand, crb overexpression in a broad domain of the wing epithelium
leads to tissue overgrowth (results Fig.10). This is in agreement with the definition of a
process of cell competition, according to which, interaction between ‘loser’ and
‘winner’ cells leads to elimination of the loser population, whereas ‘loser’ cells
wouldn’t die if present in a homotypical environment 73.
Endocytic internalization of survival factors could be one of the principles by
which cell competition is implemented on a population basis 176. For instance, clones
mutant for endocytic components such as Rab5, avl and vps25 seem to be eliminated
through a process of cell competition, despite the abnormal overgrowth they produce
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in a homotypical environment 61, 67, 152. Given that CP seems to be involved in endocytic
trafficking of membrane receptors, abnormal endocytic mechanisms could lead to
defective internalization of a survival factor in CP mutant cells, causing cell extrusion
and death in the clonal context (Fig.3A). It has been suggested that, in the wing
imaginal disc, the limiting ligand for endocytosis is the TGF‐β homologue Dpp 177.
However, a defect in Dpp uptake is not sufficient to explain the region preferential
extrusion of CP mutant cells, since overexpression of a constitutive active form of tkv
in CP mutant clones does not rescue the extrusion phenotype (Janody, unpublished).
Nevertheless, this does not rule out the possibility that endocytosis defective cells
might not respond identically along the proximal‐distal axis of the wing epithelium. In
fact, Rab5 mutant clones seem to be preferentially eliminated in the presumptive wing
blade 68,178. Therefore, it is possible that internalization of other factors, besides Dpp, is
critical to prevent cell extrusion in the presumptive blade.
Another possible mechanism by which cells could initiate cell competition is by
exchanging information through cell‐cell communication mechanisms 179. A
mathematical model predicts that build‐up of negative pressure around a group of
cells leads to their distortion and may trigger apoptosis in the clonal context 180. In this
sense, stronger adhesive interactions in the distal wing epithelium could constrain
cells, preventing the flow that relieves local pressure and thereby accounting for the
specificity of the clone extrusion phenotype in this region (Fig.3B). In fact, apical cell
shapes and expression of DE‐cad are graded in response to Wg secreted from the DV
organizer, pointing to stronger adhesive interactions in the distal wing epithelium 181.
Interestingly, CP mutant clones induced in the hinge and notum are rarely
extruded but develop smooth borders, evidencing affinity differences in relation to
their WT neighbours (Supp.Fig.5). These apparently conflicting processes could
represent a continuum of responses that depends on the strength of the affinity
differences along the proximal‐distal axis of the wing disc. For instance, crb
overexpressing clones in the hinge and notum seem to round up before extruding,
possibly minimizing their adhesive contacts with WT neighbours.
Overall, my results suggest that CPs and crb could cooperate to prevent cell
extrusion, and that disruption of actin dynamics or polarity maintenance respectively
mediated by these two proteins can lead to cell competition.
Gaspar P. Master Thesis
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Fig.3 ‐ Cell extrusion of CP mutant clones and crb overexpressing cells could be due to defective
endocytic uptake of survival factor (A). Alternativly, a mechanical stress imprinted by the disequilibrium
of adhesive tension forces between these cells and there WT neighbours, could trigger cell extrusion
and apoptosis (B).
4. The Crb complex and the actin cytoskeleton might cooperate to prevent growth
distortions in the wing imaginal disc
Consistent with the existence of a proximal‐distal gradient of cell affinities, crb
overexpression and CP depletion seems to induce hinge specific overgrowth, which
could be due to reduced resistance of this tissue to cell contact inhibition, such as
mediated by the Hippo pathway.
To activate Hippo signalling, CPs could link actin dynamics at the cell periphery
to cortical tension sensing between adhesive neighbours, thereby transducing a
growth contact‐inhibitory signal. For instance, CP mutant and depleted cells, as well as
crb overexpressing cells, look rounder then WT, possibly pointing to a role in regulating
A B
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cortical stiffness. Interestingly, increased membrane stiffness seems to be required for
isometric cortex contraction and microtubule spindle attachment during mitosis 182, 183.
Therefore, increased cell roundness in CP depleted and crb overexpressing cells could
disturb the maintenance of uniform disc growth. Indeed, upon CP depletion or actin
overexpression, wing imaginal discs seem to grow disproportionally, showing size
enhancement of the peripheral regions belonging to the distal hinge (introduction
Fig.6E; results Fig.10). A mathematical model explains that growth in the centre of the
disc could be induced by the combined activity of Dpp and a second growth factor
coming from the D‐V boundary, whereas growth in the peripheral regions would be
induced by stretching due to central pouch growth 184. In this sense, autonomous and
non‐autonomous growth mechanisms seem to operate along the wing epithelia, the
non‐autonomous being highly dependent on cell‐cell interactions, possibly through
adhesion molecules, such as Fat 164.
Surprisingly, I’ve shown that crb overexpression in a broad tissue context drives
hinge specific neoplastic transformation. This situation is associated with expression of
the proximal fate determinant Hth and with disturbance of the Wg morphogen ring in
the distal hinge (results Fig.11). Upregulation of Wg in this region could be due to
defective Fat signaling activity, since Wg is upregulated in Fat mutant clones in the
distal hinge 185, and disruption of Fat activity leads to the transcriptional upregulation
of glypincans 186, which are able to increase the extracellular diffusion of Wg 187, 188.
Furthermore, crb overexpression seems to induce a dramatic expansion of the
presumptive hinge (results Fig.11), supporting the idea that the Wg morphogen
promotes growth in this region 189. Alternatively, distal cells overexpressing crb could
dedifferentiate and reacquire the ability to express Hth, therefore contributing to
hinge proliferation. Indeed, Hth can be looked as a primordial transcription factor,
being expressed in proliferating and undifferentiated imaginal discs and being
ultimately repressed by distal specific transcription factors, such as Vg in the wing disc 116, 117.
Interestingly, my results show that neoplastic transformation of the
presumptive blade upon crb overexpression seems to be prevented or delayed near
the D‐V boundary, since loss of the normal epithelial structure is less apparent in the
cells apposed to it (results Fig.11). This could mean that some factor emanating from
Gaspar P. Master Thesis
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the D‐V organizer prevents neoplastic transformation by reinforcing polarity and
adhesion. Interestingly, Wg limits cell proliferation in the presumptive wing blade,
despite its growth promoting activity in the wing hinge 189. Therefore I hypothesize
that, despite the potential deregulation of Fat signalling in CP depleted and crb
overexpressing cells, the growth inhibitory function of Wg at the level of the D‐V
boundary is unaffected, leading to resistance of the presumptive blade region to
overproliferation.
5. The actin cytoskeleton might be crucial to reinforce the maintenance of epithelial
surface integrity
Despite the striking differences in the resulting cell morphology, similar growth
patterns of CP depleted and crb overexpressing tissue, suggested that CP and crb could
genetically interact to regulate tissue growth. Instead, by combining CP depletion with
expression of a kinase‐dead aPKC to reduce Crb polarizing activity, the adult wing
phenotype was severely enhanced. Although this does not rule out a possible genetic
interaction between crb and CP, it revealed a previously unreported genetic
interaction between CP and aPKC in the regulation cell size and shape.
The role of aPKC during the establishment and maintenance of cell polarity is
likely complicated. For instance, embryos maternally and zygotically mutant for
aPKCk06403, affecting the kinase domain, show apical clustering and punctated
accumulation of DE‐cad and Par‐3, which is concomitant with embryonic epithelial
polarity defects at the onset of gastrulation 190. This was attributed to abnormal
cytoskeleton interactions that arise from unattachment of microtubules from the
centrosome during cellularization 190. However, another aPKC mutant, also affecting
the kinase domain, has been isolated that shows no evidence of a zygotic phenotype
given the maternal contribution of a ‘WT’ aPKC allele, although it recapitulates the
gastrulation‐defective phenotype if ‘WT’ contribution is not provided by the female
germline (Ferreira, Prudêncio and Martinho, unpublished). This seems to indicate that
some function of aPKC is required for the establishment of epithelial polarity and
adhesion during early embryonic development, but is not longer crucial for
maintaining it during later stages. Accordingly, my results show that clonal expression
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of a kinase‐dead aPKC during the 2nd and 3rd larval instar doesn’t seem to affect the
polarity and adhesion of wing imaginal disc cells.
Interestingly, expression of a kinase‐dead aPKC in cpa (data not shown) or cpb
mutant clones leads to cell extrusion and death in all regions of the wing imaginal disc
(results Fig.12), suggesting that polarity maintenance still relies on aPKC during larval
stages. In fact, it has been suggested that the actin cytoskeleton plays a crucial role in
providing tension at junction remodelling sites, such as during cell rearrangements that
establish the ommatidial cores in the Drosophila retina 191. As such, CPs could be
crucial to promote the formation of an actin network that provides tension and
prevents cell extrusion whenever AJ stability is compromised, such as in the case of
aPKC loss of function.
Wings depleted of CP and expressing aPKCCAAXDN show an increased density of
wing hairs and margin bristles as well as shortening of the cells’ apical‐basal length,
while expression aPKCCAAXDN alone gives no obvious phenotype in the adult wing
(results Fig.13‐14). This suggests a synergistic role of CPs and aPKC in regulating cell
size and shape. At least in some cases, the apical Baz/Par‐3/PAR‐6/aPKC complex is
thought to locally regulate cytoskeletal dynamics, through the Rho‐like GTPase Cdc42
and Rac. Furthermore, Rac seems to suppress Rho activity, thereby preventing
excessive acto‐myosin contractility 192. In the Drosophila wing epithelium, Rac1 is
essential for actin polymerization at the level of the AJ, and Cdc42 is important for cell
elongation and reorganization of the basal actin network 193. In detail, localized apical
activity of Cdc42 and Rac could promote the activity of the Arp2/3 complex 194, 195,
thereby promoting actin filament polymerization and cell elongation. On the contrary,
Rho1 has recently been shown to promote isometric cortex contraction thereby
promoting transition from the columnar to cuboidal cell shape (Widmann and
Dahmann, unpublished). In cells depleted of CP, the aPKC complex might be sufficient
to promote the columnar shape, through activation of the Arp2/3 complex (Fig.4).
However, if CP is lost in cells expressing a domainat negative aPKC, the actin network
typical of anisotropic columnar cells might be disrupted, favouring the cuboidal shape
(Fig.4). In these sense, CP activity in concert with the Arp2/3 complex would be
sufficient to produce the actin network typical of columnar epithelial cells, preventing
transitions to the cuboidal shape that would lead to tissue shape distortions (Fig.4).
Gaspar P. Master Thesis
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Fig.4 – Synergy between aPKC and CP in the definition of epithelial cell shape. aPKC regulates actin
dynamics through the Rho‐like GTPases Cdc42 and Rac, which, in turn, activate the Arp2/3 co‐factors,
SCAR/WAVE and WASP, respectively. Rho activity might antagonize the Arp2/3 complex and CPs in the
production of anisotropic cortical tension, promoting isometric cortex contraction of the acto‐myosin
network and transition to the cuboidal cell shape (A). Upon expression of a dominant negative aPKC
(DN‐aPKC), Arp2/3 modulation by Cdc42 and Rac might become interrupted, which could lead to a
columnar‐to‐cuboidal cell shape transition, unless CP activity is able to compensate for the generation of
anisotropic tension (B, C).
A B
C
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7. Concluding remarks
Overall, the data presented in this thesis has contributed to understand the
role of CPs in endocytic vesicle trafficking of membrane surface receptors.
Furthermore, this work has contributed to clarify a role of the actin cytoskeleton and
the Crb polarity complex in the control of uniform and coordinated tissue growth
during development, the disruption of which could result in tumorigenesis. This work
has pointed to a significant synergy between between the actin cytoskeleton and
polarity/adhesion complexes in achieving structural support for cell shape changes and
adhesion during epithelial morphogenesis.
In the near future, understanding the signal transducing capacity of the actin
cytoskeleton, from the regulation of vesicle trafficking to mechanotransdution of
cortical tension, will help us understand how this cellular constituent impinges on
growth, and possibly differentiation pathways. In particular, combining new
biophysical approaches with genetic and biochemical data will help us understand how
cells sense the physical aspects of their environment, thereby triggering signaling
pathways through force‐bearing actin filaments.
Gaspar P. Master Thesis
55
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Appendix I
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Drosophila melanogaster as a model organism in developmental biology – a brief
historical background
Drosophila melanogaster, commonly known as the ‘fruit fly’, has become one
of the most widely used animal models in biological research. Dating from the first
decade of the twentieth century, Thomas Hunt Morgan’s laboratory set the basic
techniques for breeding and identifying large collections of mutants, allowing for the
development of a new era in genetics and developmental biology 196.
Due to its short life cycle, spanning only 12 days at normal room temperature,
and to the ease and low cost of its culturing techniques, Drosophila became a favourite
model in the utmost years of genetic research. The field of Drosophila genetics had its
major development taking advantage of the use of balancer chromosomes, phenotypic
markers and non‐occurrence of meiotic recombination in the male sex. For instance,
balancer chromosomes were artificially generated by multiple inversions and
translocations of the wild‐type chromosomes, allowing for the inhibition of
homologous recombination in mutant and transgenic stocks, so that these can be
maintained without selection. On the other hand, phenotypic markers, either
dominant or recessive, allow tracking of their associated chromosomes over multi‐
generation crosses, so that certain genotypes of interest can become easily selected
and scored.
Since the first large scale generation of mutants, many phenotypic aspects,
such as the denticle pattern of the embryonic cuticle, turned out to be an excellent
read‐out to support systematic screens, pertaining to identify new genes and their
epistatic processes 197. However, it was not until the 1980s that recombinant DNA
technology developed in bacteria and phage opened new ways to target genes and
regulatory sequences and to further observe patterns of expression. The ingenious
domestication of wild transposable elements has allowed the use of vector mediated
transformation by transposon sequences, such as P‐elements in Drosophila, and
became the major technique in the generation of animal transgenics through many
forms of genome engineering 198, 199.
Even more, mosaic tissue analysis of particular mutations, through the artificial
induction of mitotic recombination, has allowed for the study of complex interactions
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between genetically heterogeneous tissues, giving new insights on the specificity and
instructive nature of phenotypes, which are not easily evaluated in the drastic context
of whole mutant animals. More recently, powerful genetic tools, such as the use of the
UAS‐Gal4 and MARCM systems, allowed for the conditional expression of genes in
Drosophila, giving us a better understanding of gene function in time and space132.
Since the publication of its euchromatic genome in 2000, Drosophila became
known as an important reference for human biological research, for instance sharing
over 70% of the proteins involved in human disease 200‐202. Additionally, in this new
century, Drosophila research continues to expanded its use of technological advances,
such as knockdown RNA interference, microarrays, confocal imaging, laser ablation
and many others, therefore being a major subject in biological research and leading to
the discovery of evolutionarily conserved developmental mechanisms in other
metazoans.
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Appendix II
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Suplementary Figures Sup.Fig.1 – Overexpressing both act42A and act5C in the wing imaginal disc leads to additional wing
defects, as compared to the phenotypes of wings overexpressing act5C or act42A alone (C‐D).
Ovexpressing either act5C or act42A leads to notching of the wing margin (B‐C), although this is slightly
more evident in the case of act5C overexpression (C), as compared to WT (A); simultaneous
overexpression of both act5C and act42A leads to more severe defects, including: the appearance of
necrotic patches (D – red arrowhead), loss of hinge structures and the differentiation of ectopic blade
tissue, disrupting the proximal‐anterior margin (D – arrow).
Sup. Fig.2 – CP depleted cells accumulate the Notch receptor and Crb in the wing imaginal disc. cpb
depleted cells are marked by the presence of GFP (green) and Crb appears in red/gray (A‐A’’’); Crb
accumulates at the apical cell surface (A’’) and at the level of punctated structures (A’‐A’’’ – arrow).
A WT B nub>UAS‐act42A C nub>UAS‐act5C D nub>UAS‐act42A, UAS‐act5C
sd>UAS‐cpbRNAiC10
A A’
A’’
A’’’
Crb GFP Crb
Crb
Crb
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Sup. Fig.3 – Overexpression of act42A and act5C leads to accumulation of Crb in the wing imaginal disc.
Cells overexpressing act42A (A), act5C (B), or both act42A and act5C (C,D) are marked by the presence
of GFP (green), and Crb appears in red/gray (A‐D’’); Crb accumulates at the apical cell surface in wing
discs overexressing either act42A (A’), act5C (B’) or both act42A and act5C (C’), as indicated by anti‐Crb
(gray); Crb also accumulates at the level of puncated structures, as indicated by anti‐Crb (gray), in wing
discs overexpressing both act42A and act5C (D’‐D’’).
A’
Crb
B’
Crb
C’
Crb
D D’ D’’
Crb Crb Crb GFP
nub>UASact42A::GFP A
Crb At42A‐GFP
nub>UASact5C::GFP B
Crb Act5C‐GFP
nub>UASact5C::GFP UASact42A::GFP C
Crb Act42A‐GFP Act5C‐GFP
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Sup. Fig.4 – aPKC positively regulates the intracellular domains of Crb, during wing morphogenesis.
Driving crbintra and DN‐apkc in the wing imaginal disc gives rise to adult wings that show some mild
defects: loss of cross‐vein differentiation and general broadening of the blade surface (B), as compared
to WT (A).
Sup. Fig.5 – cpa107E and cpbM143 mutant clones induced in the wing imaginal disc have reduced contact
with their tissue surroundings, suggesting differences in cell‐cell affinity towards their WT neighbouring
cells. Measurement of clonal Ferret’s circularity is based on the estimation of a single distance, the
Ferret’s diameter, which corresponds to the largest diameter of an irregular object (the clone). This
formula gives the ratio between the object’s area (A) and the area of a disc reference with the same
Ferret’s diameter (π(dFer./2)2 (varies between 0>Fer. Circ. >1, such that values close to 0 approach the
perfect square circularity, and values close to 1 approach the perfect disc circularity). cpa107E and cpbM143
are significantly rounder than ‘WT’ clones induced in mutation‐clear FRT42 and FRT40 isogenic lines,
respectively (p‐value cpa107E = 0,0028, N = 32; p‐value cpbM143 = 0,0099, N=36). Clones of each
genotype are shown in B‐E, being marked by the absence of GFP (green). NOTE: clones were induced at
1st and 2nd larval instars. A – area, dFer – Ferret’s diameter.
D FRT42D clones
GFP
B FRT42D, cpa107E clones
GFP
A WT B nub>UAS‐crbintra; UAS‐aPKCCAAXDN
A C FRT40A, cpbM143 clones
GFP
E FRT40A clones
GFP
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Experimental genotypes
Results Fig.1 ‐ GFP‐ cpa‐ clones: w‐; FRT42D, tub‐GFP/FRT42D, cpa107E; T155‐Gal4, UAS‐
FLP .
Results Fig.2‐4, 8 ‐ express UAS‐cpbRNAiC10 in the en domain: w‐; en‐Gal4, dll‐lacZ/
UAS‐cpbRNAiC10; UAS‐mCD8::GFP .
Results Fig.5 ‐ express UAS‐act5C::GFP in the nub expression domain : w‐; nb‐Gal4/
UAS‐act5C::GFP .
Results Fig.6 ‐ express UAS‐act5C::GFP in the nub expression domain : w‐; nb‐Gal4/
UAS‐act5C::GFP (A); express UAS‐act42A::GFP in the nub expression domain: w‐; nb‐
Gal4/ UAS‐act42A::GFP (B); express UAS‐act5C::GFP, UAS‐act42A::GFP in the nub
expression domain: w‐; nb‐Gal4/ UAS‐act42A::GFP, UAS‐act5C::GFP (C).
Results Fig.7 ‐ clones expressing HA‐cpa: y‐,w‐,hsFLP122; FRT42D, tub‐Gal80/FRT42D,
UAS‐HA‐cpa (A); clones expressing rab5::GFP: y‐,w‐,hsFLP122; FRT42D, tub‐
Gal80/FRT42D, UAS‐rab5::GFP (B); (C) clones expressing rab7::GFP: y‐,w‐,hsFLP122;
FRT42D, tub‐Gal80/FRT42D, UAS‐rab7::GFP; (D‐D’’’) express UAS‐rab5::GFP and UAS‐
HAcpa in the nub expression domain: w‐; nb‐Gal4/ UAS‐rab5::GFP; UAS‐HAcpa; (E‐E’’’)
express UAS‐rab7::GFP and UAS‐HAcpa in the nub expression domain: w‐; nb‐Gal4/
UAS‐rab7::GFP; UAS‐HAcpa.
Results Fig.9 – clones expressing UAS‐crbintra: y‐,w‐,hsFLP122, UAS‐GFP; FRT42D, tub‐
Gal80/ FTR42D, UAS‐crbintra; tub‐Gal4.
Results Fig.10‐11 ‐ express UAS‐crb in the nub expression domain: w‐; nb‐Gal4/ UAS‐
crbweak (A); w‐; nb‐Gal4/ UAS‐crbstrong (B); w‐; nb‐Gal4/ UAS‐crbintra (C‐C’).
Results Fig.12 ‐ (A‐A’) clones expressing UAS‐aPKCCAAXDN: y‐,w‐,hsFLP122, UAS‐GFP;
FRT40D, tub‐Gal80/ FRT40A; tub‐Gal4/ UAS‐aPKCCAAXDN; (B‐B’) cpb‐ clones expressing
UAS‐aPKCCAAXDN: y‐,w‐,hsFLP122, UAS‐GFP; FRT40D, tub‐Gal80/ FRT40A, cpbM143; tub‐
Gal4/ UAS‐aPKCCAAXDN.
Results Fig.13 ‐ (B‐B’’) express UAS‐aPKCCAAXDN in the nub expression domain: w‐; nub‐
Gal4/sp; UAS‐aPKCCAAXDN; (C‐C’’) express UAS‐cpbRNAiC10 in the nub expression
domain: w‐; nub‐Gal4/ UAS‐cpbRNAiC10; (D‐D’’) express UAS‐cpbRNAiC10 and UAS‐
aPKCCAAXDN in the nub expression domain: w‐; nub‐Gal4/ UAS‐cpbRNAiC10; UAS‐
aPKCCAAXDN.
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Results Fig.14 ‐ (A) express UAS‐cpbRNAiC10 in the en expression domain: w‐; nb‐Gal4/
UAS‐cpbRNAiC10 ; (B) express UAS‐cpbRNAiC10 and UAS‐aPKCCAAXDN in the en
expression domain: w‐; nb‐Gal4/ UAS‐cpbRNAiC10; UAS‐aPKCCAAXDN.
Sup. Fig. 1 ‐ express UAS‐act42A in the nub expression domain: w‐; nub‐Gal4/UAS‐
act42A (B); express UAS‐act5C in the nub expression domain: w‐; nub‐Gal4/UAS‐act5C
(C); express UAS‐act42A and act5C in the nub expression domain: w‐; nub‐Gal4/UAS‐
act42A, UAS‐act5C (D).
Sup. Fig.2 ‐ express UAS‐cpbRNAiC10 in the sd expression domain: sd‐Gal4; UAS‐
cpbRNAiC10; UAS‐mCD8::GFP
Sup. Fig.3 –express UAS‐act42A in the nub expression domain: w‐; nub‐Gal4/UAS‐
act42A (A‐A’); express UAS‐act5C in the nub expression domain: w‐; nub‐Gal4/UAS‐
act5C (B‐B’); express UAS‐act42A and act5C in the nub expression domain: w‐; nub‐
Gal4/UAS‐act42A, UAS‐act5C (C‐D’’).
Sup. Fig.4 ‐ express UAS‐crbintra and UAS‐aPKCCAAXDN in the nub expression domain: nub‐
Gal4; UAS‐crbintra; UAS‐aPKCCAAXDN.
Sup. Fig.5 ‐ GFP‐ cpa‐ clones: y‐,w‐,hsFLP122; FRT42D, tub‐GFP/FRT42D, cpa107E (B) ; GFP‐
cpb‐ clones: y‐,w‐,hsFLP122; FRT40A, tub‐GFP/FRT40A, cpbM143 (C); GFP‐ WT clones y‐,w‐
,hsFLP122; FRT42D, tub‐GFP/FRT42D or y‐,w‐,hsFLP122; FRT40A, tub‐GFP/FRT40A (D‐E).