Major elements Nuclear lamina Desmosomes 10 nm25 nm7 nm
centrosome
Slide 3
Desmosome A desmosome, also known as macula adherens (plural:
maculae adherentes) is a cell structure specialized for
cell-to-cell adhesion. A type of junctional complex, they are
localized spot-like adhesions randomly arranged on sides of plasma
membranes. They help to resist shearing forces Intermediate
filaments supply mechanical strength to the nuclear envelope
Intermediate filaments strengthen desmosomes
Slide 4
Intermediate filaments Intermediate filaments are stable They
are formed by fibrous protein subunits The monomer of the subunit
is alfa-spiral The basic subunit is a tetramer Intermediate
filaments are non-polar
Slide 5
Intermediate filament types Epithelial cell intermediate
filaments are made of keratin In other cell types: vimentin,
desmin, neurofilaments Nuclear lamina are made of lamins
Slide 6
Intermediate Filament Associated Proteins Cross-link
intermediate filaments with one another forming a bundle (also
called a tonofilament) and with microtubules, microfilaments and
desmosome Plectin 500 kDa Striated muscle, epithelia Nuclear
envelop
Slide 7
Nuclear lamina Nuclear lamina is intranuclear mesh of IF
Farnesylation of lamin is used to bind NL with nuclear membrane The
genetically governed loss of NL causes progeria
Slide 8
Nuclear lamina rearrangements are regulated by phosphorylation
(disassembly)/dephos phorylation (assembly)events Other PTMs are
sumoylation, glycosylation and farnesylation
Slide 9
Microtubules Pairs of alfa-tubulin and beta- tubulin interact
via non- covalent bounds Microtubules are polar= they have + and
ends +end is where beta-tubulin +end is fast-growing
Slide 10
Centrosome includes a pair of centrioles Centrosome matrix
include gamma- tubulin ring complex that is associated with end of
microtubules Centrosome
Slide 11
Microtubules Microtubules are very dynamic structures The
dynamic instability of microtubules is determined by a ratio
GTP/GDP bound tubulins
Slide 12
Microtubules as a target for anticancer drugs Colchicine blocks
cell division by distrupting microtubules and the spindle
microtubules are more sensitive to colchicine than the interphase
microtubules. Colchicine inhibits tubulin polymerization Taxol
prevents microtubule depolymerization
Slide 13
Cell polarization: nerve cells Microtubules are involved in
cell polarization: when one end of the cell is different from
another end
Slide 14
Microtubules in axon has uniform orientation with their plus
ends facing the axon tip and its bundled up together by the protein
called tau protein. Tau is important in the case of neurite
outgrowth Microtubules in dendrites have multiple orientations with
their plus ends facing either the cell body or the dendritic tips
and the bundling protein here is the MAP2. Microtubule Associated
proteins (MAPs)
Slide 15
The role of microtubules in intracellular traffic Saltatory
movement of organelles along microtubules ATP-dependent Kinesins
move toward microtubule +end Dyneins move toward microtubule end
Different organelles could have either dynein or kinesin attached
to their sirface
Slide 16
Anterograde and retrograde transport Motor proteins move
vesicles and organelles along microtubules that extend from the
MTOC near the Golgi, with (+) ends pointing to the cell periphery.
Kinesin dependent anterograde transport moves mitochondria,
lysosomes and vesicles to the cell periphery. Dynein retrograde
transport moves vesicles from the ER to the Golgi.
Slide 17
Cilia and flagella Cilia are on the surface of respiratory
tract epithelial cells Flagellum on spermatozoon Microtubelus of
cilia and flagella are different in structure and much more
stable
Slide 18
Flagellum bending Flagellum bending is due to dynein and other
accessory proteins that bind microtubules
Slide 19
Rulers and motors RulersMotors Tropomyosin Calponin nebulins
Myosin (>17 classes) Muscles myosin II Cell movements - Myosin
I
Slide 26
Spectrin forms membrane-bound skeleton of erythrocytes Vann
Bennett, and Anthony J. Baines Physiol Rev 2001;81:1353-1392 2001
by American Physiological Society Cell cortex is a layer beneath
the plasma membrane Cell cortex includes actin and spectrin
meshworks Spectrin is a filament forming accessory protein that
forms a erythrocyte membrane bound cytosceleton (cell cortex)
Slide 27
Cell movement (crawling, migration) Major stages of cell
crawling 1. Cell first acquires a characteristic polarized
morphology in response to extracellular signals. At the cell front,
actin assembly drives the extension of flat membrane protrusions
called lamellipodia and fingerlike protrusions called filopodia. 2.
At the leading edge of the lamellipodium, the cell forms adhesions
that connect the extracellular matrix to the actin cytoskeleton to
anchor the protrusion and tract the cell body. 3. To move forward,
the cell retracts its trailing edge by combining actomyosin
contractility and disassembly of adhesions at the rear
Slide 28
Cell movement The movement occurs at the same rate as actin
assembly Actin dynamics controls cell migration via Maintaining of
rapid actin assembly at steady state in the lamellipodium during
cell migration Constant initiation of actin assembly in a
site-directed fashion Mechanical coupling of actin assembly to
adhesion to enable protrusion and traction of the cell body
Slide 29
Cell movement Microfilaments that push filaments are
straightforward and bundles by fascin etc. At the tip of filopodia,
formins like mDia2 catalyze the processive assembly of
microfilaments. Adhesion is mediated by integrins that are
associated with a cortex proteins
Slide 30
lamellipodia and filopodia The formation of different
protrusions, such as lamellipodia and filopodia, in the same
cellular environment, can be explained by the activation of two
different nucleation machineries (Arp2/3 and forminsm,
respectively) that generate actin arrays with different
architecture and dynamics
Slide 31
Lamellipodia Lamellipodium is driven by a growing network of
actin microfilaments (branched actin nucleation) Arp2/3 and WASP
are nucleators ADF, profilin, and capping proteins cooperate to
accelerate the treadmilling rate
Slide 32
Regulation of actin treadmilling Christophe Le Clainche, and
Marie-France Carlier Physiol Rev 2008;88:489-513 ADF (cofilin)
induces pointed-end depolymerization to increase the concentration
of monomeric actin Profilin enhances the exchange of ADP for ATP to
recycle actin monomers and the directionality of treadmilling. By
blocking the majority of actin filament barbed ends, capping
proteins increase the concentration of monomeric actin to row
faster individual filaments grow faster.
Slide 33
Arp2/3 complex Arp2/3 (actin related protein) complex is a
stable complex of seven conserved subunits The Arp2/3 complex
localizes at the leading edge of migrating cells where it nucleates
branched actin filaments The Arp2/3 complex is a nucleator, and a
branching agent Arp2/3 regulates actin dynamics in endocytosis,
phagocytosis, cell migration, intracellular traffic, and
internalization and propulsion of pathogens Arp2/3 is activated by
WASP WASP is activated by Cdc42(Rac)/Src Christophe Le Clainche,
and Marie-France Carlier Physiol Rev 2008;88:489-513
Slide 34
Filopodia Filopodia contain 1520 parallel filaments tightly
packed into a bundle with their barbed ends facing the
membrane
Slide 35
Filopodium growth Extension of filopodia is controlled by
formins that regulate actin assembly at the tip Formins are
activated by Cdc42 Profilin enhances the exchange of ADP for ATP to
recycle actin monomers Fascin controls filament bundling
Slide 36
Cell adhesion Adhesions connect cytosceleton to extracellular
matrix to convery the force generated by actin assembly at the
leading edge into protrusion Adhesions control the Mechanical
Coupling Between the Actin Cytoskeleton and the Substrate by
regulated molecular interactions at different levels:
integrin-substrate, integrin-actin binding proteins, and actin
filaments-actin binding proteins. Christophe Le Clainche, and
Marie-France Carlier Physiol Rev 2008;88:489-513
Slide 37
Cell adhesion structures Adhesion structures are classified
into focal complexes, focal adhesions, and fibrillar adhesions.
Focal complexes are dotlike structures of 1 m 2 at the leading edge
of migrating cells In slow moving cells, focal complexes mature
into focal adhesions of 25 m long Fibrillar adhesions arise from
focal adhesions. They are elongated structures associated with
fibronectin fibrils and located more centrally in cells. Christophe
Le Clainche, and Marie-France Carlier Physiol Rev
2008;88:489-513
Slide 38
Stress fibers Stress fibers are higher order cytoskeletal
structures composed of cross-linked actin filament bundles, and in
many cases, myosin motor proteins, that span a length of 1-2
micrometers The presence of motor proteins in stress fibers enables
contractility - an important factor in stress fiber function and in
cell motility. In most cases, stress fibers connect to focal
adhesions, and hence are crucial in mechanostransduction.
Slide 39
Kaksonen et al. Nature Reviews Molecular Cell Biology 7, 404414
(June 2006) | doi:10.1038/nrm1940 Arp2/3 complex plays the central
role in lamellipodium formation, endocytosis, and intracellular
parasite invasion and movement
Slide 40
Regulation of cytosceleton rearrangements Cell cortex
cytosceleton rearrangements are controlled by small GTPases of the
Rho- family Rho influences cell adhesion assembly and maturation
and controls contractile activity. Rac1 primarily controls actin
assembly and nascent adhesion formation in the lamellipodium Cdc42
controls the cell polarity and the formation of filopodia and
nascent focal adhesions. Hall et al., 1998
Slide 41
Cell body translocation is mediated by actomyosin contractility
Qingjia Chi et al. J. R. Soc. Interface 2014;11:20131072
Slide 42
Rac and CDC42 are active during an early spreading Rho is
active during late spreading
Slide 43
Qingjia Chi et al. J. R. Soc. Interface 2014;11:20131072
Asymmetric accumulation of myosin II at the leading and trailing
edges. Rear actin-myosin filaments associate with intracellular
sites of focal complexes
Slide 44
Myosin II structure Qingjia Chi et al. J. R. Soc. Interface
2014;11:20131072 Myosin II consists of myosin heavy chains (MHCs)
of 230 kDa four myosin light chains (MLCs); two 20 kDa regulatory
light chains (RLCs), two 17 kDa essential light chains (ELCs),
Slide 45
Muscle contraction Muscle contraction is dependent on
interactions between actin and myosin II filaments 1 ATP per 5 nm
15 mkm per sec
Slide 46
Ca 2+ - dependent changes in troponin/tropomyosin/myosin
interactions Tropomyosin (ruler) prevents actin/myosin interactions
Troponin binds Ca 2+ Changes in troponin conformation upon binding
cause changes in tropomyosin that results in opening of
microfilament groove and promotes actin-myosin interactions
Slide 47
Ca 2+ -driven actin-myosin interactions
Slide 48
Signal transduction/Chemotaxis/Cytosceleton rearrangements/Cell
movement Physiological and pathological processes that involve cell
movement - Immune response (leucocytes) - GPCR - Wound healing
(fibroblasts) - TKRs - Cancer (metastasis)
Slide 49
Leukocyte chemotaxis is regulated by a number of
chemoattractants include bacterial signals (pathogen- associated
molecular patterns), complement proteolytic fragment C5a, and the
superfamily of small (8 10 kDa), inducible, secreted, pro-
inflammatory cytokines called chemokines Cells respond to
chemoattractant gradients as shallow as a 2-5% difference across
the anterior and posterior PIP3 phosphatase PTEN and RhoA
accumulate at the rear end CDC42 and Rac are recruited to the
leading edge
Slide 50
Chemoattractants such as chemokines bind to their specific cell
surface receptors and activate the trimeric G-proteins (G i ) in
leukocytes i -subunit inhibits adenylyl cyclase i -subunits
activate PI3K PtdIns (3,4,5)P 3 recruits CDC42 and Rac CDC42/Rac
Rho
Slide 51
Hijacking of actin machinery by intracellular parasites:
invasion Pizarro-Cerda et al., 2012 Listeria monocytogenes uses 2
ways 1 st is mediated by interactions of InlA with E-cadherin and
initiates caveolar endocytosis or CME E-cadherin is a single-pass
transmembrane protein that functions as a Ca 2+ - dependent,
homophilic cell cell adhesion molecule InlA/E-cadherin interactions
result in polyubiquitination of E-cadherin and recruitment of
clathrin and bacterial internalization.
Slide 52
Hijacking of actin machinery by intracellular parasites:
invasion 2 nd is mediated by interactions of InlB with HFGR (Met)
and initiates CME Met binding results in Met dimerization,
autophosphorylation and downstream signaling involving
phosphatidylinositol 3- kinase and MAP kinase (MAPK) Hijacking of
actin machinery by intracellular parasites: invasion
Slide 53
The bacterial surface protein ActA mimics the host protein
WASP. ActA binds actin/ profilin, VASP and activates Arp2/3 In
addition to the factors that act at the bacterial surface,host
capping protein binds to the barbed end of actin filaments to
prevent elongation of older filaments, -actinin crosslinks
filaments to stabilize the tail structure, and ADF/cofilin
disassembles old filaments. Hijacking of actin machinery by
intracellular parasites: intracellular movement