Cytoskeleton and cell movement
Major elements
Desmosomes
centrosome
Nuclear lamina
10 nm
25 nm
7 nm
 Intermediate filaments
supply mechanical strength
to the nuclear envelope
 Intermediate filaments
strengthen desmosomes
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
 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
Intermediate filament types
 Epithelial cell intermediate filaments are made of keratin
 In other cell types: vimentin, desmin, neurofilaments
 Nuclear lamina are made of lamins
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
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
 Nuclear lamina
rearrangements are
regulated by
phosphorylation
(disassembly)/dephos
phorylation
(assembly)events
 Other PTMs are
sumoylation,
glycosylation and
farnesylation
Microtubules
 Pairs of alfa-tubulin and beta-
tubulin interact via noncovalent bounds
 Microtubules are polar= they
have + and – ends
 +end is where beta-tubulin
 +end is fast-growing
Centrosome
 Centrosome
includes a pair
of centrioles
 Centrosome
matrix include
gammatubulin ring
complex that
is associated
with –end of
microtubules
Microtubules
 Microtubules are very dynamic structures
 The dynamic instability of microtubules is
determined by a ratio GTP/GDP bound tubulins
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
Cell polarization: nerve cells
 Microtubules are
involved in cell
polarization: when one
end of the cell is
different from another
end
Microtubule
Associated
proteins
(MAPs)
 Microtubules in axon has uniform orientation with their plus ends facing the axon tip
and it’s 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.
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 kinesi
attached to their sirface
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.
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
Flagellum bending
 Flagellum bending is due to
dynein and other accessory
proteins that bind
microtubules
Actin filaments=microfilaments
 Microvilli
 Contractile bundles
 Filopodia
 Contractile ring
 Actin filament
assembly is ATPdepdendent
 Actin monomers
(G-actin) are
polarized molecules,
with a positive (+)
“barbed end” and a
negative (-)
“pointed" end.
Actin-binding proteins
 Monomer binders and capping proteins control rate G-actin/F




actin turnover = regulate actin polymerization
Bundlers, cross-linkers B, rulers etc. control microfilamen
stability
Nucleators involved in branch formation
Membrane anchors
Cytosceletal linkers
Molecular motors (myosines)
Actin-binding proteins: monomer
binders and cappers
 Nucleotide exchange: profilin
 Monomer capping/sequestration: thymosins
 Cappers gelsolin fragmin, vilin, capZ
 Depolymeriizing: ADF/cofilin, AIP1
Actin nucleators: branch formation
 ARP2/3 complex
 WASP
F-actin bundlers and cross-linkers
 Microvilli – fimbrin, villin, espin
 Filopodia – fascin, alfa-actinin
 Strees bibers - – fascin, alfa-actinin
 Spectrin, transgelin - cross-linking
proteins influence the packing and
organization of actin filaments into
secondary structures.
Linkers
Cytosceletal linkers
 Actin to IF – spectrin
 Actin to microtubules – tau
 Actin to IF and microtubules –
MAP2, plectin, BRAG
Membrane linkers
 α-actinin
 α-catenin
 Plectin
 Spectrin
 Annexin II
Rulers and motors
Rulers
Motors
 Tropomyosin
 Myosin (>17 classes)
 Calponin
 Muscles – myosin II
 nebulins
 Cell movements - Myosin I
Spectrin is a filament forming accessory protein that forms a
erythrocyte membrane bound
cytosceleton (cell cortex)
Vann Bennett, and Anthony J. Baines Physiol Rev
2001;81:1353-1392
 Cell cortex is a layer beneath the
plasma membrane
 Cell cortex includes actin and
spectrin meshworks
 Spectrin forms
membrane-bound
skeleton of erythrocytes
Cell movement (crawling, migration)
 Major stages of cell crawling
1. Cell first acquires a characteristic
2.
3.
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.
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.
To move forward, the cell retracts its
trailing edge by combining actomyosin
contractility and disassembly of
adhesions at the rear
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
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
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
 Lamellipodium is driven by a
Lamellipodia
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
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.
 Arp2/3 (actin related protein)
Arp2/3 complex



Christophe Le Clainche, and Marie-France Carlier Physiol
Rev 2008;88:489-513


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
 Filopodia contain 15–20 parallel
Filopodia
filaments tightly packed into a
bundle with their barbed ends facing
the membrane
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
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
Christophe Le Clainche, and Marie-France Carlier Physiol
proteins.
Rev 2008;88:489-513
Cell adhesion structures
 Adhesion structures are
Christophe Le Clainche, and Marie-France Carlier Physiol
Rev 2008;88:489-513
classified into focal
complexes, focal adhesions,
and fibrillar adhesions.
 Focal complexes are
dotlike structures of 1 μm2
at the leading edge of
migrating cells
 In slow moving cells, focal
complexes mature into
focal adhesions of 2–5 μm
long
 Fibrillar adhesions arise
from focal adhesions. They
are elongated structures
associated with fibronectin
fibrils and located more
centrally in cells.
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.
 Arp2/3 complex
plays the central role
in lamellipodium
formation,
endocytosis, and
intracellular parasite
invasion and
movement
Kaksonen et al. Nature Reviews Molecular Cell Biology 7, 404–414 (June 2006) | doi:10.1038/nrm1940
Regulation of cytosceleton
rearrangements  Cell cortex cytosceleton
Hall et al., 1998
rearrangements are controlled
by small GTPases of the Rhofamily
 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 .
Cell body translocation is mediated by
actomyosin contractility
Qingjia Chi et al. J. R. Soc. Interface 2014;11:20131072
 Rac and
CDC42 are
active during
an early
spreading
 Rho is active
during late
spreading
 Asymmetric accumulation of
myosin II at the leading and
trailing edges.
 Rear actin-myosin filaments
associate with intracellular
sites of focal complexes
Qingjia Chi et al. J. R. Soc. Interface 2014;11:20131072
Myosin II structure
 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),
Qingjia Chi et al. J. R. Soc. Interface 2014;11:20131072
 Muscle contraction is
Muscle contraction
dependent on interactions
between actin and myosin
II filaments
 1 ATP per 5 nm
 15 mkm per sec
Ca2+- dependent changes in
troponin/tropomyosin/myosin interactions
 Tropomyosin (ruler) prevents actin/myosin interactions
 Troponin binds Ca2+
 Changes in troponin conformation upon binding cause
changes in tropomyosin that results in opening of
microfilament groove and promotes actin-myosin
interactions
Ca2+ -driven actin-myosin interactions
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)
 Leukocyte chemotaxis is regulated
by a number of chemoattractants
include bacterial signals (pathogenassociated molecular patterns),
complement proteolytic fragment
C5a, and the superfamily of small (8–
10 kDa), inducible, secreted, proinflammatory cytokines called
chemokines
 Cells respond to chemoattractant
gradients as shallow as a 2-5%
difference across the anterior and
posterior
 CDC42 and Rac are recruited to
the leading edge
 PIP3 phosphatase PTEN and RhoA
accumulate at the rear end
CDC42/Rac
Rho
 Chemoattractants such as chemokines bind to their specific cell surface
receptors and activate the trimeric G-proteins (Gi) in leukocytes
 α i-subunit inhibits adenylyl cyclase
 βγi-subunits activate PI3K
 PtdIns (3,4,5)P3 recruits CDC42 and Rac
Hijacking of actin machinery by
intracellular parasites: invasion
 Listeria monocytogenes uses
2 ways
 1st 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 Ca2+dependent, homophilic cell–
cell adhesion molecule
 InlA/E-cadherin interactions
result in polyubiquitination of
E-cadherin and recruitment
Pizarro-Cerda et al., 2012
of clathrin and bacterial
internalization.
Hijacking
of actin
machinery
by by
Hijacking
of actin
machinery
interactions of InlB
intracellular
parasites:
invasion
intracellular
parasites:
invasion
with HFGR (Met) and
 2nd is mediated by
initiates CME
 Met binding results in
Met dimerization,
autophosphorylation
and downstream
signaling involving
phosphatidylinositol 3kinase and MAP kinase
(MAPK)
Hijacking of actin machinery by intracellular
parasites: intracellular movement
 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.