Cytoskeleton and cell movement. Major elements Nuclear lamina Desmosomes 10 nm25 nm7 nm centrosome.

Cytoskeleton and cell movement

Major elements Nuclear lamina Desmosomes 10 nm25 nm7 nm centrosome

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

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 non- covalent bounds Microtubules are polar= they have + and ends +end is where beta-tubulin +end is fast-growing

Centrosome includes a pair of centrioles Centrosome matrix include gamma- tubulin ring complex that is associated with end of microtubules Centrosome

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

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)

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

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

Rulers and motors RulersMotors Tropomyosin Calponin nebulins Myosin (>17 classes) Muscles myosin II Cell movements - Myosin I

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)

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

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

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

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 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

Filopodia Filopodia contain 1520 parallel 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 proteins. Christophe Le Clainche, and Marie-France Carlier Physiol Rev 2008;88:489-513

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

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.

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

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

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

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

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),

Muscle contraction Muscle contraction is dependent on interactions between actin and myosin II filaments 1 ATP per 5 nm 15 mkm per sec

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

Ca 2+ -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 (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

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

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.

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

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

Cytoskeleton and cell movement. Major elements Nuclear lamina Desmosomes 10 nm25 nm7 nm centrosome.

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