Cytoskeleton Locomotion Kohidai Laszlo MD Ph D Med

Cytoskeleton - Locomotion Kohidai, Laszlo MD, Ph. D Med. habil. , Assoc. Professor Dept. Genetics, Cell & Immunobiology, Semmelweis University Lecture ED 2017 http: //gsi. semmelweis. hu

Main functions of cytoskeleton • • Determines the shape of the cell Anchores organelles Movement of organelles Tensile strength Movement of chromosomes Polarity Motility !

Cytoskeleton Microfilaments (actin) l Microtubuli (tubulin) l Intermedier filaments l Motor proteins l Actin and microtubule associated proteins l !

! Microfilaments Microtubuli Intermedier filaments

SLIDING ! Globular proteins Ca 2+ ATP Fibrillar proteins Motor proteins

Microfilaments

Polymerization of actin ADP ATP ! + Depolymerization ADP ATP cytochalasin – inh. phalloidin - stabilizer Pi Polymerization - slow

Actin - still in Prokaryots ! ((Ent et al. Nature 2001, 413, 39)

Other actin homologues ((Roeben A et al. J Mol. Biol 2006, 358, 145)

Comparison of homologues l Polymerization in both forms l Opposite chirality !!! ((Wickstead and Gull J Cell. Biol 2011, 194, 513)

! Cyclosis Moving cytoplasm Stationary (cortical) cytoplasm Plasma membrane Cell-wall Actin filaments Chloroplasts l Transitional connections between actin and myosin l Ca 2+, temperature- and p. H-dependent (Lodish, H. et al. Mol. Cell Biol. 2000, 767)

„Fountain” mechanism ! Ca 2+-dep. requires ATP Formation of pseudopodium stress-fibrillums integrins Mono. Poly. Lobo- podial Filo. Reticulo-

Cross-linking proteins of actin ! contractile bundle a actinin – in stress fibr. gel-like network filamin - cortex „tight” parallel bundle fimbrin – in filopodium

Migrating keratinocyte 15 mm/sec Formation of lobopodium actin-network microtubuli

! Regulator proteins of actin polymerisation g. CAP 39 Tropomodulin + - Cofilin Severin Gelsolin Villin Cap. Z

Actin polymerization – acrosomal-reaction (Lodish, H. et al. Mol. Cell Biol. 2000, 767)

Listeria monocytogenes • local actin polymerization • speed: 10 mm/min • high ability to transmit in tissues actin (Fred Soo & Julie Theriot Laboratory

Model of actin nucleation ! WASP = Wiscott-Aldrich syndr. prot.

Structure of cortical region (Svitkina, TM, Borisy GG J. Cell Biol. 1999, 145, 1009)

Actin – membrane links membrane Myosin I. Arp 2/3 F-Actin Profilin - G-actin Filamin Integrin !

Profilin-mechanism Tb 4 = thymosin b 4 Proline-rich protein (Lodish, H. et al. Mol. Cell Biol. 2000, 767)

Filamin – Membrane link filamin actin

Structure of focal contact actin filament a actinin vinculin + integrin paxillin talin fibronectin !

A plasma membrane – cortex links Thrombocyte Glycophorin Ankyrin Spectrin tetramer Muscle Epithel ((Lux SE, 1979 Nature 281: 426)

E Electromagnetic field induces the transformation of cytoskeleton and formation of pseudopodia - Adhesion plaque ++ + -

head ! Myosin Ca 2+-dependent phosphorylation and its effect on the 3 D strcture light chain heavy chain a helix ATP - ADP Pi myosin I. 150 k. D monomer myosin I I. 260 k. D dimer Head: - ATP-ase - motor

Distribution of myosines in the migrating Dyctiostelium and in dividing cell myosin I. (green) myosin II. (red) (Fukui, Y. Mol. Cell Biol 2000, 785))

- + ! Main types of interactions between the globular and fibrillar components of cytoskeleton membrane

Non-treated F-actin blocked MT-blocked

Microtubules

Tubulin – still in Prokaryotes ! Fts. Z Tubulin (Margolin Laboratory, University of Texas)

Comparison of homologues l Polymerization in both forms l Monomers build helical structure vs. dimers build tubulus ((Wickstead and Gull J Cell. Biol 2011, 194, 513)

Polymerization of tubulin GTP GTP Polymerization - fast Protofilament (strait) GDP GDP Protofilament (curved) GDP Depolymerization !

Dynamics of microtubule-assembly Nucleation Elongation + incorporation balanced release - !

Role of g-tubulin in nucleation (Wiease et al. Curr. Opin. Struct. Biol. 1999, 9, 250) !

Microtubular systems in the cells centrosome Cilla Basal body - Centrosome Dividing cell spindle - Cilia / flagellum - Mitotic system - Vesicular transport ! Interphase cell Neuron centrosome axon

MTOC = Microtubule organizing center g-tubulin specific region of the cortex ((Brinkley, B. R. Encyclop. Neurosci. 1987, 665) !

Network of microtubuli 24 nm a-b dimer Protofilaments alpha tubulin beta tubulin Fibroblast

! Cilia cilia flagellum Paramecium

! tubulin (13 ill. 11 protofilaments) B A dynein-arms nexin

Composition of dynein-arms ATP-independent binding ATP-dependent hydrolysis The arm moves toward the - pole

The role of dynein arms in beating of cilia Bending „Telescoping” Proteolysis

Molecules composing the cilia more than 250 types of molecules 70% a and b tubulin l dynein arms outer - 9 polypeptides - ATP-ase inner – composition varies l radial spokes - 17 polypeptides l

Microtubules of mitotic spindle and kinetochore

Arrangement of actin during cell-division

Intermedier filaments

Crescentin

Mechanical characterization of cytoskeleton components ! intermedier filament i. e. vimentin deformation microtubule = rupture actin filament force

Role of intermedier filaments ! Buffer against external mechanical stress Tissue specificity Nucleus – lamines (lamina fibrosa) Epithel – keratin Connective tissue Muscles Neuroglia } vimentin Neurones - neurofilaments

Structure of intermedier filamentums (Lodish, H. et al. Mol. Cell Biol. 2000, 767)

Domain structures of intermedier filamentums H 2 N- a helical domain keratins vimentin neurofilam. prot. nuclear prot ! -COOH

Intermedier filaments Keratin filaments Vimentin-like filaments ! They DO NOT co-polymerize !

Microvilli actin myosin I. ! villin „terminal web” • a rigid bundle composed by 20 -30 actin mol. s • actin + on the apical part • villin is the linker molecule of actins • „terminal web” = intermedier fil. + spectrin • anchoreb by myosin I. and calmodulin to the surface membrane

SEM structure of microvilli actin bundle linker molecules „terminal web”

Intermedier filaments Glial filaments Neuro-filaments – many cross-linkers – few cross-linkers The number of protein cross-links between the intermedier filaments varies in different tissues

Microtubuli associated proteins (MAP-s)

Groups of MAP-s • Structural MAP-s - MT-assembly - links to MF and to IF • Motor proteins - sliding on MT • ! Shape and polarity of the cell Membrane transports Enzymes, signal molecules - glycolytic enzymek - kinases Assembly of molecules

Motor-proteins

Structure of motor-proteins assoc. motor domain polypeptides motor domain „stalk” assoc. polypeptides Kinesin „stalk” assoc. polypeptides Myosin Dynein !

Motor proteins - + microtubule heavy chain light chain kinesin dynein

! kinesin - + dynein c. AMP pigment cells c. AMP

! Kinesin ADP ATP ADP-Pi

MT-motor proteins and the transported elements (Hirokawa, N. Science 1998, 279: 519

There are other mechanisms over sliding …

Locomotion – with spasmoneme of Vorticella

! Spasmoneme spring Contracts 40% in few msecs Velocity: 8 cm˛/sec Negative charges Neutralization with Ca 2+

Actin spring in sperm of horseshoe crab Limulus polyphemus acrosome actin bundle • The extension does not involve a myosin motor or actin polymerization • The bundle is crystalline in its coiled and uncoiled states !