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 Ecelctive Course / 2017 www. dgci. sote. 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 l Microtubule associated proteins (MAP-s) Motor proteins

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)

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 polymerisation – 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 Myozin I. Arp 2/3 F-Actin Profilin - G-aktin Filamin Integrin

Profilin-mechanism Tb 4 = timozin 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 myozin I. (green) myozin 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)

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 Interphase cell centrosome Cilla Basal body - Centrosome Dividing cell spindle - Cilia / flagellum - Mitotic system - Vesicular transport Neuron centrosome axon

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

Network of microtubuli 24 nm ab dimer Protofilaments a tubulin b 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 hydrolisis 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

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-polymerise !

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” = intermed. fil. + spectrin • myosin I. and calmodulin anchore 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 asszoc. motor domain polypeptides motor domain „stalk” asszoc. polypeptides Kinesin „stalk” asszoc. polypeptides Myozin 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

Dynein – membrane relations (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 !

Signalling mechanisms – In a nutshell –

Polymerization of microtubules Significance of motor proteins

Ameboid movement and microtubules

Complex effects of focal adhesion kinase (FAK)

LIM domains of proteins

Central role of vimentin in signalling mechanisms of cytoskeleton

Phosphatases and cell migration