Cytoskeleton and locomotion Láng, Orsolya MD, PhD Dept. Genetics, Cell & Immunobiology, Semmelweis University Lecture EPh 2016 www.gsi.semmelweis.hu

Main components of the eukaryotic cytoskeleton Microfilaments: actin 7nm Microtubules: tubulins (a, b) 25 nm Intermediate filaments: lamin cell specific prot. (e.g. vimentin) 8-12 nm + Associated proteins (e.g. motor proteins)

Filamentous structures in the cytoplasm of walled bacteria weak sequence homology but crystallography appreciate their striking structural properties and similarity to eukaryotic cytoskeletal elements Microfilament like: MreB Microtubule like: FtsZ, TubZ Intermediate filament like: Crescentin, Parm Nature Cell Biology12, 731–733 (2010)

Function of the cytoskeleton Tissue level: muscle movement Cellular level: determines shape of the cell motility of the cells cell adhesion mitosis, meiosis Subcellular level: anchors organelles organization of organelles provides tensile strength movement of chromosomes organizing cell polarity Intracellular movement of vesicles - Endocytosis – clathrin-mediated endocytosis and phagocytosis Dynamic Adaptable Stable Strong

Cytoskeletal filaments are dynamic and adaptable

Stability of cytoskeletal filaments

Strong cytoskeletal filaments Intermediate filaments- resistant to stretching forces

Microfilaments

Molecular structure of actin G-actin ( Garland Science Molecular Biology of the Cell 2008)

Polymerazition of actin + ATP ADP Depolymerization - ADP ATP Pi Polymerization - slow Drugs: cytochalasin – inhibition (cap at + end) phalloidin - stabilizer

Treadmilling of actin filament Video: http://csls-text.c.u-tokyo.ac.jp/flash/0611_1.html

Actin and accessory proteins Molecular Biology of the Cell (© Garland Science 2008)

Actin binding proteins (ABPs) 3 groups: banding and cross linking proteins regulatory proteins: polymerization/depolymerization, severing proteins,capping proteins Motor proteins - sliding on MF (myosin) Organisation of actin filaments Sliding

Organisation of actin filaments Molecular Biology of the Cell (© Garland Science 2008)

Cross-linking proteins I. Contractile bundle Parallel bundle Molecular Biology of the Cell (© Garland Science 2008)

Cross-linking proteins II. plasma membrane Gel-like network Molecular Biology of the Cell (© Garland Science 2008)

 Regulatory proteins - + gCAP39 Tropomodulin Severin Gelsolin Villin CapZ Tropomodulin - +  Cofilin Severin Gelsolin Profilin: G actin pool Thymosin: actin sequestring Formin: actin polymerizing protein

Actin polymerisation – moition I. Acrosomal reaction (Lodish, H. et al. Mol. Cell Biol. 2000, 767)

Actin polymerisation – motion II. Listreia Monocytogenes infection

F-Actin Myozin I. Arp2/3 Profilin - G-aktin Filamin Integrin Actin – membrane links membrane Myozin I. Arp2/3 Profilin - G-aktin F-Actin Integrin Filamin

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

Profilin-mechanism Tb4 = thymozin b4 Proline-rich protein (Lodish, H. et al. Mol. Cell Biol. 2000, 767)

Filamin – Membrane link actin

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

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

Microvilli At bottom: (spectrin, myosin, intermediate filaments) It is called: terminal web

Motor proteins: myosins General structure: Globular head and fibrillar tail Heavy chains and light chains Head: motor domain with ATP-ase activity ADP- straight Direction: + end motors

Myosin II molecule Molecular Biology of the Cell (© Garland Science 2008)

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

Microtubules

Molecular structure of tubulin dimers and microtubules GTP Molecular Biology of the Cell (© Garland Science 2008)

Polymerisation of microtubules Drugs: Colchicine and colcemid– inhibition (binds subunit) Taxol - stabilizer Molecular Biology of the Cell (© Garland Science 2008)

Polymerisation of microtubules Dynamic instability and treadmilling Video: http://csls-text.c.u-tokyo.ac.jp/flash/0612_1.html Molecular Biology of the Cell (© Garland Science 2008)

Role of g-tubulin in nucleation of microtubules at - end Molecular Biology of the Cell (© Garland Science 2008)

Microtubule associated proteins (MAPs) Structural MAP-s - MT-assembly links to MF and to IF (eg. tau, MAPs1 and MAPs2) Motor proteins - sliding on MT (e.g. kinesin and dynein family) Enzymes, signal molecules - glycolytic enzymes - kinases Shape and polarity of the cell Vesicular transports Assembly of molecules

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

- + ? Motor proteins + kinesin dynein http://www.nature.com/nrn/journal/v14/n3/fig_tab/nrn3380_F1.html ?

kinesin - + dynein cAMP cAMP pigment cells

Kinesin ADP ATP ADP ATP ADP ATP ADP ADP-Pi

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

In cells: MTOC = Microtubule organizing center g-tubulin 9x3 microtubules A,B,C 2 centrioles at a right angle Organisator: Gamma-tubulin in pericentriolar matrix - end of forming microtubule (Brinkley, B.R. Encyclop. Neurosci. 1987, 665)

Centrosome Molecular Biology of the Cell (© Garland Science 2008)

Cilia cilia flagellum Paramecium

Structure of cilia B A tubulin (13 ill. 11 protofilaments) dynein-arms nexin tubulin (13 ill. 11 protofilaments)

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

Role of the dynein arms in beating cilia Telescopic effect Beating

Microtubules of mitotic spindle and kinetochore

How motor proteins can organise the position of cell organelles (ER, Golgi) ? (Hirokawa, N. Science 1998, 279:519

Dynein – membrane relations (Hirokawa, N. Science 1998, 279:519)

Intermediate filaments

Structure of an intermediate filaments Antiparallel tetramer Monomer Parallel dimer Antiparallel tetramer Protofilaments Intermedaite filaments Molecular Biology of the Cell (© Garland Science 2008)

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

Role of intermedier filaments Buffer against external mechanical stress Tissue specificity !!! Epithel – keratin Connective tissue Muscles Neuroglia Neurones(axon) - neurofilaments } vimentin-like Vimentin Desmin Glial protein Exception: Nucleus – lamines (A,B,C) →(lamina fibrosa)

Domain structures of intermedier filamentums Head Tail a helical domain -COOH H2N- keratins vimentin neurofilam. prot. nuclear prot.

Axon of a neuron Glial cell Axon cross-section Neurofilament (NF-H) Glial cell Glial filaments Axon cross-section Cross bridges are formed by non-helical C terminus few cross bridges Molecular Biology of the Cell (© Garland Science 2008)

Intermediate filaments and the cytoskeleton In the hypothetical epithelial cell depicted, the three key filament systems of the cytoskeleton, microfilaments (MFs), microtubules (MTs) and intermediate filaments (IFs), are connected to each other by dimeric complexes of plakin-type molecules such as plectin and BPAG1. In addition, a multitude of MT-associated proteins and actin-binding proteins, including motor proteins, are thought to increase the complexity of these interactions. IFs are coupled to IF-anchoring plaques of cell–cell junctions (desmosomes) by desmoplakin, a prototype plaque molecule (plakin), and to those of cell–matrix junctions (hemidesmosomes) by plectin and BPAG1. The transmembrane proteins that mediate the contact with the neighbouring cells and with the extracellular matrix (ECM) are desmosomal cadherins and integrins, respectively. IFs are furthermore coupled to the outer nuclear membrane (ONM) by plectin and nesprin-3, whereas nesprin-2 anchors the MF system to the nucleus. On the inner side of the nuclear envelope, a layer of nuclear IF proteins (lamins) is attached to pores and inner nuclear membrane (INM) proteins as well as to chromatin. The membrane proteins of the INM might be linked to those of the ONM and thereby provide a mechanical continuum reaching from the ECM to chromatin. The number of newly identified INM and ONM proteins is increasing steadily and is represented here only in a schematic manner. ER, endoplasmic reticulum; MTOC, microtubule-organizing centre; NPC, nuclear pore complex. outer nuclear membrane protein inner nuclear membrane protein http://www.nature.com/nrm/journal/v8/n7/fig_tab/nrm2197_F1.html

Cell locomotion/ movement Cellular level: Ciliar movement Amoeboid mocevent Mesenchymal migration Collective migration Tissue level: muscle movement – skeletal muscle

Cilia - flagellum

Amoeboid movement chemoattractant

Composition of thick filament in a sarcomer

Composition of sarcomer Working unit bordered by two Z-lines/disc Z line – a-actinin + desmin Thin filament – a-helical actin molecules + tropomyosins + troponin Thick filament – myosin II molecules + MBP (myosin binding proteins) Other proteins: titin, distrophin Troponin: Tn-T binds tropomyosin Tn-C binds Ca2+(4 Ca2+/mol = calmodulin) Tn-I inhibitor

Skeletal muscle - Sarcomere

Skeletal muscle - Sarcoplasmic reticulum

X-linked recessive inheritance Duschenne muscular distrophy

Useful videos and linkes: http://csls-text.c.u-tokyo.ac.jp/active/06_01.html http://www.microscopyu.com/moviegallery/livecellimaging/index.html http://www.cellmigration.org/science/