Transcript In cells
Cytoskeleton and locomotion
Láng, Orsolya MD, PhD Dept. Genetics, Cell & Immunobiology, Semmelweis University
Lecture EPh 2014 www.dgci.sote.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
Dynamic Subcellular level:
anchors organelles organization of organelles provides tensile strength
Adaptable Stable Strong
movement of chromosomes organizing cell polarity Intracellular movement of vesicles Endocytosis – clathrin-mediated endocytosis and phagocytosis
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 F-actin
( Garland Science
Molecular Biology of the Cell
2008)
Depolymerization Polymerazition of actin ADP ATP ADP ATP
P
i 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)
Tropomodulin
-
Regulatory proteins +
g
CAP39 Severin Gelsolin Villin CapZ 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
Actin – membrane links
membrane
Myozin I.
Arp2/3 F-Actin Profilin - G-aktin Filamin Integrin
Model of actin nucleation WASP = Wiscott-Aldrich syndr. prot.
Profilin-mechanism
T
b
4 = thymozin
b
4
(Lodish, H. et al. Mol. Cell Biol. 2000, 767)
Proline-rich protein
Filamin – Membrane link
filamin actin
Structure of focal contact
actin filament a actinin
+
vinculin paxillin talin integrin fibronectin
A plasma membrane – cortex links
Thrombocyte:
filamin Glycophorin Ankyrin Spectrin tetramer
Muscle:
dystrophin
Epithel:
ezrin
(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
assoc.
polypeptides motor domain motor domain „stalk”
Kinesin
assoc.
polypeptides „stalk” assoc.
polypeptides
Myozin Dynein
-
Motor proteins
dynein heavy chain light chain + kinesin +
?
-
cAMP
dynein kinesin
cAMP
+ pigment cells
ADP ATP ADP
Kinesin
ATP ATP ADP-Pi ADP ADP
Microtubular systems in the cells
-Centrosome
Interphase cell
-Cilia / flagellum
centrosome Cilla Basal body Dividing cell spindle
-Mitotic system - Vesicular transport
Neuron centrosome axon
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
Paramecium
flagellum
Structure of cilia
tubulin (13 ill. 11 protofilaments)
B A
dynein-arms nexin
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 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 force actin filament
Role of intermedier filaments
Buffer against external mechanical stress
Tissue specificity !!!
Epithel – keratin Connective tissue Muscles Neuroglia }
vimentin-like
Neurones(axon) - neurofilaments Exception: Nucleus – lamines (A,B,C) →(lamina fibrosa)
Vimentin Desmin Glial protein
Domain structures of intermedier filamentums
H 2 N-
Head
a
helical domain Tail
COOH keratins vimentin neurofilam. prot.
nuclear prot.
Axon of a neuron
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)
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 Ca 2+ (4 Ca 2+ /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/