1 Cytoskeleton A cytoplasmic system of fibers -> critical to cell motility (movement) Macrophage cytoskeleton Cytoskeleton of a lung cell in mitosis

2 The cytosol: w% of cytosol are proteins -> ¼ - ½ of total protein is in cytosol Protein conc mg/ml -> complexes of protein It is believed that cytosol is highly organized -> Most soluble proteins are - bound to filaments - localized in specific regions

3 Cytoskeleton of an epithelial cell and a migrating cell

4 Eukaryotes Bacteria Actin filaments (AF) Intermediate filaments (IF) microtubule (MT)

5 Cytoskeleton is made out of 3 different types of filaments

6 Filaments differ in Size, Shape and Flexibility

7 Filament network in the cell Filament network (fluorescence) From the nucleus to the plasma membrane Microtubules network Starting from the MT center near nucleus

8 Cytoskeleton supporting the plasma membrane in human red blood cells

9 Cell Signaling Regulates Cytoskeleton Function

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11 1. Microfilaments and Actin Structures Actin cytoskelet in a moving cell

12 Actin monomers assemble into long helical polymers with polarity 1. Microfilaments and Actin Structures

13 Actin Filament Assembly

14 Actin Filaments grow faster at (+) end than at (-) end The rate of addition of ATP-G-actin is much faster at the (+) end than at the (-) end (rate of dissociation is similar) -> lower critical concentration (Cc) at (+) end in steady state -> filament grows preferentially at the (+) end If actin conc. is between Cc - and Cc + (steady state) -> actin subunits flow through filaments by attaching to (+) end and dissociating from (-) end Treadmilling phenomen -> involved in movement of cells

15 Capping Proteins Block Assembly and Disassembly at Actin Filament Ends The presence of these 2 proteins at opposite ends prevent actin from dissociating during muscle contraction

16 Actin Filament Branching Nucleation of branching mediated by Arp2/3

17 Movement of invaders inside the cell Most infections are spread by lysed cells. Some Bacteria (Listeria monocytogenes) or viruses (vaccinia – related to smallbox virus) escape from cell on the end of a polymerizing actin filament. These organisms or viruses move through the cytosol at rates of 11μm/min. Actin generates the force necessary for movement

18 Proteins that organize Microfilaments into networks Filamin forms networks Forms bundles

19 Filaments attached to Membranes Microvilli on an epithelial cell showing polarity of actin filaments

20 Myosins - Cellular Motor Proteins Tail: -> Locatized to cellular membranes -> vesicle attached (cargo) Form thick filaments in muscles S1 motor domain Head -> Motor domain (S1) -> ATP depentend

21 Myosin heads walk along actin filaments -> towards (+) end Sliding-filament assay: Myosin tail absorbed onto glass surface -> a solution of actin filaments allowed to flow through In presence of ATP myosin heads walk towards (+) end of actin filaments -> sliding of filaments -> Movement of labeled actin filaments

22 Myosins – Motor proteins responsible for cell movement These are the most important 3 myosins (out of ca. 40 we have in humans) Loss of more specialized ones -> causes deafness/blindness

23 Myosin motion along actin Length of the neck domain -> determines rate of movement Step size -> Moves in 72 nm steps

24 Actin fibers in the muscle

25 Skeletal muscle contraction is regulated by Calcium and actin binding proteins Tropomyosin (TM) Troponin (TN)

26 Cell Locomotion Coordination of motions generated by different parts of the cell Movement of fish epidermal cell Cell locomotion mechanism: Includes actin polymerization and branching- generated movement at the edge, assembly of adhesion structures, and contractions mediated by myosin II

27 Myosin V Carries Many Cargoes Myosin V: -> carries secretory vesicles, organelles,... -> Used to prepare nucleus for mitosis -> used to segregate organelles

28 2. Intermediate Filaments Keratin and lamin IF IF differ in stability, size and structure from other cytoskeleton fibers: - IF are extremely stable (hair, nails, wool) -10 nm diameter - α-helical rods -> assemble into ropelike filaments - assemble from different IF proteins - assembly through several intermediate structures Intermediate structures in the assembly of IF

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30 Cross-links between Microtubules and Intermediate Filaments in Fibrioblast cells Microtubules (red), Intermediate Filaments (blue), connection between fibers (green)

31 3. Microtubules Kinesin-powered movement of a vesicle along a microtubule Microtubules are involved in cell movement: - Beating of cilia and flagella - transport of vesicles in the cytoplasm Microtubules organizing center (MTOC)

32 Microtubules organization 2 type of MT in cells: - Stable and long-lived (found in non-replication cells) -> in cilia, flagella, neurons - unstable and short-lived (found in mitosis) -> spindle-shaped apparatus that partitions chromosomes equally to daughter cells

33 Microtubules Arrangement Flagella

34 Microtubules assemble from Microtubule Organizing Centers (MTOCs) MTOCs in non-mitotic cells -> centrosomes

35 Microtubules Assembly preferably at (+) end Nucleation of microtubule assembly -> Treadmilling

36 Microtubules Assembly/Disassembly Colchicine and Taxol: Drugs that interfere with Microtubules Assembly/Disassambly Colchicine: 2500 years ago Egyptians treated heart problems Nowadys: treatment of gout, skin and joint diseases Taxol: (stabilizes Microtubules) Anticancer agents -> treatment of ovarian cancer

37 Microtubules Dynamic Instability Presence of GTP-β-tubulin cap determines stability

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39 Kinesin and Dynein – two Families of Motor Proteins Responsible for Transport along Microtubules Microtubules based vesicle transport

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41 Kinesin-catalysed Vesicle Transport Carries cargo

42 Kinesin-1 uses ATP to walk down a microtubule to the (+) end

43 Dynein-catalysed Vesicle Transport Moves towards the (-) end of microtubules

44 Microtubule motors in a Cell Kinesins -> transport to cell periphery (+) Dyneins -> transport to cell center (-)

45 Cooperation of Myosin and Kinesin at the cell cortex Secretory vesicles are handed over from Kinesin to Myosin -> last part of secretory pathway

46 Rotory Motors – Cilia and Flagellar Sperm Bacterial Flagella (E. coli, Salmonella) Rotation of Flagellar -> Motion

47 The Flagellar Motor

48 Bacterial Flagellum is made out of subunits

49 Motion of E. coli The points show the locations of the bacterium at 80 ms intervals. Changing of direction: Tumbling is caused by an abrupt reversal of the fagellar motor A second reversal restores smooth swimming -> almost always in a different direction

50 Reversal of the direction of Flagellar Rotation is obtained by Proton Transport through Motor Proton flow drives flagella rotation !!!

51 Chemotaxis Signaling Pathway Direction of bacterial movement depends on chemical substances: -> Bacteria swim towards high concentrations of glucose - chemoattractans -> Bacteria swim away from harmful substances, such as phenol - chemorepellants Receptor in plasma membrane initiate signal pathway -> Phosphorylation of CheY protein -> P-CheY binds to flagellar motor -> clockwise rotation favored Attractant binds to receptor -> pathway blocked -> smooth swimming Repellant binds to receptor -> pathway stimulated -> more frequent clockwise rotation -> tumbling