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Cytoskeleton

A cytoplasmic system of fibers -> critical to

cell motility (movement)

Macrophage cytoskeleton

Cytoskeleton of a lung cell in mitosis

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The cytosol:

20-30 w% of cytosol are proteins -> ¼ - ½ of total protein is in cytosol

Protein conc. 200-400 mg/ml -> complexes of protein

It is believed that cytosol is highly organized

-> Most soluble proteins are

- bound to filaments

- localized in specific regions

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Cytoskeleton of an epithelial cell and a migrating cell

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Eukaryotes

Bacteria

Actin filaments (AF) Intermediate filaments (IF) microtubule (MT)

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Cytoskeleton is made out of 3 different types of filaments

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Filaments differ in Size, Shape and Flexibility

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Filament network in the cell

Filament network (fluorescence)

From the nucleus to the plasma membrane

Microtubules network

Starting from the MT center near nucleus

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Cytoskeleton supporting the plasma membrane in human red blood cells

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Cell Signaling Regulates Cytoskeleton Function

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1. Microfilaments and Actin Structures

Actin cytoskelet in a moving cell

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Actin monomers assemble into long helical polymers with polarity

1. Microfilaments and Actin Structures

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Actin Filament Assembly

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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

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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

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Actin Filament Branching

Nucleation of branching mediated by Arp2/3

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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

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Proteins that organize Microfilaments into networks

Filamin forms networks

Forms

bundles

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Filaments attached to Membranes

Microvilli on an epithelial cell showing polarity of actin filaments

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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

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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

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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

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Myosin motion along actin

Length of the neck domain -> determines rate of movement

Step size

-> Moves in 72 nm steps

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Actin fibers in the muscle

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Skeletal muscle contraction is regulated by Calcium and actin binding proteins

Tropomyosin (TM)

Troponin (TN)

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Cell Locomotion

Coordination of motions generated by different parts of the cellMovement 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

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Myosin V Carries Many Cargoes

Myosin V: -> carries secretory vesicles, organelles,...

-> Used to prepare nucleus for mitosis

-> used to segregate organelles

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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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Cross-links between Microtubules and Intermediate Filaments in Fibrioblast cells

Microtubules (red), Intermediate Filaments (blue), connection between fibers (green)

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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)

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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

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Microtubules Arrangement

Flagella

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Microtubules assemble from Microtubule Organizing Centers (MTOCs)

MTOCs in non-mitotic cells -> centrosomes

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Microtubules Assembly preferably at (+) end

Nucleation of microtubule assembly

-> Treadmilling

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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

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Microtubules Dynamic Instability

Presence of GTP-β-tubulin cap determines stability

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Kinesin and Dynein – two Families of Motor Proteins

Responsible for Transport along Microtubules

Microtubules based vesicle transport

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Kinesin-catalysed Vesicle Transport

Carries cargo

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

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Dynein-catalysed Vesicle Transport

Moves towards the (-) end of microtubules

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Microtubule motors in a Cell

Kinesins -> transport to cell periphery (+)Dyneins -> transport to cell center (-)

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Cooperation of Myosin and Kinesin at the cell cortex

Secretory vesicles are handed over from Kinesin to Myosin -> last part of secretory pathway

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Rotory Motors – Cilia and Flagellar

Sperm

Bacterial Flagella (E. coli, Salmonella)

Rotation of Flagellar -> Motion

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The Flagellar Motor

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Bacterial Flagellum is made out of subunits

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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

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Reversal of the direction of Flagellar Rotation is obtained by Proton Transport through Motor

Proton flow drives flagella rotation !!!

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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