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
Microtubules network
Starting from the MT center near nucleus
Filament network (fluorescence)
From the nucleus to the plasma membrane
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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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1. Microfilaments and Actin Structures
Actin monomers assemble into long helical polymers with polarity
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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
Forms bundles
Filamin forms networks
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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 cell
Movement of fish epidermal cell
Cell locomotion mechanism:
Includes actin polymerization and branchinggenerated 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
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
Keratin and lamin IF
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
Bacterial Flagella (E. coli, Salmonella)
Rotation of Flagellar -> Motion
Sperm
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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
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
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
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