Cytoskeleton

cytoskeleton

a network of fibers that holds the cell together, helps the cell to keep its shape, and aids in movement

-specific to eukaryotes

cytoskeleton filaments

actin, microtubules, intermediate filaments

actin

thin cytoskeletal filaments

-double helix, associated with integrin, ATP-dependent, cortex

actin cortex

dense region of actin underlying the plasma membrane

-outside of cells, microvilli

actin and integrin

integrin cannot function without actin

actin conformation examples

dorm rigid gels, networks, and linear bundles

intermediate filaments

a component of the cytoskeleton that includes filaments intermediate in size between microtubules and actin

-great tensile strength; keratin filaments

microtubules

spiral strands of cytoskeletal protein molecules that form a tubelike structure

-GTP-dependent

deformation vs. deforming force of cytoskeletal filaments

comparing applied stretching force with the ability of filaments to withstand the force

Intermediate filaments>>>>>>>>>>actin>microtubules

nuclear connection among cytoskeletal filaments

can connect with laminin filaments to keep nucleus stationary

G-actin

a globular subunit of F actin with an active site for binding S1 myosin head

G-actin requirements

ATP and Mg2+ to add to F-actin

derivation of G-actin

derived from ancestral genes in prokaryotes, yeasts, and other cells that do not have G-actin

F-actin

A fibrous protein made of a long chain of G actin molecules twisted into a helix; main protein of the thin myofilament

S1 myosin

coats actin (part of G-actin); acts as a nucleating site for actin polymerization

F-actin decoration

decorated with proteins that bind it to S1 myosin head --> pointed end (minus), barbed end (plus)

G-actin experiment with ATP

G-actin + ATP + Mg2+ --> nucleation --> elongation --> steady state

G-actin nucleation

two G-actins form initial F-actin filament

-formin

steady state of actin

number of G-actin of F-actin remains stable, with dynamic addition/subtraction at plus/minus ends still occuring

treadmilling

the process by which an actin filament is maintained at constant length by addition of protein subunits at one end and loss of subunits at the other

critical concentration of actin

concentration of which the rate of actin monomers added to the polymer are the same as the rate of monomers removed

-no net growth

critical concentration of ends of actin

plus end: 0.12 uM

minus end: 0.60 uM

-net addition is greater at plus end

alpha, beta, gamma actin

alpha: contractile structures

beta: cell movement (at leading edge of cells)

gamma: stress fibers (integrin-associated)

examples of actin functions

microvilli, cell cortex, adherens belt, stress fibers, phagocytosis, contractile rings, moving endocytic vesicles

Thymosin beta-4

actin sequestering protein

-regulates availability of G-actin

profilin

binds actin subunits and speeds elongation

-ADP/ATP exchange protein

cofilin

Breaks F-actin into smaller segments by removing G-actin units from minus end

actin formation cycle (starting with cofilin)

cofilin binds to ADP-actin on minus end --> dissociation --> cofilin goes back to minus end; profilin binds to ADP-actin --> phosphorylation of ADP actin to ATP-actin --> Thymosin-beta-4 cycle --> ATP-actin addition to plus end; profilin recycled

capping protein

prevents assembly and disassembly at plus end (barbed end) of actin

-capZ, tropomodulin

CapZ

protein that caps the plus end of actin filaments

tropomodulin

capping protein

-prevents assembly and disassembly at minus end of actin

Arp2/3 complex

caps minus end of actin

-initiates site binding, nucleation

formin

dimer nucleating protein that brings two G-actins together

actin drugs

Cytochalasin: fungal-derived product that inhibits polymerization at plus end of F-actin (more active end)

Phalloidin: promotes polymerization of F-actin

-angel of death mushroom

useful dye for F-actin

phalloidin

molecular tweezer microscope

can be used to measure interaction of F-actin with myosin

-uses infrared light: actin molecule bound to bead, turn down IR to measure activity

Listeria monocytogenes

bacteria causing very serious food poisoning and death and can pass into placental barrier

-builds actin comet tail

Listeria monocytogenes pathway

nonmotile bacteria enters cell via endocytosis --> triggers formation of F-actin comet tail on bacteria (ActA, Arp2/3) --> exocytosis of bacteria, now motile

ActA

F-actin/G-actin-facilitating protein

-triggered by Listeria monocytogenes to build F-actin comet tail

analyzing Listeria monocytogenes

rhodamine-labeled transferrin and fluorescent-labeled actin

opsonization

coating antigen with antibody enhances phagocytosis

-phagocytosis of bacteria, F-actin involvement

diaphanous gene

codes for actin-supported hair cells in ear

-tectorial membrane in ear vibrates, hair bundles recognize movement and send signal to brain

defect in diaphanous gene

defect in microfilament supporting hair cell prohibits recognition of tectorial membrane vibration

intermediate filaments characteristics

tough (rope-like), 70 different genes, relatively less dynamic than F-actin/microtubules, tetramer main part of structure (toughness)

intermediate filaments structure

fibrous proteins supercoiled into thicker tetramer

classes of intermediate filaments

Classes I and II: keratins (epithelial cells)

-tissue strength and integrity

Class IV: neurofilaments (neurons)

-axon organization

Class V: lamins (nucleus)

-nuclear structure and organization

Epidermolysis bullosa

genetic skin disease characterized by blisters caused by any mechanical friction, resulting in large open sores and extreme skin fragility

-keratin defect (intermediate filament)

Ortec International

created substitute skin for EB treatment

-Dr. Mark Eisenberg and his son

microtubules structure

star-like array, must have 13 protofilaments

-alpha and beta-tubulin dimers; single, doublet, and triplet; can undergo treadmilling

building blocks of microtubules

tubulin heterodimers

-alpha: non-exchangeable GTP; on minus end

-beta: exchangeable GTP/GDP; on plus end

rescue/catastrophe

periods of persistent microtubule growth (rescue) interrupted by occasional switching to rapid shrinkage (catastrophe)

fish pigment cells

thin structure allows for adaptation to color background of environment

-useful in microtubule experiments

vesicle and organelle movement by microtubules experiment

fish pigment cells

dark adapted cell --> decrease cAMP concentration and add microtubular inhibitor --> cells still dark-adapted

fish pigment cells and melanosomes

high cAMP concentration: melanosomes situated across microtubules (dark imaging)

low cAMP concentration: melanosomes resort towards center of cell using microtubules --> cell looks lighter

microtuble-associated proteins (MAPs)

very tightly-bound proteins on microtubules that are functionally important

-XMAP215/CLASP, Kinesin-13, Stathmin, Katamin

XMAP215 and CLASP

decreases number of catastrophes

-stabilizes plus ends and accelerates assembly

Kinesin-13 and Stathmin

increases number of catastrophes

-accelerates disassembly

Katanin

severing protein that cuts longer microtubules during neuronal development

-embryos

dynein and kinesin

motor proteins that control organelle and vesicle movement along microtubules

molecular motors in axon

Kinesin 1,2: anterograde travel (towards synaptic membrane)

Dynein: retrograde travel (away from synaptic membrane)

microtubule drugs

colchicine; taxol and taxotere

colchicine

depolymerizes microtubules

-first used to treat gout (disrupts microtubules transferring WBCs)

-derivatives

Taxol (paclitaxel) & Taxotere (docetaxel)

Microtubular inhibitors

-disrupts mitotic spindle in cell division --> apoptosis

-blocks Bcl-2