Chapter 9 Cytoskeleton

Microtubule assembly and dynamic instability

GTP required for microtubule assembly. Microtubule disassembly is a rapid process that does not require an input of energy (this spontaneous disassembly is referred to as dynamic instability) DI occurs when incorporation of new-GTP bound dimers is slower than the rate of GTP hydrolysis

Cytoplasmic dynein

Huge protein with a globular,force-generating (ATP hydrolyzing) head. Stalks that interact with microtubule. Stems connected to intermediate and light chains that bind necessary accessory proteins to interact with membrane-bounded cargo

3 kinds of molecular motors

Kinesin and Dynein- move along microtubule tracks. Myosin-moves along microfilament tracks

Major functions of cytoskeleton

Scaffold providing structural support and maintaining cell shape. Internal framework to organize and position organelles. Directs cellular locomotion and movement of materials within a cell. Dynamic change in cell shape and principle force of cell motility

Sliding filament model of muscle contraction

Skeletal muscles contract by myosin motors sliding antiparallel actin filaments; oppositely oriented heads of myosin pull toward the opposing plus ends; A bands remain constant in length; H and I bands decrease in width as Z discs are pulled toward sarcomere center. Muscles do not contract until neuronal impulse is received, releasing calcium from sarcoplasmic reticulum stores

WASP proteins

activated by stimulus at cell surface; recruit and activate Arp2/3 protein complex; initiates nucleation and polymerization of actin; diverse actin-binding proteins, such as profilin, are also activated

Z line

anchor proteins that form the boundaries b/n sarcomeres; where actin filament plus ends are bound, minus ends extend toward sarcomere center

Centrosome

animal specific structures that initiate microtubule nucleation and regulate their dynamics. Each contains two barrel shaped centrioles which are surrounded by pericentriolar material (PCM)

Tropomyosin

associated with resting state actin filaments; occupies gap b/n two actin strands and inhibits myosin from biding to actin filaments. In unstimulated state, it blocks the myosin binding sites on actin monomers

treadmilling

at low ATP concentrations, filaments reach a steady state where addition to the plus end equals loss from minus end

Actin filament depolymerizing proteins

bind actin-ADP subunits at the minus end promoting rapid turnover of actin filaments

Axoneme

central microtubule core found in both cilia and flagella; consists of microtubules in a 9+2 arrangement with diverse accessory proteins. They are nucleated by basal bodies (that produce gammaTURK complexes) and plus end extends into membrane surrounded cilia or flagella

basal bodies

centrioles found at the base of eukaryotic cilia and flagella

Neuromuscular junction

contact between nerve and muscle

plectin

cross bridging protein, connects IFs to other components of cytoskeleton, organelles, and nucleus

Microtubule-Associated Proteins (MAPs)

diverse group of proteins that attach to microtubules an function to increase or decrease stability by promoting either polymerization or disassembly

filopodia

dynamic probing extensions whose membrane tips possess receptor proteins that bind chemoattractants and repellants; this stimulation leads to regulatory changes in cytoskeleton and direction of axonal growth

protofilaments

each microtubule contains an arrangement of 13 of these aligned longitudinally along its length. It is assembled from heterodimers of non-identical alpha and beta-tubulin subunits

Cilia and flagella

hair-like motile organelles that project from PM of diverse eukaryotic cells; distinguished by the differences in their beat processes. Cilia tend to occur in large #s on cell surface, while flagellated cells usually contain only one flagellum. Cilia promote motility in many single-celled organisms, but in multicellular animals they function to circulate extracellular fluids

intermediate filaments

heterogeneous group of proteins that assemble as strong, flexible, rope-like fibers that provide mechanical strength; far less dynamic than microtubules and microfilaments, and much less sensitive to chemical agents that disrupt their structure-highly stable. Five major classes, 1-4 are cytoplasmic while type 5 (lamins) are present in the inner lining of nuclear envelope. Assembly of IFs is spontaneous does not require ATP or GTP hydrolysis . Formed by two antiparallel dimers which causes IFs and tetramers to lack polarity-cannot be used as tracks for motor proteins

Axonemal dynein

hydrolyzes ATP, providing the mechanical forces that promote both cilia and flagellar movement

cortex

in non-muscle motile cells, majority of actin is restricted to this thin region just beneath the PM; here dynamic changes in actin polymerization facilitate cell shape changes that promote crawling and phagocytosis; diverse actin-binding proteins affect localized assembly/disassembly of actin filaments for this purpose

Intraflagellar transport

kinesin and dynein transport materials into and out of both growing and mature structures promoting growth and maintenance, as well as cilia mediate cell-cell signaling

lamellipodium

leading edge along the substrate

Membrane-binding proteins

link microfilaments to proteins in the to PM

gamma-tubulin

localized to PCM and is critical for MT nucleation. Forms ring structures in the PCM known as gamma-TURKs (gamma-tubulin ring complexes that are same diameter as a microtubule. Serves as protective cap to the minus end, preventing its disassembly. gamma-TURKs can be assembled on established microtubules in order to promote branching

M line

middle of the sarcomere where tails of antiparallel myosin fibers overlap

myosins

molecular motors of actin filaments; share a characteristic motor head for binding actin and hydrolyzing ATP; tail is divergent, allowing binding to diverse structures/molecules; approx. 40 are encoded by human genome; two groups

processive movement

motor protein moves in a hand-over hand manner along an individual microtubule for a long distance w/o falling off

Axonal transport

movement of neurotransmitters along length of cell, movement away from cell body and toward cell body, establish tracks for a variety of unidirectional motor proteins

Sarcomere

muscle contractile units with a stereotypical arrangement of actin and myosin fibers; each has a distinct banding pattern due to thick filaments (myosin) and thin filaments (actin)

Cytoskeleton

network of filamentous protein structures: microtubules, microfilaments, and intermediate filaments

Unconventional myosins

often have single head and do not assemble into filaments, but dimers; function with microtubule motors to deliver targets to correct locations; similar to kinesin, unconventional myosin involved in vesicle transport moves in hand over hand fashion

traverse (T) tubules

plasma membrane involutions that connect to the SR; action potential in muscles is propagated into cell interior by this

Cross-linked proteins

possess 2 or more actin binding sites and join microfilaments into 3D networks

Conventional type II myosins

principle motor proteins that promote muscle contraction; all migrate toward barbed plus end pf microfilaments; non-muscle activities include cell division, tensile strength of focal adhesions, cell migration, and direction of axonal growth cone movement. All motor activity is confined to head domain. Each composed of 4 regulatory light chains and two heavy chains associated with catalytic heads

Monomer-polymerizing proteins

promote the growth of actin filaments; the polymerizing protein profiling binds to actin-ADP-monomers and facilitates replacement with ATP, enhancing polymerization

titin

protein and is a giant elastic fiber that extends from the Z line to the M band providing elasticity, resting tension and preventing sarcomere form being pulled apart during muscle stretching

+TIPS

regulated expression and targeting of proteins; can stabilize the plus ends of microtubules and inhibit catastrophe either directly or by facilitating the connection of microtubules to various cellular structures

Dynein

retrograde minus end-directed microtubule movement of vesicles and organelles. Responsible for movement of cilia and flagella

rhodamine

small, fluorescent dye that can be covalently linked to proteins

Microtubule-Organizing Centers( MTOCs)

specialized structures for nucleation and assembly of microtubules

minus end

terminates with alpha-subunits and is static-no growth or disassembly; pointed and has lower affinity for monomers

A band

thick filaments; myosin filaments engaging with actin filaments

growth cones

tip of migrating neuronal axons; within these lamellipodia reside probing extensions known as filopodia (microspikes)

Sliding-microtubule mechanism

tubules are fixed in place by radial spokes and the basal body; when dynein hydrolyzes ATP and attempts to "walk" toward the minus ends of associated B tubules, this causes adjacent microtubules to slide past one another. Through precise controlling of when and which dyneins hydrolyze ATP, distinct patterns of ciliary and flagellar movements are coordinated

Two groups of myosin

1.) Conventional (type II) 2.) Unconventional myosins (types 1, 3-18)

motor proteins

ATP-hydrolysis protein machine that bind specific cargo, like vesicles or organelles, via adapter proteins

Troponin

associated with resting state actin filaments; binds and links actin and tropomyosin; following impulse it binds to released Ca2+, causing a conformational change that shifts tropomyosin exposing myosin binding sites

how is microfilament cytoskeleton organized?

by controlling equilibrium between assembly and disassembly of microfilaments

Filament-severing proteins

cleave filaments internally

Myofibril

composes muscle fibers; consists of repeating array or sarcomeres

plus-end

end of microtubule terminates with beta-subunits. This is the dynamic end where growth and disassembly occurs; higher affinity for monomers and incorporates them about 10x as fast as other end

Focal adhesions

formed via localized integrals, providing anchorage support

microtubules

hollow, cylindrical rod-like structures

Keratin IFs

major structural proteins of epithelial cells establishing a protective barrier in such tissues

neurofilaments

major supporting network in neuronal axons

Kinesin

member of a superfamily called Kinesin-related proteins. Each is heterotetramer of two identical heavy and light chains. It is a plus end-directed micro tubular motor (only moves toward plus end of microtubules (away from nucleus))

muscle fibers

multinucleate cells resulting from embryonic fusion of myoblasts;

H zone

myosin tails only (center or sarcomere)

situs inversus

syndorme in which the left-right body symmetry is reversed; often results from mutations in genes encoding ciliary proteins

in vivo

taking place in a live organism

I band

thin filament; actin filaments only

Monomer-sequestering proteins

(negative regulation) bind to actin-ATP monomers and prevent them from polymerizing

End-blocking (capping) proteins

(negative regulation), but also stabilizing. Regulate the length of actin filaments

Nucleating proteins

(positive regulation) microfilament assembly occurs rapidly if filament "seed" is present; these proteins bind monomers and catalyze their polymerization into small filaments through regulation of the Arp2/3 protein complex

Actinomyosin contractile cycle

ATP binds myosin head, conformational change causes detachment of the head from the actin filament. Hydrolysis of ATP changes conformation increasing affinity for actin monomers • Release of Pi further enhances affinity and promotes conforma6onal change on myosin that produces the "power stroke" that moves the thin filament toward the center of the sarcomere. • Release of the ADP allows binding of ATP and repeating the cycle. • In the absence of ATP, myosin remains tightly bound - rigor mortis