Key Points in Constitution of Cytoskeleton Network

1. Polymerization of monomer

1. Regulation of assembly and disassembly

1. Formation of network by associated protein

1. Perform function by motor proteins

Cytoskeleton functions are difficult to assay due to

1. Formation of large protein complex

2. Force generation without chemical change

Components of cytoskeleton

Microtubule is made up of 13 protofilaments

Tubulin dimers constitute protofilament

Nucleation of microtubule is GTP and Mg++- dependent

A threshold concentration of tubulin (critical concentration, Cc)Is indispensibe for microtubule elongation

Flourescence– labeled microtubule demonstrates that microtubue is a dynamic structure

Dynamic instability of microtubule: consequence of delay hydrolysis of GTP after assembly

MTOC (microtubule organization center, centrosome)

Centrosome is the place within a cell where microtubule minus end is protected

Centrosome is located at peri-nuclear region

Centrosome is the center of a cell

Centrosome comprises centriole and pericentriole material

Structure of a centriole

Cilia: derivative of centrioles

-tubulin is the major component of pericentriole material

Microtubule dynamic can be manipulated experimentally

Microtubule associated proteins (MAP) enhance microtubule stability

Microtubules interact with other cytoskeleton via MAPs

Pigment granules move along the microtubule tracks

Dynein and kinesin are microtubule motors

Identification and characterization of microtubule motors

Dynein and kinesin move cargos along opposite direction

Molecular structure of kinesin

A model for moving microtubule cargo through motor proteins

Intracellular organelles distribute along microtubule networks

GolgiER

MT MT

Intermediate filaments occur only in multicellular organisms

Intermediate filaments are the bona fide cellular skeleton

Intermdiate filament proteins are made uIntermdiate filament proteins are made up of helical rod domain and non-helical p of helical rod domain and non-helical

domaindomain

Intermediate filament is a non-polar structure aIntermediate filament is a non-polar structure and its basic structural unit is the staggered tetrand its basic structural unit is the staggered tetramer of two coiled coil dimersmer of two coiled coil dimers

Assembly of Intermediate filaments Assembly of Intermediate filaments (also called 10 nm filaments) is (also called 10 nm filaments) is

dependent on rod domain dependent on rod domain aggregationaggregation

Nuclear lamin disassembly is dependent on phoNuclear lamin disassembly is dependent on phosphorylation of non-helical domainsphorylation of non-helical domain

Intermediate filaments function as intercellular bridgIntermediate filaments function as intercellular bridge to link neighboring cells via desmosomese to link neighboring cells via desmosomes

Keratins are divided into 2 groups based on their pI

Expression of keratin pairs are tissue Expression of keratin pairs are tissue specificspecific

Keratins are more amenable to deforming force Keratins are more amenable to deforming force than other cytoskeletal proteinsthan other cytoskeletal proteins

Keratins are the first cytoskeletal protein founKeratins are the first cytoskeletal protein found to be related to human diseasesd to be related to human diseases

Numerous hot spots are found in Numerous hot spots are found in human keratin geneshuman keratin genes

Keratin mutation affects its assembly and mechanical property

Methods to generate transgenic Methods to generate transgenic micemice

Transgenic mice overexpressing k14 mutaTransgenic mice overexpressing k14 mutant display Epidermolysis bullosa Simplex nt display Epidermolysis bullosa Simplex

(EBS) phenotype(EBS) phenotype

Ultrastructure of epidermal cells displaying EBS

Actins occupy up to 5% total cellular protein

F actin is made up of G (globular) actinsF actin is made up of G (globular) actins

Polymerization of actin is ATP and KPolymerization of actin is ATP and K++, M, Mgg2+2+- dependent- dependent

Amount of actin within a cell is kept dynaAmount of actin within a cell is kept dynamically equilibriummically equilibrium

S1 myosin fragment can be used to deteS1 myosin fragment can be used to detect actin threadmillingct actin threadmilling

Barbed end (plus end) of actin contains newly Barbed end (plus end) of actin contains newly decorated actindecorated actin

Profilin and thymosin compete with each Profilin and thymosin compete with each other for actin monomer bindingother for actin monomer binding

Cytochalasin inhibits growth of actin fiberCytochalasin inhibits growth of actin fiber

Cellular structures made up of actin fibersCellular structures made up of actin fibers

Cell cortex contains actin fibers arranged in Cell cortex contains actin fibers arranged in different array and densitydifferent array and density

Stress fibers are not culture Stress fibers are not culture artifactartifact

Actin binding proteinsActin binding proteins

-actinin and fimbrin are bundling proteins

Filamin is an actin-binding gel forming protein

Myosins are actin motor proteinsMyosins are actin motor proteins

Figure 16–51 Myosin II.

(A) A myosin II molecule is composed of two heavy chains (each about 2000 amino acids long (green) and four light chains (blue). The light chains are of two distinct types, and one copy of each type is present on each myosin head. Dimerization occurs when the two a helices of the heavy chains wrap around each other to form a coiled-coil, driven by the association of regularly spaced hydrophobic amino acids (see Figure 3–11). The coiled-coil arrangement makes an extended rod in solution, and this part of the molecule is called the tail. (B) The two globular heads and the tail can be clearly seen in electron micrographs of myosin molecules shadowed with platinum. (B, courtesy of David Shotton.)

Mysoin derivatives

Actin moves along myosinActin moves along myosin

Myosin mutant mice displays defective Myosin mutant mice displays defective heartheart

Figure 16–75 Effect on the heart of a subtle mutation in cardiac myosin.

Left, normal heart from a 6-day old mouse pup. Right, heart from a pup with a point mutation in both copies of its cardiac myosin gene, changing Arg 403 to Gln. Both atria are greatly enlarged (hypertrophic), and the mice die within a few weeks of birth. (From D. Fatkin et al., J. Clin. Invest. 103:147, 1999.)

Summary of actin binding proteins

Figure 16–50 The dramatic effects of Rac, Rho, and Cdc42 on actin organization in fibroblasts.

In each case, the actin filaments have been labeled with fluorescent phalloidin, and focal contacts have been located with an antibody against vinculin. (A) Serum-starved fibroblasts have actin filaments primarily in the cortex, and relatively few focal contacts. (B) Microinjection of a constitutively activated form of Rho causes the rapid assembly of many prominent stress fibers and focal contacts. (C) Microinjection of a constitutively activated form of Rac, a closely related monomeric GTPase, causes the formation of an enormous lamellipodium that extends from the entire circumference of the cell. (D) Microinjection of a constitutively activated form of Cdc42, another Rho family member, causes the protrusion of many long filopodia at the cell periphery that form adhesive contacts with the substratum. The distinct global effects of these three GTPases on the organization of the actin cytoskeleton are mediated by the actions of dozens of other protein molecules that are regulated by the GTPases. Many of these target proteins resemble the various actin-associated proteins that we have discussed in this chapter. (From A. Hall, Science 279:509–514, 1998. © AAAS.)

Figure 16–90 A model for protrusion of the actin meshwork at the leading edge.

Two time points during advance of the lamellipodium are illustrated, with newly assembled structures at the later time point shown in a lighter color. Nucleation is mediated by the ARP complex at the front. Newly nucleated actin filaments are attached to the sides of preexisting filaments, primarily at a 70° angle. Filaments elongate, pushing the plasma membrane forward because of some sort of anchorage of the array behind. At a steady rate, actin filament plus ends become capped. After newly polymerized actin subunits hydrolyze their bound ATP in the filament lattice, the filaments become susceptible to depolymerization by cofilin. This cycle causes a spatial separation between net filament assembly at the front and net filament disassembly at the rear, so that the actin filament network as a whole can move forward, even though the individual filaments within it remain stationary with respect to the substratum.