Lecture 14 Cytoskeleton: components

Cytoskeleton proteins revealed by Commassie staining Cytoskeleton: filament system Internal order Shape and remodel surface Move organelles Movement Cell division Three types of filaments and accessory proteins (assembly of cytoskeleton, motor proteins that move organelles or filaments)

Intermediate filaments: mechanical strength and resistance to shear stress Microtubules: positions of membrane- enclosed organelles, intracellular transport Actin filaments: shape of the cell’s surface and whole cell locomotion Dynamic and adaptable 5-9 nm diameter 25 nm diameter 10 nm diameter

Cytoskeletal filaments are all constructed from smaller protein subunits Intermediate filaments: smaller elongated and fibrous subunits Actin and microtubule filaments: compact and globular subunits All form as helical assemblies of subunits Noncovalent interactions: rapid assembly and disassembly

Multiple protofilaments give strength and adaptability Ends are dynamic

Intermediate filaments are resistent to bending or stretching forces

The time course of actin polymerization in a test tube

GTP GTP! The structure of a microtubule and its subunits 13 parallel protofilaments hollow and cylindrica and polar heterodimer

monomer ATP polar two parallel protofilaments that twist around each other in a right-handed helix The structure of an actin monomer and actin filament Flexible but cross-linked and bundled together by accessory proteins in a living cell

The preferential growth of microtubules at the plus end Plus end: polymerize and depolymerize faster than minus end Actin filaments Plus end- barbed end Minus end- pointed end Microtubules: Plus end-  subunit Minus end-  subunit

The treadmilling of an actin filament D form polymer leans towards disassembly Structural difference between the two ends

Treadmilling behavior of a microtubule as in a living cells The extent of treadmilling inside the cell Is uncertain. Actin treadmilling is observed in vitro. A treadmilling-like phenomenon is seen in living cells for microtubules Tubulin conjugated with fluorescent dye 1/20 subunit is fluorescent “Microtubule lattice”

Dynamic instability:predominant in microtubules GTP hydrolysis “catch up” Treadmilling: predominant in actin filaments

GTP hydrolysis causes filament to curve

Lateral bonds force GDP-containing protofilaments into a linear conformation

Direct observation of the dynamic instability of microtubules in a living cell Dynamic instability of individual actin filaments cannot be observed readily-difference between two ends are not so extreme However the actin filament turn over is rapid: individual filament persists for a only few minutes

The dynamic behavior of filaments allows cells to change structures rapidly and Giant multinuclear cell of a fly early embryo Actin filaments:red Mircotubules:green One division per 10 minutes

Actin and tubulin are highly conserved: they have to bind to many proteins directly and indirectly Accessory proteins and intermediate filament proteins are not as conserved

Intermediate filaments are only found in some metazoans:vertebrates, nematodes,molluscs Not required in every cell type Ancesters: nuclear lamins Parallel Antiparrel “subunit” No polarity! 8 parallel protofilaments Easily bent Hard to break A model of intermediate filament construction

Mechanical properties of actin, tubulin and Intermediate filament polymers viscometer Microtubules: easily deformed and then rupture Actin filaments are more rigid and also rupture easily Intermediate filaments: easily deformed and don’t rupture--maintain cell integrety

Keratin filaments in epithelial cells “desmosomes” The most diverse family 20 in human epithelial cells 10 more in hair and nails Intermediate filaments impart mechanical stability to animal cells Diagnosis of epithelial cancers (carcinomas)

Blistering of the skin caused by mutant keratin genes Epidermolysis bullosa simplex:the skin blisters in response to very slight mechanical stress Other blistering diseases: mouth, esophageal lining and cornea of the eye-- mutations of different keratins Truncated keratin (missing both the N- C- domains) Tg mice

Two types of intermdiate filaments in cells of the nervous system Neurofilaments:axons NF-L, NF-M, NF-H proteins coassemble NF-M and NF-H have long C-terminal tails That bind to neighboring filaments:uniform spacing When axons grow, subunits are added at the filament ends and along the filament length; axon diameter increase 5 fold In ALS (Lou Gehrig’s Disease), there is an accumulation and abnormal assembly of Neurofilaments in motor neuron cell bodies and axon--interfere with normal axon transport Regular spacing axon glia

Actin filaments and microtubules are targets of many plant toxins Amanita Phalloids (death cap) (Eat raw meat)

Effect of the drug taxol on microtubule organization treatment of cancers

Summary 1.Three types of cytoskeletal filaments, protofilaments; 2.Subunits, polymerization, treadmilling, dynamic instability; 3.Intermediate filaments, cell integrity, diseases caused by mutations in the intermediate filament genes 4.Natural toxins and cytoskeleton