Last Class 1. GPCR signaling: 2. Enzyme-linked Receptor signaling: PKA and Calcium 2. Enzyme-linked Receptor signaling: RTK->Ras and PI3K 3. Proteolysis-mediated signaling pathways NF-KB pathway

The Cytoskeleton

Cytoskeletal Filaments Microtubules: intracellular traffic Actin Filaments: cell shape and locomotion Intermediate Filaments: mechanical strength

Cytoskeleton and changes in cell shape

Multi-filament formation of cytoskeletal filaments

Actin Monomers and Filaments

The Time Courses of Actin Polymerization

The treadmilling of an actin filament

Microtubules

Microtubules: Plus end and Minus end Dynamic Instability

Microtubules: Plus end and Minus end Experimental Recording

Microtubules: Dynamic Instability

Microtubules: GDP-bound and bending

Microtubules: GDP-bound and bending

Intermediate Filaments

Intermediate Filaments Coiled coil formation

Intermediate Filaments Difficult to break

Mutation of Keratin (one kind of intermediate filament) gene causes skin blistering

Cytoskeletal Regulation

Microtubules

g-tubulin nucleation for microtubule polymerization

Centrosome and Microtubule Network (movie)

Centrosome and its center-seeking function (movie: MT-red and ER-green)

Microtubules can find its center

Stathmin on Microtubule depolymerization (sequestering)

Katanin on Microtubule depolymerization cleavage

Protein binding and the effects on microtubule stability (movie, MT binding proteins and dynamics)

Organization of Microtubule bundles MAP: microtubule associated protein

Actin Filaments

Actin Filament Regulation occurs at plasma membrane Green: Phalloidin staining of existing actin fibers Red: new actin filaments incorporating rhodamine actin

ARP proteins and actin nucleation

ARP proteins and actin nucleation

Profilin and Thymosin on Actin polymerization

Profilin and Thymosin on Actin polymerization

Cofilin on Actin Depolymerization

Gelsolin severs actin filaments

Platelet Activation

Capping and Stability

Different Actin Arrays in a cell Bundles and gel-like networks

Actin Cross-Linking Proteins Bundles: Fimbrin: filopodia; a-actinin: stress fibers Gel-like network: spectrin and filamin

Different Bundles with different cross-linking Proteins

ERM Linking Actin to Plasma Membrane (ezrin, radixin, moesin)

Actin Filaments and Cellular functions Focal Adhesions

Actin Filaments Regulations

Molecular Motors

Myosins (movie: heart contraction)

Motor Activity of Myosin Heads

Myosin Structural Changes walking along an actin filament

Movie: summarize actomyosin contraction cycle

Skeletal Muscle Cell, myoblasts

Skeletal Muscle Myofibrils

Sliding Acto-Myosin filaments

Protein Structures of Sarcomere Tropomodulin/troponin: minus end Z-disc/a-actinin: plus end

T-tubules and sarcoplasmic reticulum

Calcium/Troponin and skeletal muscle contraction T, I, C subunits of troponin: tropomyosin binding, inhibitory, calcium binding

Regulation of Myosin

Kinesin and dynein bind to microtubules Kinesin and Myosin Kinesin and dynein bind to microtubules

Kinesin: - to +; Dynein: + to – Both on microtubules (movie, MT transportation)

Cell Behaviors and Cytoskeleton

Cell Migration Model

Migratory Keratocytes

Lamellipodial Fragments Blue: Actin Pink: myosin II

ARP protein and Lamellipodia: Actin: Red ARP: Green

Lamellipodial Fragments Actin: Red Cofilin: Green

Traction forces on the substratum

Chemotaxis and Polarization External signals can control cell behavior

Summary 1. Cytoskeleton components: Microtubules Actin Filaments Intermediate Filaments 2. The regulation of microtubules and Actin filaments 3. Molecular Motors 4. Cell behaviors related to cytoskeleton