Day 35 Announcements Please remove tests, etc. from your folders. Friday, April 6: Microtubules and microfilaments, pp (quiz material), Monday, April 9: Muscle contraction, cell motility, intermediate filaments, pp Best example of genetic defect related to apoptosis: p53 tumor suppressor.

Outline/Learning Objectives A.Microtubules B.Microtubule-based motility C.Microfilaments After reading the text, attending lecture, and reviewing lecture notes, you should be able to: Describe microtubule structure and assembly/disassembly, including nucleation and dynamic instability. Explain the function of MAPs, kinesin, and dynein. Give examples of microtubule- based motility. Describe microfilament structure and assembly. Analyze experiments involving microtubule or microfilament inhibitors.

Microtubule organization in cells

Dynamic Instability of MTs: GTP cap stabilizes + end of MTs Recent review: Howard and Hyman. (2003). Nature 422:

Microtubule Poisons (and Anti-Cancer Drugs) Colchicine –Alkaloid from meadow saffron –Binds tubulin monomers, prevents assembly  processes that require assembly are blocked –Colcemid, nocadazole, etc. also (-) assembly Taxol –Alkaloid from Pacific yew tree, effective chemo- in ovarian cancer –Can now be synthesized in vitro –Binds microtubules, stabilizes, prevents disassembly  processes that require disassembly are blocked

MAPs regulate MT Function in the Cell Don’t memorize this list

Microtubule Motor Proteins Cytoplasmic dynein moves toward the - end (retrograde transport towards nucleus in neurons). Kinesin moves toward the + end (anterograde transport towards synaptic bulb in neurons). Both are ATPases, which use energy to move organelles and macromolecules along MTs.

Intracellular Trafficking on MTs Microtubules are like intracellular railroad tracks, on which vesicles move. Microtubules also help maintain structure of Golgi (nocadazole reversibly causes collapse of Golgi).

Axonemal Microtubules In cilia and flagella MTOC: basal body microtubules Axonemal dynein Linkage proteins Sliding microtubule model

Sliding MT Model  Bending of Cilium

Clinical Relevance Human males with Kartegener syndrome are sterile because they have non-motile sperm. –Defect is in one or more genes which code for the axonemal structure, including axonemal dynein. Situs inversus in humans: left-right asymmetry of organs is partially or completely reversed. –Defect due to non-functional axonemal dynein, required to establish asymmetric flow of signaling molecules at the gastrula stage.

Summary: Microtubule-based motility Cilia and flagella Organelle transport along microtubules –Plus-end directed –Minus-end directed Chromosome movements in meiotic, mitotic spindles

Videos of Microfilament-based Motility 1.Organelle transport along microfilaments 2.Cytoplasmic streaming in plant cells 3.Cytoplasmic streaming in Chara and Nitella 4.Cell cleavage following mitosis

Monomer of Microfilaments is G-actin bound to ATP 5% of most cell protein, 20% of muscle 42 kD Bound to ATP Polymer is F-actin in two strands (microfilament) Evolved from prokaryotic actin-like proteins.

Microfilament Assembly + end fast growing, - end slow No organizing center as for microtubules ATP on monomer converted to ADP in polymer, becomes less stable (analogous to GTP cap on MTs)

Ways of labeling microfilaments 1.Actin antibodies 2.Phalloidin 3.S1 myosin fragments

Actin-binding proteins regulate actin function in the cell [actin] in cell = 200  M; this spontaneously polymerizes in vitro, why not in the cell? Actin-binding proteins regulate MF structure in cells.

Regulation of actin polymerization and types of membrane protrusions + Rho + Rac + Cdc42 Stress fibers filopodia lamellipodia

Microfilament Poisons MFs generally more stable than MTs Cytochalasins destabilize, cause disassembly Phallotoxins stabilize, prevent disassembly Both are toxins from Amanita mushroom.