1. CHAPTER 9 The Cytoskeleton and Cell Motility

2. Introduction The cytoskeleton is a network of filamentous structures: microtubulues, microfilaments, and intermediate filaments.

3. Properties of cytoskeletal components

4. 9.1 Overview of the Major Functions of the Cytoskeleton (1) The cytoskeleton has many roles: –Serves as a scaffold providing structural support and maintaining cell shape. –Serves as an internal framework to organize organelles within the cell. –Directs cellular locomotion and the movement of materials within the cell.

5. Structure and functions of the cytoskeleton

6. Overview of the Major Functions of the Cytoskeleton (2) Provides anchoring site for mRNA. Serves as a signal transducer. An essential component of the cell’s division machinery.

7. 9.2 Study of the Cytoskeleton (1) The Use of Live-Cell Fluorescence Imaging –Can be used to locate fluorescently-labeled target proteins. –Molecular processes can be observed (live- cell imaging). –Used to reveal the location of a protein present in very low concentrations.

8. Applications using fluorescence imaging

9. Study of the Cytoskeleton (2) The Use of In Vitro Single-Molecule Assays –They make possible to detect the activity of an individual protein molecule in real time. –Can be supplement with atomic force microscopy to measure the mechanical properties of cytoskeletal elements.

10. Using video microscopy to follow activities of molecular motors

11. 9.3 Microtubules (1) Structure and Composition –Microtubules are hollow, cylindrical structures. –The microtubule is a set of globular proteins arranged in longitudinal rows called protofilaments. –Microtubules contain 13 protofilaments. –Each protofilament is assembled from dimers of α- and ß-tubulin subunits assembled into tubules with plus and minus ends.

12. The structure of microtubules

13. Microtubules (2) Microtubule-Associated Proteins (MAPs) –MAPs comprise a heterogeneous group of proteins. –MAPs attach to the surface of microtubules to increase their stability and promote their assembly. –MAPs are regulated by phosphorylation of specific amino acid residues.

14. MAPs

15. Microtubules (2) Microtubules as Structural Supports and Organizers –The distribution of microtubules determines the shape of the cell. –Microtubules maintain the internal organization of cells.

16. Microtubules (3) Microtubules as Structural Supports and Organizers –Microtubules function in axonal transport. –Microtubules play a role in axonal growth during embryogenesis.

17. Microtubules (4) Microtubules as Structural Supports and Organizers –In plant cells, microtubules help maintain cell shape by influencing formation of the cell wall.

18. Microtubules (5) Microtubules as Agents of Intracellular Motility –They facilitate movement of vesicles between compartments. –Axonal transport: Movement of neurotransmitters across the cell. Movement away from the cell body (anterograde) and toward the cell body (retrograde). Mediate tracks for a variety of motor proteins.

19. Axonal transport

21. Visualizing axonal transport

22. Microtubules (6) Motor Proteins that Traverse the Microtubular Cytoskeleton –Molecular motors convert energy from ATP into mechanical energy. –Molecular motors move unidirectionally along their cytoskeletal track in a stepwise manner. –Three categories of molecular motors: Kinesin and dynein move along microtubule tracks. Myosin moves along microfilament tracks.

23. Microtubules (7) Kinesins –Kinesin—member of a superfamily called KLPs (kinesin-like proteins). –A kinesin is a tetramer of two identical heavy chains and two identical light chains. –Each kinesin includes a pair of globular heads (motor domain), connected to a rod-like stalk. –Kinesin is a plus end-directed microtubular motor based on its movement.

24. Kinesin

25. Microtubules (8) Kinesins (continued) –They move along a single protofilament of a microtubule at a velocity proportional to the ATP concentration. –Movement is processive, motor protein moves along an individual microtubule for a long distance without falling off. –KLPs move cargo toward the cell’s plasma membrane.

26. Kinesin-mediated organelle transport

27. Microtubules (9) Cytoplasmic Dynein –Dynein – responsible for the movement of cilia and flagella. –Cytoplasmic dynein – Huge protein with a globular, force-generating head. –It is a minus end-directed microtubular motor. –Requires an adaptor (dynactin) to interact with membrane-bounded cargo.

28. Cytoplasmic dynein

30. Microtubules (10) Microtubule-Organizing Centers (MTOCs) –MTOCs – specialized structures for the nucleation of microtubules. –Centrosome – structures responsible for initiating microtubules in animal cells. It contains two barrel-shaped centrioles surrounded by pericentriolar material (PCM). Centrioles are usually found in pairs.

31. The centrosome

33. Microtubules (11) Centrosomes (continued) –Responsible for initiation and organization of the microtubular cystoskeleton. –Microtubules terminate in the PCM.

34. Microtubules (11)

35. Microtubules (12) Basal Bodies and Other MTOCs –Basal body – structure where outer microtubules in a cilia and flagella are generated. –Plant cells lack MTOCs and their microtubules are organized around the surface of the nucleus.

36. Microtubules (13) Microtubule Nucleation –MTOCs control the number of microtubules, their polarity, the number of protofilaments, and the time and location of their assembly. –The protein  -tubulin is found in all MTOCs and is critical for microtubule nucleation.

37. The role of  -tubulin in centrosome function

38. Microtubules (14) The Dynamic Properties of Microtubules There are four distinct arrays of microtubules in a dividing plant cell: –Widely distributed throughout the cortex. –Making a single transverse band. –In the form of a mitotic spindle. –As a phargmoplast assisting in the formation of the cell wall of daughter cells.

39. Four arrays of microtubules in a plant cell

40. Microtubules (15) The Dynamic Properties of Microtubules –Newly formed microtubules branch at an angle of pre-existing microtubules. –The changes in spatial organization of microtubules are a combination of two mechanisms: Rearrangement of existing microtubules. Disassembly of existing microtubules and reassembly of new one in different locations.

41. Nucleation of plant microtubules

43. Microtubules (16) The Underlying Basis of Microtubule Dynamics –Insight into factors that influence microtubule assembly and disassembly came from studies in vitro. –GTP is required for microtubule assembly. –Hydrolysis of GTP leads to a replacement of bound GDP by new GTP to “recharge” the tubulin dimer.

44. Microtubule assembly in vitro

45. Structural cap model of dynamic instability