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Molecular Cell Biology Microtubules and their Motors Cooper.

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Presentation on theme: "Molecular Cell Biology Microtubules and their Motors Cooper."— Presentation transcript:

1 Molecular Cell Biology Microtubules and their Motors Cooper

2 Microtubules and their Motors n Intro n Vesicle Trafficking n Cilia n Mitosis

3 Microtubule Structure n Cross-section Hollow tube 24 nm wide 13-15 protofilaments n Helical structure n Polar Plus ends generally distal Minus ends generally proximal (at MTOC) Composed of Tubulin  Heterodimer

4 Microtubule Structure & Assembly

5 Microtubule Motors n Definition Microtubule-stimulated ATPase Motility along MT’s Sequence of known motor n Dynein Moves to Minus End of Mt Large, multi-subunit protein n Kinesin Moves to Plus End of Mt Exception - Ncd/Kar3

6 Discovery of Kinesin n Search for Motor for Axonal Transport Development of Video-enhanced DIC Imaging n Movement Requires ATP n AMPPNP Freezes Particles n Microtubule Affinity Chromatography Bind in AMPPNP, Release in ATP

7 Kinesin Structure

8 Kinesin Movement and Processivity

9 Kinesin Superfamily Structures

10 Kinesin Superfamily Phylogenetic Tree

11 Cytoplasmic Dynein n Discovered Biochemically n Minus End Motor for Vesicle Transport n Requires Dynactin Complex for Function n Moves the Mitotic Spindle

12 Dynein and Kinesin Motor Domain Structures

13 Dynein Motor Subunit Architecture

14 Model for Interactions between Dynein, Dynactin Complex, Microtubules, and Cargo

15 Membrane Trafficking - ER and Golgi n Positioning ER & Golgi Golgi near MTOC –Minus Ends are at MTOC –Golgi Position Requires Dynein ER –Tubular network spread about the cell –Kinesin moves the tubules peripherally

16 Microtubules (Red) and ER (Green)

17 Vesicle Traffic: Trans-Golgi to Plasma Membrane n Kinesin - “KIF13A” Discovered by sequencing Plus-end Directed, fast (0.3 µm/s) Binds AP-1 (affinity chromatography) and mannose 6-P receptor Inhibit function (express tail as dominant negative) -> less M6PR at cell surface

18 Xenopus Melanophore Pigment Granule Movement n Vesicle Move Along Microtubules n Vesicles Carry Dynein, Kinesin & Myosin-V n Regulation of the motors accounts for the dispersion / aggregation Inward Motion (Movie Loops)

19 Xenopus Melanophore Pigment Granule Movement Outward Motion (Movie Loops) n Vesicle Move Along Microtubules n Vesicles Carry Dynein, Kinesin & Myosin-V n Regulation of the motors accounts for the dispersion / aggregation

20 Cilia in Action

21 Chlamydomonas Cilia Sperm Flagellum

22 Cilia on Surface of Epithelial Cells

23 Structure of Axoneme: Cross-section

24 Axonemes are Anchored at their Base in Basal Bodies

25 Conversion of Sliding to Bending to Wave Formation n Slide on only side of axoneme n Propagate down the long axis

26 Rotation of Central Pair Whole Chlamydomonas Cell w/ Two Flagella Axonemes Isolated from Chlamydomonas Dark-Field Microscopy

27 Experimental Approaches to Study Cilia in Chlamydomonas n Axoneme 2-D gel - 250 polypeptides! n Mutants - Collect & Characterize n What Structures and Polypeptides Missing?

28 Missing Structures in Mutant

29 Missing Polypeptides in Mutant

30 Primary Cilium n Kidney Tubule Epithelium n Defective in Polycystic Kidney Disease 4th most common cause of kidney failure Autosomal Dominant n How does loss of the cilium cause the disease?

31 Mitosis Background n Names of Stages: Interphase, prophase, metaphase, anaphase, telophase n Interphase MTs disassemble then reassembly as Spindle MTs

32 Mitosis Stages: Spinning-Disk Confocal Images of Microtubules and DNA Early Anaphase Late Anaphase MetaphasePrometaphase Cytokinesis OnsetLate Cytokinesis

33 Onion Root Tip c/o KU Med Ctr

34 Boveri: Centrosome and Centriole

35 Centrosomes n Animals: Centriole Pair in Amorphous Cloud n Ends of MT’s in Cloud.No Relationship to Centrioles. Different from Relationship of Basal Body and Axoneme MT’s. n Flowering Plants: Lack Centrioles

36 Centrosome Ultrastructure

37 Centriole Fine Structure

38 Mitotic Spindle Assembly n Centrosome duplicates and separates n Nuclear envelope breakdown in animals n MT’s rearrange via dynamic instability

39 Spindle MT’s

40 Dynactin RNAi Control Mitotic Spindle Rotation in C. elegans Embryo

41 Chromosome Congression to Metaphase Plate n Kinetochores capture MT’s n Chromosome pulled to Pole Force at Kinetochore n Chromosome pushed away from Pole Forces on arms Force at Kinetochore

42 Microtubule / Kinetochore Attachment

43 Metaphase Normal

44 Types of Mt / Kc Attachment

45 Metaphase - Merotelic Chrom

46 Metaphase to Anaphase

47 Metaphase/Anaphase Lagging

48 Anaphase

49 n Centromere splits and Chromosomes Move Anaphase A: Chromosome to Pole GFP-labeled Centromeres

50 Models for Chromosomes Moving to the Pole n Treadmilling? Depolymerization at Pole n Depolymerization at Kinetochore How remain bound while end shrinks? n Motors at Kinetochore or Pole

51 Pac-Man and Poleward Flux Models for Anaphase A

52 Poleward Tubulin Flux in Anaphase A Movement to Pole... Blue: Photobleach Mark, 0.7 µm/min Yellow: Edge of Chromosome, 1.2 µm/min

53 Kinetochore as a slip-clutch mechanism High tension: Switch to polymerization to prevent detachment Low tension: Depolymerization generates force and movement

54 Anaphase B Pole - Pole Separation

55 End

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