Cytoskelet Prednáška 6 Mikrotubuly a mitóza

Transcription

1 Cytoskelet Prednáška 6 Mikrotubuly a mitóza

2 Polarity of tubulin polymerization Nuclei Tubulin > C C Preferential addition of tubulin ar (+) ends Tubulin < C C Preferential loss of tubulin ar (+) ends

3 Kinesin another molecular motor Heavy chain Coiled-coil α helix Globular head 10 nm Light chain ATP hydrolysis; Binding to MT Binding to transported vesicle

4 Kinesin versus myosin cycle

5 Dynein [(-)-end directed motor] and dynactin complex Heavy chains Intermediate chains Light chains

6 Mitosis in an animal cell

7 A course of mitosis in a typical animal cell Animation mitosis

8 A typical time course for mitosis and cytokinesis (M phase) in a mammalian cell CYTOKINESIS time (minutes)

9 The cytoskeleton in M phase

10 Three sets of MTs in the mitotic apparatus (polar)

11 Three sets of MTs in the mitotic apparatus

12 A model for formation of bipolar mitotic spindle by the selective stabilization of microtubules Astral MT Polar MT centrosome

13 Mitotic apparatus in Saccharomyces cerevisiae Chromosome Kinetochore MT Spindle pole body (SPB) Polar MT Nucleus

14 The spindle pole body in yeast

15 A chromosome in metaphase

16 Kinetochore microtubules

17 Centromeric attachment of MTs Chromatids Kinetochore Centromeric chromatin Fibrous corona Microtubule Outer plate Inner plate

18 The kinetochore DAPI kinetochore kinetochore anaphase chromatid direction of chromatid movement MTs embedded in kinetochore 1 µm

19 Kinetochores in cultured cells 10 µm Scleroderma patients produce anti-kinetochore antibodies

20 Three problems of prometaphase: 1. Spindle pole formation 2. Chromosome capture 3. Chromosome line-up at the equator

21 Centriole replication

22 A newly replicated pair of centrioles

23 Fluorescence-recovery after photobleaching (FRAP)

24 FRAP can be used for understanding dynamics of protein complexes McNally et al. (2000). Science 287:

25 Microtubules in an M-phase cell are much more dynamic than the microtubules at interphase

26 Dynamic instability and capture of chromosomes kinetochore Growing MTs SPB Shrinking MTs end capture

27 Dynamic instability and capture of chromosomes Side capture Chromosome slides to the (+) end

28 The dynamic behavior of microtubules in the metaphase spindle

29 The dynamic behavior of microtubules in the metaphase spindle Blue-DNA; Red-rhodamine-tubulin; Yellow-caged fluorescein-tubulin

30 The dynamic behavior of microtubules in the metaphase spindle CENP-E Dynein (+) (-)

31 Demonstration of the astral exclusion (pushing) force

32 Demonstration of the astral exclusion (pushing) force

33 Two possibilities for how chromosomes line up at the metaphase plate PULL Pulling force proportional to length of kinetochore MTs PUSH Astral exclusion force decreases with distance from pole ANIMATION: MT behavior during prometaphase

34 The two processes that separate sister chromatids at anaphase

35 Separation of chromatids at anaphase A

36 Movement of chromosomes during anaphase A

37 Movement of chromosomes during anaphase A is mediated by shortening of MTs at (+) ends

38 Involvement of kinetochores in chromosome movement during anaphase A

39 The two processes that separate sister chromatids at anaphase

40 How microtubule motor proteins act in anaphase B ONWARD PUSH ON SPINDLE POLES cell cortex ONWARD PULL ON SPINDLE POLES

41 Sliding of overlap microtubules at anaphase

42 Anaphase checkpoint important tool ensuring a healthy cell cycle Mitosis in animal cells video

43 How do two blind people equally split 46 pairs of socks?

44 Cell ensures bipolar attachment of each chromosome Incorrect MT capture Correct MT capture Monopolar attachment Unipolar attachment Bipolar attachment Kinetochores occupied Tension not established Kinetochores unoccupied Tension not established Kinetochores occupied Tension established Cell 109: 9-12 (2002)

45 Cohesins and anaphase Cohesin dimer Sister chromatid cohesion by cohesin

46 Cohesins and anaphase nm Gruber et al. (2006). Evidence that loading of cohesin onto chromosomes involves opening of its SMC hinge. Cell 127: SMC: Structural Maintenance of Chromosomes

47 Anaphase-promoting complex (APC) Ub APC Mad2 cohesin Mad2 Ub ubiquitin Pds1 (securin; inhibitor of separase) separase Nature 434: (2005); Cell 120: (2005) Sun, Y.,Kucej Martin et al. (2009). Separase Is Recruited to Mitotic Chromosomes to Dissolve Sister Chromatid Cohesion in a DNA-Dependent Manner. Cell 137,

48 Meiosis: even more challenging type of cell division Meiosis Mitosis Metaphase I Metaphase Anaphase I Anaphase Metaphase II Anaphase II Journal of Cell Science 117, (2004)

49 Meiosis: even more challenging type of cell division Juraj Gregáň : Gregan, J., et al (2007). Current Biology, 17(14): Petronczki, M., Matos, J., Mori, S., Gregan, J. et al. (2006). Cell, 126(6): Riedel, C.G., Katis, V.L., Katou, Y., Mori, S., Itoh, T., Helmhart, W., Galova, M., Petronczki, M., Gregan, J. et al. (2006). Nature, 441(7089): Gregan, J. et al. (2005). Current Biology, 15(18):

50 Novel role(s) of cohesin S. cerevisiae: SMC1 & SMC3 are required to prevent spreading of heterochromatin from silenced HMR locus H. sapiens: Mutations in Scc and SMC cause Cornelia de Lange Syndrome (autosomal dominant; 1-10,000-1:30,000) (some cases (SMC1A exhibit X-linked inheritance) NO DEFECTS IN SISTER CHROMATID COHESION Peric-Hupkes & van Steensel (2008). Linking Cohesin to Gene Regulation. Cell 132,

51 Novel role(s) of cohesin Read-through transcription in G1 leads to RNAi-dependent formation of heterochromatin and recruitment of cohesin Nonrandom distribution of cohesin throughout the chromosomes (acummulation of cohesin between convergent genes in G2 phase) Gullerová Monika & Proudfoot, N.J. (2008). Cohesin Complex Promotes Transcriptional Termination between Convergent Genes in S. pombe. Cell 132, Peric-Hupkes & van Steensel (2008). Linking Cohesin to Gene Regulation. Cell 132,

52 So similar Santino (Furuwik ZOO) yet so different Osvald. M. (2009). Spontaneous planning for future stone throwing by a male chimpanzee Curr. Biol. 19: R190 Sci. Amer. May 2009 issue

53 How to identify loci in human genome, which are under positive selection?

54 Examples of human loci under apparent positive selection HAR1, brain, possible affects size of cerebral cortex FOXP2, transcription factor, speech production AMY1, digestion of starch, exploration of novel food (?) wt ASPM, control of brain size (mutated in microcephalic patients) aspm LCT, digestion of lactose provided by domesticated animals har1 HAR2, development of wrist and thumb

55 Spindle rotation during development

56 Neuroeipithelial (NE) cells: primary neural progenitors BP NE RG N Lineage relationships between neuroepithelial cells (NE), radial-glial cells (RG), basal progenitors (BP) and neurons (N). Huttner & Kosodo (2005). Curr. Opinion Cell Biol. 17:

57 Types of division: Symmetric proliferative: NE NEUROGENIC: Asymmetric self-renewing: NE Asymmetric bi-differentiative: NE radial glial cell + neuron NE + NE NE + neuron

58 Ventricular germinal zone: A layer of intensive division of neuroepithelial cells Marginal zone Intermediate zone Ventricular germinal zone neural tube Purves et al. (2001). Neuroscience, 2 nd Edition, Sinauer Associates, Inc.

59 Symmetric versus asymmetric division of neuroepithelial and radial-glial cells with vertical cleavage plane orientation apical plasma membrane Aspm symmetric asymmetric Huttner & Kosodo (2005). Curr. Opinion Cell Biol. 17: Fish a kol. (2007). Proc. Natl. Acad. Sci. USA 103: