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CytoskeletPrednáška 6
Mikrotubuly a mitóza
Polarity of tubulin polymerization
Nuclei
Tubulin > CC
Tubulin < CC
Preferential addition of tubulin ar (+) ends
Preferential loss of tubulin ar (+) ends
Kinesin – another molecular motor
Globular head
Heavy chain Coiled-coil α helix
ATP hydrolysis;Binding to MT
Binding to transported vesicle
10 nm Lightchain
Kinesin versus myosin cycle
Dynein [(-)-end directed motor] and dynactin complexHeavy chains
Intermediate chains
Light chains
Mitosis in an animal cell
A course of mitosis in a typical animal cell
Animation mitosis
A typical time course for mitosis and cytokinesis (M phase) in a mammalian cell
time (minutes)
CYTOKINESIS
0 20 40 60 80
The cytoskeleton in M phase
Three sets of MTs in the mitotic apparatus
(polar)
Three sets of MTs in the mitotic apparatus
A model for formation of bipolar mitotic spindle by the selective stabilization of microtubules
Astral MT
Polar MT
centrosome
Mitotic apparatus in Saccharomyces cerevisiae
Chromosome
Polar MT
Kinetochore MT
Nucleus
Spindle pole body (SPB)
The spindle pole body in yeast
A chromosome in metaphase
Kinetochore microtubules
Centromeric attachment of MTsChromatids
Kinetochore
Outer plate
Inner plate
Centromeric chromatin
Fibrous coronaMicrotubule
The kinetochoreDAPI kinetochore
anaphase chromatidkinetochore
MTs embedded inkinetochore
direction of chromatidmovement
1 µm
Kinetochores in cultured cells
Scleroderma patients produce anti-kinetochore antibodies
10 µm
Three problems of prometaphase:
1. Spindle pole formation2. Chromosome capture
3. Chromosome line-up at the equator
Centriole replication
A newly replicated pair of centrioles
Fluorescence-recovery after photobleaching (FRAP)
McNally et al. (2000). Science 287: 1262-1265.
FRAP can be used for understanding dynamics of protein complexes
Microtubules in an M-phase cell are much more dynamic than the microtubules at interphase
Dynamic instability and capture of chromosomes
end capture
kinetochoreGrowing MTs
Shrinking MTs
SPB
Dynamic instability and capture of chromosomes
Side capture
Chromosome slidesto the (+) end
The dynamic behavior of microtubules in the metaphase spindle
The dynamic behavior of microtubules in the metaphase spindle
Blue-DNA; Red-rhodamine-tubulin; Yellow-caged fluorescein-tubulin
The dynamic behavior of microtubules in the metaphase spindle
CENP-E
Dynein
(+) (-)
Demonstration of the astral exclusion (pushing) force
Demonstration of the astral exclusion (pushing) force
Two possibilities for how chromosomes line up at the metaphase plate
PUSH Astral exclusion force decreases with distance from pole
Pulling force proportional to length of kinetochore MTsPULL
ANIMATION:MT behavior during prometaphase
The two processes that separate sister chromatids at anaphase
Separation of chromatids at anaphase A
Movement of chromosomes during anaphase A
Movement of chromosomes during anaphase A is mediated by shortening of MTs at (+) ends
Involvement of kinetochores in chromosome movement during anaphase A
The two processes that separate sister chromatids at anaphase
How microtubule motor proteins act in anaphase B
ONWARD PUSH ON SPINDLE POLES
ONWARD PULL ON SPINDLE POLES
cell cortex
Sliding of overlap microtubules at anaphase
Anaphase checkpoint – important tool ensuring a “healthy” cell cycle
Mitosis in animal cells video
How do two blind people equally split 46 pairs of socks?
Cell 109: 9-12 (2002)
Cell ensures bipolar attachment of each chromosome
Monopolar attachment Unipolar attachment Bipolar attachment
Kinetochores occupiedTension not established
Kinetochores unoccupiedTension not established
Kinetochores occupied Tension established
Incorrect MT capture Correct MT capture
Cohesins and anaphase
Cohesin dimer Sister chromatid cohesion by cohesin
Gruber et al. (2006). Evidence that loading of cohesin onto chromosomes involves opening of its SMC hinge. Cell 127: 523-537.SMC: Structural Maintenance of Chromosomes
Cohesins and anaphase
∅30-40 nm
Anaphase-promoting complex (APC)
cohesin
separasePds1 (securin; inhibitor of separase)
Ub
Ub ubiquitin
APC
Mad2
Mad2
Nature 434: 575-576 (2005); Cell 120: 739-746 (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, 123–132.
Meiosis: even more challenging type of cell divisionMitosis
Meiosis
Metaphase
Anaphase
Metaphase I
Metaphase II
Anaphase I
Anaphase II
Journal of Cell Science 117, 4017-4023 (2004)
Juraj Gregáň :
http://www.mfpl.ac.at/index.php?cid=441
Gregan, J., et al (2007). Current Biology, 17(14): 1190-1201.
Petronczki, M., Matos, J., Mori, S., Gregan, J. et al. (2006). Cell, 126(6): 1049-64.
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): 53-61.
Gregan, J. et al. (2005). Current Biology, 15(18): 1663-69.
...
Meiosis: even more challenging type of cell division
Peric-Hupkes & van Steensel (2008). Linking Cohesin to Gene Regulation.Cell 132, 925-928.
Novel role(s) of cohesinS. cerevisiae: SMC1 & SMC3 are required to prevent spreading of heterochromatin from silenced HMR locusH. 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
Gullerová Monika & Proudfoot, N.J. (2008). Cohesin Complex Promotes Transcriptional Termination between Convergent Genes in S. pombe. Cell 132, 983–995.
Peric-Hupkes & van Steensel (2008). Linking Cohesin to Gene Regulation. Cell 132, 925-928.
Novel role(s) of cohesin
Nonrandom distribution of cohesin throughout the chromosomes(acummulation of cohesin between convergent genes in G2 phase)
Read-through transcription in G1 leads to RNAi-dependent formation of heterochromatin and recruitment of cohesin
So similar…
… yet so different
Sci. Amer. May 2009 issue
Santino(Furuwik ZOO)
Osvald. M. (2009). Spontaneous planning for future stone throwing by a male chimpanzee Curr. Biol. 19: R190
How to identify loci in human genome, which are under positive selection?
Examples of human loci under apparent positive selectionwt
aspm
har1
HAR1, brain, possible affects size of cerebral cortex
FOXP2, transcription factor, speech production
AMY1, digestion of starch, exploration of novel food (?)ASPM, control of brain size (mutated in microcephalic patients)
LCT, digestion of lactose provided by domesticated animals
HAR2, development of wrist and thumb
Spindle rotation during development
Neuroeipithelial (NE) cells: primary neural progenitors
Lineage relationships between neuroepithelial cells (NE), radial-glial cells (RG), basal
progenitors (BP) and neurons (N).
Huttner & Kosodo (2005). Curr. Opinion Cell Biol. 17: 648-657
NE RG
BP
N
Types of division:
Symmetric proliferative: NE à NE + NENEUROGENIC:Asymmetric self-renewing: NE à NE + neuronAsymmetric bi-differentiative: NE à radial glial cell + neuron
Ventricular germinal zone: A layer of intensive division of neuroepithelial cells
Purves et al. (2001). Neuroscience, 2nd Edition, Sinauer Associates, Inc.
neural tube
Ventricular germinal zone
Intermediate zoneMarginal zone
Symmetric versus asymmetric division of neuroepithelial and radial-glial cells with vertical cleavage plane orientation
Huttner & Kosodo (2005). Curr. Opinion Cell Biol. 17: 648-657Fish a kol. (2007). Proc. Natl. Acad. Sci. USA 103: 10438-10443
apical p
lasm
a mem
bran
e
Aspm
symmetric asymmetric