Mitosis vs. microtubule
Anaphase-promoting complex/cyclosome (APC/C)
Relation of centrosome duplication to the cell cycle.
Parent centrioles
Daughter centrioles
Grow complete
Centrosome cycle or centriole cycle
Duplicated centrosomes align and begin separating in prophase
G1 Phase Æ 1st growth phase S Phase Æ DNA duplicated
G2 Phase Æ Final growth phase Mitosis
Cytokinesis
The mitotic spindle contains three class of microtubules
1. Astral microtubule, from spindle poles to the cell cortex
2. Kinetochore
microtubule, attaches to chromatid
3. Polar microtubule,
pushing the duplicated centrosomes, and
maintaining the
structure
Electron microscopy visualizes
components of the mitotic apparatus in a metaphase mammalian cell.
交叉
Connected chromosome
Prophase signals must convert interphase array to mitotic apparatus - increase in short dynamic microtubules - mitotic MT turnover 5-10 fold faster than interphase MT - less polymer and more monomer tubulin during M phase than at other times
動粒
紡錘體 Chromosomes align at the 星狀體
equatorial plane
Shortening at the (+) end of
kinetochore MTs moves chromosomes pole ward in anaphase A
Shortening at + end (attach kinetochores) of microtubule by disassembly.
In vivo fluorescent-tagging experiment Kinetochore associated kinesin, MCAK →
promotes disassembly at the + end, CENP- E (also at kinetochore) binds to
progressively shortening end.
Chromosome move toward to “-” Loss fluorescent
Shortening at the + end of kinetochore microtubules moves chromosomes poleward in anaphase A
Microtubule dynamics increase dramatically in mitosis
XMAP215( xenopus MAP of 215 kDa): stabilizing microtubule
Microtubule dynamic increases in mitosis due to loss of a stabilizing MAP Only kinesin-13 → unstable
Microtubules treadmill during mitosis
Microtubules in mitosis treadmill toward the spindle poles GFP-tubulin
Different colors were shown different velocity
The kinetochore captures and helps transport chromosomes
Inner
kinetocho re layer
Outer kinetocho re layer Kinetochore is a centromere(著絲點)-based protein complex that mediates attachment of chromosomes to MTs.
Centromere: a constricted region of the condensed chromosome defined by centromeric DNA
Kinetochore is a centromere(著絲點)- based protein complex that mediates
attachment of chromosomes to MTs.
kinesins CENP-E: keeps the kinetochore
tethered to the kinetochore microtubule
MACK: plus ends of spindle microtubules attach to
chromosomes
Spindle poles(星狀體)
→Microtubule very
dynamic→chromosomal
attachment(1a,1b)→capture by kinetochores selctively→motor protein
(dynein/dynactin)→move toward to spindle pole (-), 2→chromosome pair bi-
oriented(3)→two kinetochores opposite→pulled and separate
Chromosome capture and congression (組織) in prometaphase
PUSH AND PULL
Can not attach
Disassembly >> assembly
Chromosome movement and spindle pole separation in anaphase Anaphase a moves chromosomes to poles by microtubules shorting
Anaphase B separates poles by the combined action knesins and dynein
Chromosome movement is powered by microtubule- shorting kenesin-13 at kinetochore and spindle pole
Chromosome arms point away spindle pole due to
chromokinesin/kinesin-4→
depolymerization force
→overcome the force
pulling the arms toward the center of spindle
Anaphase B:
1. sliding of antiparallel polar microtubule powered by kinesin-5
2.dynein/dynactin located at cell cortex
Kenesin-13 Kenesin-13
Kenesin-4
DNA
Additional mechanisms contribute to spindle formation
Centrosomes is not the only way a spindle can form Other factor cooperate to make a spindle.
egg→arrested in mitosis →centrifuging →organelles and yolk→ no centrosome→
Mitotic spindles can form in the absence of centrosome
Plant cells reorganize their MTs & build a new cell wall in mitosis.
Interphase plant cells: lack a single perinuclear microtubule-organizing center
Similar to animal:
Prophase: Bundle their cortical microtubules and reorganize, without centrosomes
Metaphase: golgi-derived vesicles are transported into the mitotic apparatus along microtubules
Telophase: vesicles line up near the center of the dividing cell and to
form the phragmoplast (microtuble formation), a membrane structure
similar to animal cell contractile ring → the plasma membrane of
daughter cells; vesicles contains cellulose pectin for cell wall
成膜體
Intermediate Filaments (IF)
Differ in stability, size, and structure from other cytoskeletal fibers
Intermediate diameter ~10 nm Subunits are fibrous
Almost all subunits are incorporated into stable intermediate filaments
No hydrolysis of ATP or GTP is required for polymerization
No known polarity of the filament
The formed fibers are not easily soluble No direct participation in cell motility
Two types of intermediate filaments
lamin intermediate
filaments: blue; nucleus Cytoplasmic keratin
cytosleleton: red
Keratin & lamin filaments
in epithelial cells.
Intermediate filament assembly spontaneous assembly
→ No need of chaperone proteins or energy (no hydrolysis of nucleotides) actin polymerization need energy
the filament has no polarity ≠ from actin or microtubule filaments
Intermediate filaments: cytoplasmic and nuclear
non-polar, tough, rope-like, less than 5% in soluble form, no nucleotide provide protection against mechanical stress,
withstand stretching forces
IF protein-specific antibodies or cDNA used for cell typing and tumour diagnosis REGULATION:
phosphorylation by PKC of the N-terminal Ser induces disassembly of IF (particularly in nuclear lamins during mitosis)
IF associated proteins (IFAPs)
All IF proteins have a conserved core domain & are organized similarly into
filaments.
Parallel dimer Antiparallel tetramer
Keratins: epithelial
Vimentin is the major IF in cells of mesenchymal and neuronal origin
Glial Fibrillary Acidic Protein forms IF in glial cells and some Schwann cells
Peripherin is a rare IF, occurring in some types of neurons
Desmin is the predominant IF in skeletal and cardiac muscle
sarcomers and in smooth muscle myofibrils
Intermediate filaments are
anchored in cell junctions
Intermediate filaments are resistent to bending or stretching forces
Intermediate filaments are
dynamic.
Disruption of keratin networks causes blistering.
Normal mouse
Keratin gene mutant
Separation between epidermis and dermis
Blistering of the skin caused by mutant keratin genes
Epidermolysis bullosa simplex EBS: the skin blisters in response to very slight mechanical stress
Other blistering diseases:
mouth, esophageal lining and cornea of the eye-- mutations of different keratins
Truncated keratin (missing both the N- C- domains) Tg mice 大皰性表皮鬆解症
IFAPs cross-link IFs to one another and to other cell structures (microtubules,
actin filaments, membranes).
Fibroblast cell.
Microtubules are red, intermediate filaments-blue, short connecting fibers is green Intermediate filament
associated protein (IFAPs):
corss link intermediated filaments with one another , forming a bundle or a network and with other cell structures, including the plasma
membrane.
plakin
Cdc42 coordinates microtubules and microfilaments during cell
migration
I. Microtubule structure
1. tubulins and microtubule structure
2. Microtubule-organizing center (MTOC) II. Microtubule dynamics & associated proteins
1. Assembly/disassembly of microtubule 2. Dynamic instability
3. Temperature influences microtubule stability 4. Drugs involved in microtubule dynamics
5. Microtubule associated protein (MAP)
III. Motor proteins and intracellular transport 1. Motor proteins
--microtubule motor proteins: Kinesin family, Dynein family