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Mitosis vs. microtubule

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(1)
(2)

Mitosis vs. microtubule

(3)

Anaphase-promoting complex/cyclosome (APC/C)

(4)

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

(5)

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

(6)

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

(7)

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

(8)

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

(9)

Microtubules treadmill during mitosis

Microtubules in mitosis treadmill toward the spindle poles GFP-tubulin

Different colors were shown different velocity

(10)

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

(11)

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

(12)

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

(13)

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

(14)

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

(15)

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

(16)

成膜體

(17)

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.

(18)

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)

(19)

All IF proteins have a conserved core domain & are organized similarly into

filaments.

Parallel dimer Antiparallel tetramer

(20)
(21)

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

(22)

Intermediate filaments are

anchored in cell junctions

(23)

Intermediate filaments are resistent to bending or stretching forces

(24)

Intermediate filaments are

dynamic.

(25)

Disruption of keratin networks causes blistering.

Normal mouse

Keratin gene mutant

Separation between epidermis and dermis

(26)

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

(27)
(28)

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

(29)

Cdc42 coordinates microtubules and microfilaments during cell

migration

(30)

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

2.Multiple motor proteins are associated with membrane vesicles

Chapter 20 summary

(31)

IV. Microtubules & motor proteins during mitosis

1. Mitotic apparatus

2. Centromere and kinetochore 3. Centrosome duplication

4. Microtubule dynamics during mitosis 5. Centrosome movement during mitosis

6. Formation of spindle poles and capture of chromosomes 7. Chromosome separation & spindle elongation

8. Cytokinesis

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