The Cell Cycle £lec 7 Flashcards by Deleted Deleted

The spindle is a highly dynamic assembly that exists at a steady state

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The spindle is a highly dynamic assembly that exists at a steady state

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that depends on the precise balance of numerous forces generated by

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that depends on the precise balance of numerous forces generated by

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various motor proteins and by microtubule polymerization and

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various motor proteins and by microtubule polymerization and

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depolymerization.

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depolymerization.

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 These forces move chromosomes once they are attached to the spindle

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 These forces move chromosomes once they are attached to the spindle

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and produce the tension that is so important for the stabilization of

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and produce the tension that is so important for the stabilization of

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correct attachments.

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correct attachments.

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 In anaphase

similar forces pull the separated chromatids to opposite

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 In anaphase

similar forces pull the separated chromatids to opposite

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ends of the spindle.

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ends of the spindle.

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 Three major spindle forces are particularly critical

although their

 Three major spindle forces are particularly critical

although their

strength and importance vary at different stages of mitosis.

 The first major force pulls the kinetochore and its associated

chromatid along the kinetochore microtubule toward the spindle pole.

 This force is generated by depolymerization at the plus end of the

microtubule.

 This force pulls on chromosomes during prometaphase and

metaphase but is particularly important for moving sister chromatids

toward the poles after they separate in anaphase.

 Notably

this kinetochore-generated poleward force does not require

 Notably

this kinetochore-generated poleward force does not require

ATP or motor proteins.

 The energy that drives the movement is stored in the microtubule and

is released when the microtubule depolymerizes; it ultimately comes

from the hydrolysis of GTP that occurs after a tubulin subunit adds to

the end of a microtubule

How does plus-end depolymerization drive the

kinetochore toward the pole?

 Ndc80 complexes in the kinetochore make multiple low-

affinity attachments along the side of the microtubule.

 Because the attachments are constantly breaking and

re-forming at new sites

the kinetochore remains

re-forming at new sites

the kinetochore remains

attached to a microtubule even as the microtubule

depolymerizes.

 In principle

this could move the kinetochore toward the

 In principle

this could move the kinetochore toward the

spindle pole.

• A second poleward force is provided in some cell types by microtubule

flux

whereby the microtubules themselves are pulled toward the spindle

flux

whereby the microtubules themselves are pulled toward the spindle

poles and dismantled at their minus ends.

• The mechanism underlying this poleward movement is not clear

although it might depend on forces generated by motor proteins and

minus-end depolymerization at the spindle pole.

• In metaphase

the addition of new tubulin at the plus end of a

• In metaphase

the addition of new tubulin at the plus end of a

microtubule compensates for the loss of tubulin at the minus end

that microtubule length remains constant despite the movement of

microtubules toward the spindle pole.

• Any kinetochore that is attached to a microtubule undergoing such flux

experiences a poleward force

which contributes to the generation of

experiences a poleward force

which contributes to the generation of

tension at the kinetochore in metaphase.

Together with the kinetochore-based forces discussed before

flux

Together with the kinetochore-based forces discussed before

flux

also contributes to the poleward forces that move sister

chromatids after they separate in anaphase.

Microtubule flux in the metaphase spindle

• A third force acting on chromosomes is the polar ejection force

or polar wind.

• Plus-end directed kinesin-4 and 10 motors on chromosome arms

interact with microtubules and transport the chromosomes away

from the spindle poles.

• This force is particularly important in prometaphase and

metaphase

when it helps push chromosome arms out from the

metaphase

when it helps push chromosome arms out from the

spindle.

• This force might also help align the sister-chromatid pairs at the

metaphase plate.

The APC/C Triggers Sister-Chromatid Separation

and the Completion of Mitosis

• The anaphase-promoting complex

or cyclosome (APC/C)

• The anaphase-promoting complex

or cyclosome (APC/C)

throws the switch that initiates sister-chromatid separation by

ubiquitylating several mitotic regulatory proteins and thereby

triggering their destruction.

 In early mitosis

the resolution of the sister chromatids is

 In early mitosis

the resolution of the sister chromatids is

accompanied by the removal of most cohesin from the

chromosome arms via a mechanism that depends on a protein that

pulls open the cohesin ring at the junction of its Smc3 and Scc1

subunits.

 When the cell reaches metaphase

cohesins remain primarily at the

 When the cell reaches metaphase

cohesins remain primarily at the

centromeric regions of the chromosomes

adjacent to the

centromeric regions of the chromosomes

adjacent to the

kinetochores

where they serve to resist the poleward forces that

kinetochores

where they serve to resist the poleward forces that

pull the sister chromatids apart.

 Anaphase begins with the abrupt removal of the remaining

cohesin

which allows the sisters to separate and move to opposite

cohesin

which allows the sisters to separate and move to opposite

poles of the spindle.

 The APC/C initiates the process by targeting the inhibitory

protein securin for destruction.

 Before anaphase

securin binds to and inhibits the activity of a

 Before anaphase

securin binds to and inhibits the activity of a

protease called separase.

 The destruction of securin in metaphase releases separase

which is then free to cleave the Scc1 subunit of cohesion.

 The cohesins fall away

and the sister chromatids separate

 The cohesins fall away

and the sister chromatids separate

The initiation of sister chromatid separation

by the APC/C

 Phosphorylation of various proteins by M Cdk promotes spindle

assembly

chromosome condensation

assembly

chromosome condensation

in early mitosis.

 It is thus not surprising that the dephosphorylation of these same

proteins is required for spindle disassembly and the re-formation of

daughter nuclei in telophase.

 Dephosphorylation of Cdk targets depends in part on the inactivation of

most Cdks in the cell

which results when the APC/C targets S- and M-

most Cdks in the cell

which results when the APC/C targets S- and M-

cyclins for destruction.

 Protein dephosphorylation also results from activation of phosphatases.

Unattached Chromosomes Block Sister-Chromatid

Separation: The Spindle Assembly Checkpoint

 Drugs that destabilize microtubules

such as colchicine or

 Drugs that destabilize microtubules

such as colchicine or

vinblastine

arrest cells in mitosis for hours or even days.

vinblastine

arrest cells in mitosis for hours or even days.

 This observation led to the identification of a spindle assembly

checkpoint mechanism that is activated by the drug treatment

and blocks progression through the metaphase-to-anaphase

transition.

 The checkpoint mechanism ensures that cells do not enter

anaphase until all chromosomes are correctly bi-oriented

on the mitotic spindle.

 The spindle assembly checkpoint depends on a sensor

mechanism that monitors microtubule attachment at the

kinetochore.

Unattached Chromosomes Block Sister-Chromatid

Separation: The Spindle Assembly Checkpoint

 Any kinetochore that is not properly attached to the spindle

sends out a diffusible negative signal that blocks Cdc20–APC/C

activation throughout the cell and thus blocks the metaphase-to-

anaphase transition.

 The negative checkpoint signal depends on several proteins

including Mad2

which are recruited to unattached kinetochores.

including Mad2

which are recruited to unattached kinetochores.

 The unattached kinetochore acts as an enzyme that catalyzes a

change in the conformation of Mad2

so that Mad2 then interacts

change in the conformation of Mad2

so that Mad2 then interacts

with other proteins to form a large multiprotein complex that

binds and thereby inhibits APC/C–Cdc20.

Unattached Chromosomes Block Sister-Chromatid

Separation: The Spindle Assembly Checkpoint

Mad2 protein on unattached kinetochores

 In mammalian cells

the spindle assembly checkpoint

 In mammalian cells

the spindle assembly checkpoint

determines the normal timing of anaphase.

 The destruction of securin in these cells begins moments

after the last sister-chromatid pair becomes bi-oriented on

the spindle

and anaphase begins about 20 minutes later.

the spindle

and anaphase begins about 20 minutes later.

 Experimental inhibition of the checkpoint mechanism causes

premature sister-chromatid separation and anaphase.

Unattached Chromosomes Block Sister-Chromatid

Separation: The Spindle Assembly Checkpoint

Chromosomes Segregate in Anaphase

A and B

The sudden loss of sister-chromatid cohesion at the

onset of anaphase leads to sister-chromatid

separation

which allows the forces of the mitotic

separation

which allows the forces of the mitotic

spindle to pull the sisters to opposite poles of the

cell—called chromosome segregation.

The chromosomes move by two independent and

overlapping processes.

The first

anaphase A

The first

anaphase A

of the chromosomes

which is accompanied by

of the chromosomes

which is accompanied by

shortening of the kinetochore microtubules.

The second

anaphase B

The second

anaphase B

spindle poles themselves

which begins after the sister

spindle poles themselves

which begins after the sister

chromatids have separated and the daughter

chromosomes have moved some distance apart

Chromosomes Segregate in Anaphase

A and B

The two processes of anaphase in

mammalian cells

 Although sister-chromatid separation initiates the

chromosome movements of anaphase A

other mechanisms

chromosome movements of anaphase A

other mechanisms

also ensure correct chromosome movements in anaphase A

and spindle elongation in anaphase B.

 Most important

the completion of a normal anaphase

 Most important

the completion of a normal anaphase

depends on the dephosphorylation of Cdk substrates

which

depends on the dephosphorylation of Cdk substrates

which

in most cells results from the APC/C-dependent destruction of

cyclins.

Chromosomes Segregate in Anaphase

A and B

 The relative contributions of anaphase A and anaphase B to

chromosome segregation vary greatly

depending on the cell

chromosome segregation vary greatly

depending on the cell

type.

 In mammalian cells

anaphase B begins shortly after anaphase A

 In mammalian cells

anaphase B begins shortly after anaphase A

and stops when the spindle is about twice its metaphase length.

 In contrast

the spindles of yeasts and certain protozoa primarily

 In contrast

the spindles of yeasts and certain protozoa primarily

use anaphase B to separate the chromosomes at anaphase

and

use anaphase B to separate the chromosomes at anaphase

and

their spindles elongate to up to 15 times their metaphase length.

Chromosomes Segregate in Anaphase

A and B

Segregated Chromosomes Are Packaged in

Daughter Nuclei at Telophase

 In telophase

the final stage of mitosis

 In telophase

the final stage of mitosis

chromosomes are packaged into a pair of daughter nuclei.

 The first major event of telophase is the disassembly of the

mitotic spindle

followed by the re-formation of the nuclear

mitotic spindle

followed by the re-formation of the nuclear

envelope. This process occurs in multiple stages.

 First

proteins on the surface of the chromosomes promote

 First

proteins on the surface of the chromosomes promote

their interaction with each other

resulting in a compact

their interaction with each other

resulting in a compact

cluster of all the chromosomes.

Segregated Chromosomes Are Packaged in

Daughter Nuclei at Telophase

 Next

fragments of endoplasmic reticulum membrane containing inner

 Next

fragments of endoplasmic reticulum membrane containing inner

nuclear envelope proteins associate with the surface of the

chromosome cluster

eventually fusing to re-form the complete nuclear

chromosome cluster

eventually fusing to re-form the complete nuclear

envelope.

 Nuclear pore complexes are incorporated into the envelope

and the

 Nuclear pore complexes are incorporated into the envelope

and the

nuclear lamina re-forms.

 The pore complexes pump in nuclear proteins

the nucleus expands

 The pore complexes pump in nuclear proteins

the nucleus expands

and the mitotic chromosomes are reorganized into their less-

condensed interphase state.

 A new nucleus has been created

and mitosis is complete.

 A new nucleus has been created

and mitosis is complete.