Module 10 - Cell cycle dynamics and checkpoints

Question 1

entry into mitosis

Answer 1

• as cells enter into prophase of mitosis, there are many changes that occur
• chromosomes condense, the nuclear envelope breaks down, the organelles fragment. and. the mitotic spindle assembles
• all of these events are initiated by the phosphoregulation of key protein
• phosphorylation is mediated by active MPF complex, a heterodimer of mitotic cyclin and CDK
• in this way, MPF can coordinate these changes to happen at the same time
• at the end of mitosis, all of these processes must be reversed, requiring the inactivation of MPF through ubiquitin-mediated degradation of cyclin B
• once MPF is inactive, cytosolic phosphatases reverse the phosphorylation of the same key proteins to initiate chains of events to ensure the cell is reset to an interphase state

Question 2

Prophase events

Answer 2

• initiated by the MPF mediated phosphorylation of target proteins at entry into mitosis
• in each. case, specific proteins are phosphorylated to set off a cascade of events that lead to each change in behaviour
  (1) mitotic spindle assembly - formation of mitotis spindle through the phosphorylation of microtubule-associated proteins that promote microtubule instability and centrosome separation
  (2) chromosome condensation - through the phosphorylation of condensing and histone proteins
  (3) chromatid cohesion - preparation for. separation of sister chromatids through phosphorylation of cohesion, also contributes to condensation
  (4) nuclear envelope breakdown - through the phosphorylation of nuclear lamins
  (5) endomembrane fragmentation - fragmentation of the Golgi and ER via the phosphorylation of GM130

Question 3

Telophase events

Answer 3

progression through mitosis and exit from mitosis requires a decrease in mitotic cyclins and inactivation of MPF

• it also requires the activity of phosphatases to dephosphorylation the MPF target proteins
• in anaphase, sister chromatid separation occurs
• at. the end of telophase, the cell reforms a nuclear envelope around each segregated set of daughter chromosomes
• the chromosomes decondense the mitotic spindle are disassembled and the membrane of the Golgi and ER resembles.

Question 4

chromosome condensation

Answer 4

while chromatin is dispersed throughout the nucleus in interphase, chromosomes are highly compared in mitosis so that individual chromosomes can be distinguished

• within each chromosome, centromeres are closely attached by cohesion complexes while the arms have separated apart. from one another slightly
• this gives the classic “X” shaped mitotic chromosome appearance
• many proteins are phosphorylated in prophase to mediate chromosome condensation including condensins, cohesion, histones and topoisomerases

Question 5

histones in chromosome condensation

Answer 5

• DNA is organized around essential chromosomal binding proteins called histones
• there are 5 types of histones
• histone H3 is part of an octomer that forms the protein core around. which DNA is wound to create the nucleosome
• H1 is a linker in between neighbouring nucleosomes
• at mitosis, the nucleosomes are packed more tightly as a part of chromosome condensation
• histone proteins H1 and H3 are both phosphorylated by a kinase called aurora B during chromosome condensation
• antibodies used to detect the histones are specific to the phosphorylated form of the protein

Question 6

cohesion proteins

Answer 6

• form the cohesion complex which is required. to hold sister chromatids together after replication until anaphase
• the release of cohesion in mitosis happens in a 2 step process
• first step in prophase is the bulk release of the cohesion from the chromosome arm while maintaining cohesion at the centromere
• this produces the “x” shaped metaphase chromosome
• phosphorylation of cohesion subunits by multiple kinases, including cyclin B CDK and aurora B allows this dissociation to occur
• cohesions at the centromere are protected from phosphorylation by phosphatases
• it is only at anaphase that the cohesion at the centromere are targetted for cleavage by the separate protein to allow sister chromatid separation
• cohesion complex unites 2 different DNA molecules (sister chromatids)

Question 7

condensin proteins

Answer 7

• release of cohesion along the chromosome arm allows the assembly of. the condensing protein complex at prophase
• condensin proteins must themselves be phosphorylated by cyclin B-CDK to allow assembly. and chromosome condensation
• multiple MPF phosphorylation sites are clustered in the carboxyl-terminal domain of one of the condensins identified in Xenopus called XCAP-D2
• condensin complex associate different regions of the same DNA molecule to cause condensation

Question 8

chromosome decondensation

Answer 8

• after successful anaphase, the cell prepares to proceed through telophase and exit mitosis
• this is accompanied by the condensation of the chromosomes. in each of the newly formed daughter nuclei
• decondensation is. necessary to allow transcription to occur in G1
• condensins and histones phosphorylated earlier in mitosis are now dephosphorylated
• this occurs in the absence of active cyclin B/CDK and in the presence of various phosphatases
• dephosphorylation allows the removal of. condensin and remodelling of DNA around the histones. to direct chromosome decondensation

Question 9

nuclear envelope disassembly

Answer 9

nuclear envelope breakdown (NEB)

• seen as cells transition. from interphase to prophase and early prometaphase and finally to metaphase
• in interphase, the intact membrane surrounds the nuclear chromosomes
• at prometaphase, the chromosomes are compacting and the nuclear envelope is fragmenting
• line surrounding the nucleus is no longer continuous
• finally, at metaphase, there is no nuclear envelope, the nuclear membrane is fragmented into small vesicles and distributed in the cytosol

Question 10

nuclear membrane composition

Answer 10

nuclear envelope / membrane is 2 lipid bilaters

• outer nuclear membrane (ONM) is continuous with the RER memrbane
• the inner nuclear membrane (INM) is associated with a scaffold of intermediate filaments called the nuclear lamina

Question 11

nuclear pore comples

Answer 11

NPC span the two membranes to allow transport between the cytosol and the nucleus

Question 12

lamins in interphase

Answer 12

• during interphase, chromosomal proteins interact with the nuclear lamina to anchor. chromatin
• the association between the chromatin and the nuclear lamina is a mechanism of regulating gene expression
• the nuclear lamina is made up of 3 nuclear lamin protein, the intermediate filament proteins, lamin A, lamin B and lamin C
• these lamin proteins polymerize to. form a network of filaments
• creates a mesh

Question 13

lamins in prophase

Answer 13

• in prophase, the 3 lamin proteins are phosphorylated at. specific serine residues by cyclin B/CDK
• this phosphorylation initiates disassemble of the nuclear lamina by causing dissociation of the lamin tetramers that formed the intermediate filaments
• the disintegration of the nuclear lamin scaffold is associated with subsequent fragmentation of the inner membrane
• only lamin B remains associated with the nuclear membrane
• phosphorylated lamin A and lamin C dimers are soluble and disperse into the cytosol

Question 14

phosphorylation of NEB experiment 1

Answer 14

• researchers followed NEB in cultured hamster cells
• added to these cultured cells was a gene for the human lamin A protein
• the lamin A proteins are so similar between these species that both hamster and human lamin A are incorporated into the nuclear laimna
• at interphase, decondensed. chromatin is surrounded by the intact nuclear. lamina and by association the intact nuclear envelope that forms a sphere around the nucleus
• at prophase, the condensing chromosomes are surrounded by a nuclear lamin that. is breaking down
• stain reveals involutions in the scaffold as it. is disassembled
• lamin A stain. can be seen diffusing into the cytosol
• at metaphase, the lamin A stain is no longer organized into a spherical ring and the chromosomes are fully compacted

Question 15

phosphorylation of NEB experiment 2

Answer 15

• experiment was repeated by adding. a variant of the human lamin A
• this time, the introduced lamin A gene codes for a protein. that cannot be phosphorylated
• target serine residues have been replaced with alanines which cannot be phosphorylated
• at interphase, the cell looks the same
• mutant lamin A is still incorporated into the nuclear lamina and the nuclear lamina can be seen surrounding the nucleus containing diffuse chromatin
• at prophase, as the chromosomes condense, the revolutions of the nuclear lamina are visible
• only lamin A cannot be phosphorylated, lamin B and C can be phosphorylated so these initial changes are occuring
• lamin A is not seen. diffusing. into the cytosol as it was earlier
• presumably, lamin A remains in the phosphorylated tetramers
• at metaphase, the chromosomes are completely condensed but there. is still a ring around the chromosomes visible with the lamin A antibody
• researchers conclude that phosphorylation of lamin A is necessary for nuclear lamina disassembly and nuclear envelope breakdown

Question 16

reassembly of the nuclear envelope

Answer 16

• at telophase, the inactivation of the cyclin B/CDK kinase and the activity of a phosphatase called cdc14 allows dephosphorylation of lamin A, B and C residues
• the lamin can reassemble into tetramers and filaments. to reform the nuclear lamina
• lamin B remained associated with small vesicles formed. from the nuclear inner membrane after NEB
• as the lamins reassemble, lamin B brings along these. vesicles which can fuse on the surface of the nuclear lamina to form the inner nuclear envelope
• in early and later anaphase, the nuclear envelope is dispersed in the cytosol. At telophase, the nuclear envelope can be seen coalescing around the decondensing chromosomes
• chromosomal proteins that interact with the nuclear lamina and anchor the chromosomes are phosphorylated in prophase. This dissociated the chromosomes from the nuclear lamina so as not to interfere with chromosome dynamics during metaphase and anaphase
• dephosphorylation permits. the nuclear lamina to assemble around the chromosomes
• nuclear pore complex proteins are also phosphorylated at NEB and phosphorylated at telophase as the nuclear envelope reforms

Question 17

karyomere fusion

Answer 17

-the steps of nuclear envelope reassembly have been replicated in a cell free sysyem
-chromosomes from xenopus sperm have been added to egg extracts
vesicles accumulate around an individual chromosome, mediated by chromosomal proteins
-these vesicles begin to coalesse to form a membrane around a single chromosome called a karyomere
-the karyomere around the individual chromosomes fuse together to form a complete nuclear envelope
-the fusion of the karyomeres is regulated using mechanisms similar to those during protein transport
-vesicle fusion to target membranes is mediated by Rab proteins

Question 18

golgi fragmentation

Answer 18

-organelles of the cell undergo reorganization. at mitosis to ensure that they are distributed to both of the daughter cells
-this is not a precise and equal separation like with chromosomes
-the process of Golgi division (dictyokinesis) was first described in 1910
-prior to cytokinesis, the vesicles and tubules of the fragmented Golgi apparatus accumulate in 2 clusters, one at each spindle pole
-this is typical of organelle division in mitosis as clustering with the spindle poles ensures that a proportion of the gradmented organelles goes to each daughter cell
0the phosphorylation of at least 1 Golgi protein is required for fragmentation
-GM130 is directly phosphorylated by cyclin B/CDK in mitosis as Golgi fragmentation begins
the protein remains phosphorylated until the end of mitosis at telophase, when the Golgi apparatus reassembled in each developing daughter cell
-this is seen using an antibody specific to GM130 phosphorylated at serine 25 (called p25)
-dephosphorylation of GM130 correlates with the inactivation of cyclin B/CDK in telophase

Question 19

organelle reorganization. during mitosis

Answer 19

• all organelles are reorganized to allow distribution. to the daughter cells in mitosis; chloroplasts, mitochondria, ER, lysosomes are all reorganized in some manner during mitosis
• targets of phosphorylation and the mechanisms or organization are not yet clear

Question 20

mitochondrial fragmentation

Answer 20

in G1/S the mitochondria are organized into a. dynamic continuous network
-in mitosis, the mitochondria are broken up into hundreds of small fragments. scattered throughout the cytoplasm

Question 21

cell cycle exit into G0

Answer 21

in multicellular systems, steps in the cell cycle are closely monitored to maintain the integrity of the genome and to coordinate individual cell division with a larger system

• in mammals, most of the cells in our body have entered into G0 and will never re-enter the cell cycle
• these cells are terminally differentiated and will eventually undergo regulated cell death or apoptosis
• replacement of the lost cells will depend upon cell division of the associated adult SC, followed by differentiation
• such post-mitotic cells include RBC, nerve cells and muscle cells
• other cells will withdraw. temporarily from the cell cycle, but will re-enter G1 when they receive. the appropriate signal

Question 22

vertebrate cell cycle

Answer 22

• there are multiple cyclin and CDK variants that are used to control the transitions between cell cycle phases
• according to the classical model of cell cycle control, D-type cyclins and CDK4 or CDK 6 regulate events in. early G1 phase, cyclin E-CDK2 triggers S-phase cyclin A-CDK2 and cyclin A-CDK1 regulate the completion of S-phase and CDK1 -cyclin B is responsible for mitosis

Question 23

regulation of entry into G1

Answer 23

in G0, there is no cyclin or CDK expression

• re-entry into the cell cycle requires the expression of D-type cyclins and CDK 4 or CDK 6
• mitogens or GFs. are signalling molecules that induce cell division. by causing. the expression of the G1 cyclin/CDK
• the restriction point (RP) is an important regulatory checkpoint in the cell cycle
• it is a time late in G1 when passage through the cell cycle becomes independent of the presence of the mitogen
• this means that a cell will continue into S-phase despite. removal or degradation of the mitogen

Question 24

transcriptional changes with entry into cell cycle

Answer 24

patterns of gene expression can be divided into 2. categories: early and late response genes

• soon after the addition of the nitrogen, early gene expression begins
• this peaks at about 1 hr and then declines to be followed by delayed gene expression

Question 25

early response gene expression

Answer 25

• regulated by TFs that are already present in. the cell but that need to be activated by phosphorylation
• this includes the SRF and TCF TFs that are activated directly or indirectly by MAP kinase, the endpoint of the r RTK signal transduction pathway
• this allows a rapid response to the addition. of GF such as EGF (epidermal growth factor)
• early response genes code for another set of TFs, c-fos and c-jun
• after transcription, translation, protein modification and folding, the early response TFs activate transcription of the delayed response gene

Question 26

delayed response gene expression

Answer 26

the delayed response genes include more TFs such as E2F

• other delayed genes encode cyclin D, cyclin E CDK2, 3 and 6
• CDK 4 and CDK 6 and the D-type cyclins are expressed first, followed by cyclin E and CDK2
• expression of some delayed response genes such as those for the cyclin D/CDK complex, must be maintained until the cell passes through the restriction point

Question 27

early gene expression regulation

Answer 27

• ealry gene expression is regulated by MAP kinase, the culmination. of the tryosine kinase receptor activation
• SRF and TCF are TFs that turn on early response gene transcription
• since these proteins are already present in the G0 cell, and only require phosphorylation, expression of the early response mRNAs is not affected by inhibitors of protein synthesis

Question 28

c-fos

Answer 28

is an early response protein / TF that will activate delayed gene expression
-intum, c-fos (and other TFs) induce the expression of. the cyclin/CDKs required for cell division

Question 29

blocking entry into G1

Answer 29

• delayed response genes are dependent upon the prior expression of the early response genes
• this is demonstrated by inhibiting protein synthesis at the same time that mitogens are added
• translation inhibitors have no effect on the initiation of early gene expression
• the TFs controlling early gene expression. are already in the cell and only need to be activated by phosphorylation
• as a result, a normal increase in normal mRNA [ ] is seen
• in contrast, turning off early response genes depends upon the expression and translation of transcriptional inhibitors encoding by early response genes, so the levels of early gene mRNAs remain high
• while early response gene transcription occurs there is no translation
• since the early response gene encode TFs necessary for delayed response gene expression
• these later genes are never transcribed

Question 30

Cyclin D and cyclin E

Answer 30

• passage through the restriction point is correlated with the end of cyclin D/CDK4-6 activity and the beginning of cyclin E/CDK 2 expression
• each cyclin CDK is responsible. for inducing the expression of the next cyclin CDK in the cycle

Question 31

functional definition of the restriction point (RP)

Answer 31

• the addition of the GF (mitogen) induces exit from G0. and entry into G1
• mitogen removal before RP results in the decrease in cyclin D/CDK 4-6 levels, a failure to. proceed into S-phase and retreating back into G0
• mitogen removal after RP: once the RP has passed, cyclin E/CDK 2 expression is sufficiently high that a decrease in cyclin D/CDK 4-6 will have no effect into progression into S-phase and the completion of cell division

Question 32

before passage through RP

Answer 32

• all of the necessary components of the RP are delayed response proteins
• E2F is a TF that regulates the expression of genes required in s-phase, these include genes coding for proteins required for nucleotide synthesis, DNA repair, DNA replication. and the ensuing cell cycle regulators CDK 1 and cdc25
• initially, E2F is inactivated by association with an inhibitory protein called the retinoblastoma protein (Rb)
• Rb is called a tumor suppressor because it has the ability to inhibit cell division
• inactivation of E2F inhibits cell cycle progression
• phosphorylation of the regulatory protein Rb releases E2F
• the cyclin D/CDK kinase phosphorylates Rb and releases E2F, however cyclin D levels are tightly regulated by the presence of the mitogen
• if the mitogen is removed before the RP, cyclin D levels decrease and Rb is phosphorylated by phosphatases in the cell
• the cell retreats back into early G1 or G0

Question 33

after passage through RP

Answer 33

• active E2F. induces expression of cyclin E/CDK2
• cyclin E/CDK2 targets Rb for phosphorylation, levels are not directly dependent on maintaining mitogen [ ]
• once enough E2F is activated, it cannot only maintain its active state by inducing expression of cyclin E/CDK2 but it is also able to induce expression of the E2F gene itself
• this (+) feedback loop is associated with passage through the RP
• even if the mitogen is removed, and cyclin D expression goes down, levels of cyclin E are maintained and the cell proceeds, uninhibited into s-phase

Question 34

cell cycle checkpoints

Answer 34

• while the RP ensures that the cell is ready for the cell cycle before committing to progression into S-phase there are a number of cell cycle checkpoints that monitor the integrity of the cell cycle in G1, S-phase, G2 and in mitosis
• these checkpoints recognize errors and delayed progression through the cell cycle until the error is corrected
• checkpoints look for damaged DNA, unreplicated DNA, assembly of the mitotic spindle and chromosome attachment at metaphase and completion of anaphase
• if the error cannot be corrected, then the cell will undergo apoptosis or regulated cell death
• in this way, a multicellular organism can rid itself of damaged cells

Question 35

DNA damage checkpoints

Answer 35

• DNA damage can occur at any time in the cell cycle, damage checkpoints are found in all cell phases
• 2 typical types of damage
  1) ionizing radiation creates double-stranded DNA breaks
  2) ultraviolet radiation creates thymine dimers
• there are 2 proteins that can recognize and bind to the damage DNA
• ATR is specific for UV damage while ATM is specific for double stranded breaks

Question 36

ATR pathway: inactivation of cdc25

Answer 36

• ATR is a kinase that is activated when bound to thymine dimers in the. affected DNA molecules
• the target of ATR is Chk1
• active phosphorylated Chk1 protein is itself a kinase
• a target of Chk1 kinase is cdc25, a regulator of CDK activity
• cdc25 is one of the proteins that regulates MPF activity at the G2 to M-phase. transition
• cdc25 phosphatase reverses the inhibitory phosphorylation at tyrosine 15 on cdc2 of MPF
• when chk1 phosphorylates cdc25, it is inactive
• as a result, MPF is not activated and this arrests the cell G2
• a long term effect of cdc25 phosphorylation is that it is sequestered in the cytosol through association with a cytosol adaptor protein called 14-3-3
• thymine dimers can occur different points in the cell cycle
• in each case, the cell cycle delay is determined by the inactivation of cdc25 and the failure to activate the cyclin/CDK associated with that particular phase
• once the damage is repaired, ATR dissociates from the DNA and is. inactivated
• this reverses the pathway, allowing progression through the checkpoint

Question 37

DNA replication checkpoint

Answer 37

the DNA replication checkpoint is also monitored through ATR

• this is because incomplete replication forks look like damaged DNA and are bound by the ATR protein
• activation of the ATR pathway and therefore inactivation of MPF delays entry into mitosis until DNA replication is complete

Question 38

ATM pathway: activation of p53

Answer 38

• ATM kinase recognizes and binds to double stranded-breaks in the DNA molecule, binding to its substrate activates ATM
• the target of the active ATM is the Chk 2 kinase
• phosphorylated Chk2 is active and its target include the. p53 protein
• p53 is a very unstable protein
• after translation it is almost immediately ubiquitinylated and degraded, however if it is phosphorylated, it becomes very stable
• p53 is a TF that activates transcription of many target genes
• one of the target genes is p21
• p21 protein is a. general cyclin/CDK inhibitor
• if the double stranded break has occurred in G1, p21 will bind to G1 cyclin CDK and inhibits its activity, causing the cell cycle to stall in G1
• this cell cycle delay gives the cell time to repair the damage, and in fact other targets of p53 include cell cycle repair genes
• once the damage is repaired, ATM dissociates from the DNA and is inactivated
• this reverses the pathway, allowing progression through the checkpoint

Question 39

targets of p53

Answer 39

p53 is very unstable and is rarely active simply because it is very quickly polyubiquitinylated

• once phosphorylated, stable p53 can activate the expression of many genes including the cell cycle inhibitor p21 (CIP) and DNA repair genes
• the goal of the cell cycle arrest is to grant the cell enough time and resources to fix the damage
• however if the DNA damage cannot be repaired, the organism needs to get rid of the faulty cell

Question 40

what happens if the damage is not repaired

Answer 40

perpetual activation of p53 will eventually induce the expression of pro-apoptotic genes and cause cell death
-if the cell cannot be fixed, it will die

Question 41

cell cycle arrest

Answer 41

can occur in any cell cycle phase

• DNA damage checkpoints are monitored by the ATM and ATR proteins
• DNA damage can occur at any point in the cell cycle, and the mechanism for the cell cycle delay is similar
• the checkpoint will function either through the activator of p53 and the synthesis of cell cycle inhibitors such as p21 (CIP) or through the inactivation of cdc25 which releases inhibitory phosphorylation on cyclin/CDK complexes
• in all cases, the checkpoint is simply providing time for the cell to repair the damage
• once the damage is corrected, the checkpoint is released or passed

Question 42

SAC

Answer 42

other steps in the cell cycle that must be monitored to ensure the integrity of the genome are the transitions from metaphase to anaphase and exit from the cell cycle

• the SAC prevents entry into anaphase prior to the proper association of chromosomes with the mitotic spindle
• even if just one chromosome is not attached, the cell. will delay in metaphase
• kinetochore proteins are found at the centromeres of replicated chromosomes
• it is the association of the kinetochore proteins to the microtubules of the mitotic spindle that must be detected

Question 43

identification of the components of the SAC: successful checkpoint arrest
-normal disjunction

Answer 43

• identified through genetic screen for mutations that disrupted the checkpoint
• in this experiment, cells were treated with a chemical called benomyl, which destabilizes microtubules and causes a cell cycle arrest b/c none of the chromosomes will be attached to spindle microtubule
• after benomyl is removed, wild type cells will reassemble a spindle and proceed into metaphase and anaphase
• after a successful checkpoint arrest, the cell corrects the problem and can enter anaphase

Question 44

nondisjunction in SAC mutant

Answer 44

• same experiment performed on cells carrying random mutations
• screen was designed to look for cells that proceeded into anaphase despite a failure to attach chromosomes to a mitotic spindle
• this would indicate that the SAC was not functioning
• this example, NDJ results
• sister chromatid separation of the unattended chromosome occurs, it splits in the 2 constituent sister chromatids, but because they are not attached to the spindle, the chromatids are not segregated to opposite poles
• results in NDJ and aneuploidy
• one daughter cell will have an extra copy of one chromosome, the other will be missing a homolog

Question 45

Mad2

Answer 45

• key regulator of the SAC
• inactivates APC-cdc20-
• in turn, in the absence of APC cdc-2o, the securin protein remains. in tact and prevents activation of separase
• as long as separate is inactive, sister chromatid separation cannot occur
• loss of function mutations in Mad2 will cause the cell to proceed into anaphase prematurely

Question 46

Mad2 open conformation

Answer 46

• newly synthesized mad2 is in the open conformation
• in the open conformation associates with the kinetochores of the chromosome that are not attached to the microtubules
• also associated with the unattached chromosomes is a protein called mad1
• when mad 1 and mad 2 associated together mad 2 is converted from open to closed

Question 47

closed mad 2 conformation

Answer 47

• leaves the chromosome
• interacts directly with cdc20
• when bound to mad 2, cdc20 cannot associate with the anaphase promoting complex (APC)
• as long as APC is inactive, cell remains in metaphase

Question 48

cdc20 open and closed

Answer 48

• closed conformation of cdc20 can be passed from one cdc20 protein to another
• when cdc20 in the open conformation and cdc20 in the closed conformation interact with one another, both assume the closed conformation
• maintains a pool of closed mad 2
• similar to prion proteins

Question 49

activation of SAC

Answer 49

as long as one kinetochore is not attached to the mitotic spindle, mad2 is converted quickly to the closed conformation
-mad2 binding to cdc20 keeps APC inactive and prevents anaphase

Question 50

passing the SAC

Answer 50

-as soon as every replicated chromosome achieves bipolar attachment to the mitotic spindle and both kinetochores are attached to microtubules, the mad1 and mad 2 proteins are released from the kinetochores
-binding of p31 to the closed conformation of mad 2 sequesters the complex away from the chromosomes, but also promotes a change in mad 2 confirmation back. to the open state
-mad2 in the open conformation has a much lower affinity for cdc20 in comparison to mad 2 in the closed conformation
0checkpoint is inactivated
-as a result, cdc20 is released and is now free to associate with APC
-APC-cdc20 is activated targeting securin for degradation and releasing the inhibition on separase
-it. is the activation of separate that initiates sister chromatid separation and therefore anaphase

Question 51

MEN

Answer 51

• another checkpoint
• chromosome segregation checkpoint
• in order to exit from mitosis, a series of events must be completed, not the least of which is the proper segregation of sister chromatids in anaphase
• the events associated with exit from mitosis include the reassembly of the nuclear envelope, chromosome condensation, re-assembly of mitochondrial Golgi and ER networks and disassembly of the mitotic spindle
• these events all require the inactivation of MPF which requires the activity of APC Cdh1 which targets cyclin B for degradation
• therefore the goal of the MEN is to monitor completion is to monitor completion of anaphase and activate APC-cdh1
• cdh1 is. inactive and unable to bind to. APC as long as it is phosphorylated (by MPF)
• dephosphorylation of cdh1 will require the activation of a phosphatase called cdc14
• key step of MEN is to activate cdc14

Question 52

cdc14 phosphatase

Answer 52

• studies of the MEN have occurred using budding yeast as a model system
• during interphase and early mitosis, cdc14 is sequestered within the nucleolus
• sequestered cdc14 is inactive since it cannot reach its target substrates
• release and activation. of cdc14 allows exit from mitosis
• as long as the sister chromatids have no segregated apart from one another, cdc14 remains in the intact nucleolus
• once anaphase occurs, cdc14 is released and activated

Question 53

cdc14 before anaphase

Answer 53

• in metaphase of a budding yeast cell, a mother cell and its daughter bud are visible
• replicated mitotic chromosomes are associated with a mitotic spindle that is aligned with bud formation
• the poles of the mitotic spindle called spindle pole bodies (SPB) in yeast are arranged so that one is t the bottom of the mother cell and the other is at the boundary with the daughter bud
• TEM1 is a small monomeric g-protein or GTPase
• Tem 1 is associated with the SPB that is. closest to the daughter bud
• also associated with the daughter SPB is a gapprotein called TEM1-gap
• this gap protein or GTPase accelerating protein, keep STEM 1 in the GDP bound form
• there is also a tem1-GEF, or guanine exchange factor, however this is associated with the membrane of the daughter cell and is not near the Tem1 protein while the cell is in metaphase
• TEM1 bound to GDP is inactive
• as long as TEM1-GDP is inactive, cdc14 remains in the nucleolus

Question 54

cdc14 anaphase

Answer 54

• at anaphase, the mitotic spindle elongates, bringing TEM1 closer to the membrane of the daughter bud
• this is where TEM 1 GEF is found
• the TEM1-GEF exchanges the GDP in the TEM 1 nucleotide binding site for GTP thus activating TEM 1
• the activation of TEM1 allows passage through the chromosome segregation checkpoint and leads to the release and activation of cdc14
• activated cdc14 phosphatase dephosphorylation cdh1
• dephosphorylayion cdh1 activates APC and polyubiquitinates cyclin B
• w/o cyclin B MPF is inactivated and all of the events of telophase and exit from mitosis can occur

Question 55

mitotic arrest

Answer 55

if chromosome segregation and anaphase do not occur, the cell cannot pass through the chromosome degradation checkpoint and the MEN is not initiated

• a failure to initiate the MEN can be induced by reorienting the mitotic spindle
• the spindle has been rotated by 90%
• now when anaphase occurs, the sister chromatids separate away from one another and. the spindle poles move apart, but the TEM 1 never enters into the daughter bud
• as a result, TEM1 protein is never proximal to TEM1-GEF and instead, TEM1 remains in the inactive GDP bound form
• without passing this checkpoint, cdc14 remains in the nucleolus, cdh1 remains inactive and MPF remains active
• the cell is unable to complete any of the events associated with telophase. and exit from mitosis

Question 56

inhibition of s-phase by cdc14

Answer 56

• a primary target of. cdc14 is the cdh1 protein
• dephosphorylation of cdh1 enables inactivation. of MPF through degradation of cyclin B
• another target of cdc14 is the sic1 protein
• by keeping sic 1 phosphorylated, the cdc13 phosphatase can prevent premature entry into the cell cycle
• unphosphorylated sic1 binds to the s-phase. cyclin/CDK and inhibits progression into s-phase
• phosphorylation of cdc14 by G1 cyclin/CDK inactivates cdc14 and allows phosphorylation of sic 1
• phosphorylated sic 1 is a target of the SCF E3 ubiquitin ligase
• degradation of Sic1 frees s-phase cyclin/CDK and allows entry into the cell cycle