Cell Bio Quiz 7

what triggers cyclin destruction near end of M phase?

"relaxation oscillator": cyclins initiate their own destruction by triggering a negative feedback loop *responsible for controlling cell cycle* but still has checkpts

what initiates mitosis in cell cycle?

*POLO kinase* initiates phosphorylation of Cdc 25, and then positive feedback loop of cyclin/Cdk complex takes over

two advantages of cyclin-Cdk complex instead of just one protein?

1. can alter specificity of complex by changing the *cyclin* family of cyclins, so different cyclins can be used during different times during cell cycle 2. increases opportunity for regulation can eliminate/replace cyclin while leaving Cdk intact

steps for cyclin-Cdk complex activation

1. cyclin binds to Cdk 2. protein kinases phosphorylate complex activating kinase (CaK) attaches activating phosphate Wee1 attaches inhibitory phosphate 3. activating protein phosphatase (Cdc 25) removes inhibitory phosphate to give active complex

what four things does the cyclin/Cdk graph indicate?

1. cyclins (likely) activate Cdk activity 2. cyclin concentration is not the only thing regulating Cdk activity (in other words, NOT a bimolecular rxn) 3. positive feedback loop (likely) regulates Cdk activity 4. positive feedback loop (likely) regulates cyclin destruction

what three questions must the answer be yes to in order for Cdk activity to be turned on?

1. is cyclin present? 2. is activating phosphate added? 3. is inhibitory phosphate removed?

what two forces get the chromosomes lined up of the metaphase plate?

1. pull longer the kinetochore microtubule, the more tension therefore, net forces will align chromosomes in middle 2. push or "astral exclusion force" microtubules reach out from centrosome closer you are to centrosome, higher the density of microtubules easier to move away from centrosomes than toward essentially, *lowest energy position is lined up in middle*

order of events required for chromosome segregation in metaphase to anaphase transition

1. sister chromatids connected by cohesin 2. kinetochores attached to mitotic spindle in bi-orientation 3. Cdc 20 activates APC 4. APC attaches ubiquitin to securin, which releases separase 5. separase cleaves cohesin 6. chromosomes free to move to opposite poles

do Cdk or cyclin levels rise/fall during the cell cycle?

CYCLIN, not Cdk

what happens to Cdk activity over time if cyclin destruction is inhibited?

Cdk activity increases, then plateaus (when runs out of components)

what is the transcription factor responsible for starting transition from G1 to S phase?

E2F

methods other than APC for protein degradation: SCF complex

F-box proteins (acting as E3) destroy Cdk inhibitors

what would happen if fused cell in G1 w/ cell in S (as defined by DNA replication) phase? why?

G1 cell would enter S phase b/c S-cyclin *feed back* to work on G1 components

cell cycle

G1 phase: growth & nucleotide synthesis S phase: DNA replication G2 phase: preparation for mitosis M (mitosis) phase: create structure for division, segregation of stuff, & division cytokinesis: physical separation of cell multiple times throughout cycle are checkpoints

how are chromosomes separated?

G1/S/G2: centrosome replicated then, microtubules (originating from gamma-tubulin rings on centrosomes) pull apart chromosomes when interpolar microtubules contact interpolar microtubules of other centrosome, interaction stabilized motors on interpolar microtubules push away from each other (b/c plus end-directed motors) until right distance, lock down, and form cage around chromosomes sometimes, astral microtubules make contact w/ plasma membrane as anchorage kinetochore microtubules actually bind to chromosomes, line them up, and separate

what triggers chromosome condensation?

M-Cdk

what triggers nuclear envelope break down?

M-Cdk

when do cyclin concentrations/Cdk activity peak in the cell cycle: mitosis or interphase?

MITOSIS

does the cell cycle occur at the same rate in all cell types?

NO

does cell require growth factor continuously throughout cell cycle to complete division?

NO, feedback loops make growth factors unnecessary later *growth factors only required during G1*

is the kinetochore the same thing as centromere?

NO, kinetochore is assembled on centromere centromere: center region of chromosome kinetochore: complex of proteins associated with the centromere of a chromosome during mitosis, to which the kinetochore microtubules attach

are kinetochore microtubules designated in advance?

NO, simply name after attaching to kinetochore

what inhibits E2F, and thus inhibits progression through cell cycle?

Rb

how does S-Cdk activity prevent re-replication of DNA during S-phase?

S-Cdk phosphorylates Cdc6 Cdc6 attached w/ ubiquitin for degradation since Cdc6 is only synthesized during G1, unable to form origin recognition complex until next cell cycle

how many positive feedback loops are involved w/ G1-Cdk activation? where does it come from?

TWO 1. transcription of more E2F, along w/ S phase genes 2. G1/S-cyclin and S-cyclin feed back to phosphorylate and inactivate Rb

how many positive feedback loops are involved w/ cyclin/Cdk activation? where does it come from?

TWO b/c so important for strong positive feedback loop during cell cycle 1. activated complex *activates* Cdc 25, which then removes inhibitory phosphates from other complexes 2. activated complex *inhibits* Wee1, which prevents attachment of inhibitory phosphates (double negative = positive)

can E3 proteins have multiple distinct substrate proteins?

YES ex: cyclin and securin

does G1 progression pathway allow cell to sense external conditions? why or why not?

YES just by being a transcription pathway, must have sufficient energy and nutrients present

how is the cell divided?

actin/myosin ring constricts membranes exocytosis to build new membrane

how does cell prevent/inactivate cyclin-Cdk complexes (say, in an emergency)?

activation of cyclin inhibitors (in particular, *p27*)

when does self-destruction of M-Cdk occur (accounting for sharp drop of Cdk activity in graph)?

after separation of chromosomes M-Cdk activates APC that results in cohesin degradation AND M-CDK ITSELF (negative feedback loop)

just to make sure, what are three types of microtubules/their functions involved in chromosome separation during mitosis?

astral microtubules: point away from the chromosomes and sometimes make contact w/ plasma membrane as anchorage kinetochore microtubules: actually bind to chromosomes, line them up, and separate interpolar microtubles: extend half way across cell and push poles away from each other does not make contact with chromosomes (forms cage)

what would happen if fused cell in G2 w/ cell in S (as defined by DNA replication) phase? why?

both stay in current phase b/c G2 does not have any directing components

what is APC?

complex that targets proteins for degradation

what do mitotic chromosomes look like?

condensed and duplicated sister chromatids are held together by cohesin rings

is cyclin or Cdk degraded?

cyclin

what is function of Cdk?

cyclin dependent KINASE, so phosphorylates

how will increasing Cdc 25 expression affect Cdk activation?

depends on situation 1. if inactive complex, NO EFFECT b/c no activation no matter how much Cdc 25 2. if complex is even a little bit active, ACCELERATES CDK ACTIVITY b/c will trigger positive feedback loop

how does cell downregulate cyclins?

destruction of cyclin by *ubiquitin proteolysis system* catalyzed by E3 enzymes, (poly)ubiquitin marks *cyclin* for degradation by proteasome Cdk NOT degraded

speckle microscopy

does not GFP-label all tubulin in microtubule, but randomly GFP-labels a few to see dynamics

mitotic spindle

football-shaped structure formed of microtubules and associated proteins that is involved in movement of chromosomes during cell division

why does cyclin/Cdk activation involve positive feedback loops?

for many cell cycle events, a yes/no decision has to be made do it to completion, or not at all allows for quick activation & then "locked" activation mode

how does DNA replication at an origin happen *once and only once* during cell cycle?

hundreds of thousands of origin recognition complexes on DNA SIMULTANEOUSLY activated by S-Cdk complexes by process of licensing: licensing factor Cdc 6 required to build origin recognition complex, but also prevent firing (safe assembly) S-Cdk phosphorylates Cdc 6, which causes it to be ubiquited and degraded origin recognition complex fires DNA replication licensing b/c cell *only makes Cdc 6 in G1*, so cannot replicate until next G1/S phase

how can we think of advancement of cell cycle?

in one phase of cell cycle, cell prepares for next phase, but also blocks next and previous phase from happening

how does a cell get chromosomes attached to kinetochore microtubules that are connected to opposite poles?

interaction will not be stable unless tension on each kinetochore coming from opposite poles multiple kinetochore microtubules b/c one is not strong enough

what is everything other than M phase called?

interphase: G1, S, and G2

do events of cell division have to occur in a particular order?

major events MUST occur in order each cell division if not, DNA replication/separation will not proceed correctly *order we learned is only order observed in life on earth*

what happens if, in addition to normal E2F, a cell expresses a lot of E2F that cannot bind DNA?

mutant E2F acts as double negative still able to bind Rb, so can remove Rb from normal E2F G1/S-Cdk activation *accelerated*

is cytoplasmic material distributed equally to the two daughter cells during cell division?

not in all cases in some cells (particularly stem cells), proteins and RNA are asymmetrically distributed so that two cells have different functions simple organisms also undergo asymmetric distribution

how is nuclear envelope broken down?

phosphorylation of nuclear pore proteins and lamins

microtubule dynamics during mitosis

prophase: capped minus end by gamma tubulin ring plus end growing and retracting while trying to stabilize interactions (between opposite interpolar microtubules and w/ kinetochores) metaphase: gaining on plus end, loss at minus end (treadmilling) anaphase A: shortening of kinetochore microtubules loss from both minus and plus end anaphase B: poles pushed apart from kinesin motors on interpolar microtubules and dyneins on astral microtubules

condensed version of what we need to know about stages of mitosis

prophase: getting ready 1. condense chromosomes 2. break down nuclear envelope (if necessary) 3. build machinery to segregate chromosomes 4. start moving chromosomes metaphase: lined up and ready to divide anaphase: separation

what actively puts ubiquitin onto cyclin?

protein complex called APC (acting as E3) APC regulated by Cdc 20

flourescence-activated cell sorter/counter

provides a method for sorting a heterogeneous mixture of cells using light-based markers, one cell at a time

during DNA replication, licensing factors ensure that what is used only once per cell cycle?

replication origin

mitogen

signal that initiates G1, and thus cell division when a mitogen binds to its receptor on plasma membrane, an intracellular signaling pathway activates G1-Cdk complex then, G1-Cdk complex phosphorylates Rb to *inactivate* inactivation of Rb allows E2F to bind to DNA, allowing transcription of S phase genes

if POLO kinase is active, and POLO kinase phosphorylation of Cdc25 activates Cdc25 even w/o phosphorylation of Cdc25 by M-Cdk, how would the cell cycle be affected by decreasing amount of POLO kinase?

significantly slow down

if CAK would normally phosphorylate almost all of M-Cdks in a cell, how would the cell cycle affected by decreasing the amount of CAK?

significantly slow down b/c less phosphorylation w/ activating protein

if Wee-1 would normally phosphorylate almost all of M-Cdks in a cell, how would the cell cycle be affected by increasing amount of Wee-1?

significantly slow down b/c since Wee-1 is opposed by Cdc25 phosphatase (unlike CAK), will be able to continue phosphorylating w/ inhibitory phosphate -> Cdc25 will have to remove -> slow down

if Wee-1 would normally phosphorylate almost all of M-Cdks in a cell, how would the cell cycle be affected by decreasing amount of Wee-1?

significantly speed up b/c no longer have to worry about inhibitory phosphate - just activating phosphate

if CAK would normally phosphorylate almost all of M-Cdks in a cell, how would the cell cycle affected by increasing the amount of CAK?

stay the same b/c everything that can be phosphorylated w/ activating protein is already phosphorylated

why does kinetochore use ring structure w/ motors instead of just connecting kinetochore microtubule end on w/ capping proteins?

take advantage of microtubule dynamics

gene expression control 101

transcription factor binds to DNA, causing transcription of mRNA mRNA goes on to synthesize proteins

what leads to sharp decline in cyclin levels during cell cycle?

ubiquitylation and degradation of cyclin

how does cell separate sister chromatids? what activates APC?

when cell is not ready protease called separase inhibited by securin when cell is ready for cytokinesis, Cdc 20 activates APC to do ubiquitylation of securin (which causes degradation) separase allowed to cleave cohesin