Cell Cycle

Question 1

most cells in the body are not proliferating

Answer 1

-these cells have exited the cycle and are in G0 or quiescence
-Ex. neurons, heart cells, muscle cells

Question 2

some cells are quiescent (in G0) but can enter G1 when stimulated

Answer 2

liver cells and lymphocytes

Question 3

opposite behavior is seen in ‘labile’ (stem) cells, which never enter G0

Answer 3

-examples include stem cells in bone marrow, gut epithelium, skin epithelium, and hair follicles
-labile cell types are the most affected by chemotherapy since they’re always proliferating

Question 4

cell division is a cycle

Answer 4

four sequential stages:
G1 –> S –> G2 –> M
stages of mitosis:
prophase –> prometaphase –> metaphase –> anaphase –> telophase –> cytokinesis
-a checkpoint occurs when you exit metaphase to anaphase –> metaphase is when the chromosomes are lined up and doubled on mitotic spindle and anaphase is when they start to separate
-mechanisms are conserved in all eukaryotes

Question 5

cell cycle fundamentals

Answer 5

-one of the 2 daughter cells and it needs to get bigger by duplicating the chromosomes and separating the chromosome pairs into 2 sides and break the big cell in half
grow big first –> divide –> separate –> divide the cell

Question 6

four stages of the cell cycle (somatic cells)

Answer 6

-arrows proportional to the amount of time eukaryotes spend in each phase
-G1 and S are long, G2 could be long but mainly variable, and mitosis is very short
-interphase just means not mitotic- when you look at cells under a microscope, it’s obvious they’re going through mitosis

Question 7

first stage G1 (gap 1)

Answer 7

-long and slow- cells are making vital decision of whether to divide or not
-go/no go decision point and cells monitor signals from outside, growth factors, fibroblast growth factors –> all the growth factors trigger signalling pathways
-also monitoring tissue integrity- if they’re epithelial cells, they’re supposed to be in tight contact with neighbors on all sides- anchorage points let them know all is well
-cancer cells- when they break these contacts, they don’t die b/c they’re already bad at signalling and signalling control
-single-celled organisms like yeast are mainly monitoring nutrient availability

Question 8

S phase (DNA synthesis)

Answer 8

-stage where entire chromosomes are replicated
-you can separate cells that have more than the usual amount of DNA using FACs –> lets you separate cells into different stages of the cell cycle without chemistry or inhibitors
-you get a tube that’s filled almost exclusively with GI cells, cells that have fully replicated are in G2/M phases with twice as much DNA
-everything with the amount of DNA in between normal and double is in the middle

Question 9

megakaryotes

Answer 9

huge by design- they duplicate, make 2 nuclei, but never divide the cell –> cells become enormously large –> have to take sections of cytoplasm off by allowing multiple S phases without mitosis

Question 10

G2 phase

Answer 10

-length is highly variable even from cell to cell
-monitoring DNA completion –> was it really completed? has it grown enough mass to be double its starting size? also looks for DNA damage
-all interphase cells are detecting DNA damage and triggering the wait/pause if need be

Question 11

amoeba experiment with G2

Answer 11

-took amoeba and watched them grow to get bigger and divide into two –> let them grow then pinch off part of the cytoplasm and they get too small to divide

Question 12

mitosis- fast phase

Answer 12

-if you’re not big enough, gigantic chromosomes when they start separating the cell has to be big enough to get them out of the middle of the cell (danger zone)
-interphase- microtubules and DNA
-prophase- DNA starts looking grainy since you have the NE starting to disassemble and chromosomes are starting to condense
-prometaphase- stage between prophase and metaphase
-metaphase- chromosomes all lined up on the mitotic spindle and this is where they will stay –> at the metaphase is arrest point
-anaphase- when permission is given, the chromosomes start separating with early separation to go towards poles in original position and then poles start moving away from the danger zone (anaphase B)
-telophase- when the microtubules are all lined up and at some point cytokinesis occurs- plasma membranes are being pulled together

Question 13

how does the nucleus disassemble during mitosis?

Answer 13

-phosphorylation of targets like lamins, NPC proteins, and nuclear membrane proteins
-make proteins let go of each other
-phosphatase comes in to clean off the phosphates and allows re-assembly of components
-starts to happen in anaphase and telophase –> as chromosomes are being pulled apart

Question 14

G0 (quiescence) is part of G1 logic

Answer 14

if cell is fated to not divide anymore, it actively chooses G0 (purposeful exit from proliferation pathway)

Question 15

total time per cycle can vary

Answer 15

-fastest dividing human somatic cells like intestinal epithelial cells is ~12 hour total cell cycle
-growing fibroblasts is a 20 hour cell cycle
-variable timing helped scientists figure system out

Question 16

early embryos have a super fast, simple cycle

Answer 16

-S phase and mitosis only
-no G1/G2 and simplicity was essential to determining system

Question 17

how fast replication takes place depends on when you fire origins of replication

Answer 17

early embryos are firing them all at once –> how you replicate large chromosomes so quickly

Question 18

proliferation is fine-tuned to the needs of each cell and tissue

Answer 18

-fingerprint Ex.- first layer of cells is stem cells that are actively dividing and give rise to 2 cells
-one stays down and the other goes up and is allowed to keep dividing and creating a lot of cells that get pushed up in the process of replicating
–> stop dividing and terminally differentiate and boot nucleus to become dead, waterproof, and connected cells

Question 19

how do cells know what phase they’re in? or when it’s safe to move on?

Answer 19

-completion of key events Is monitored by checkpoints, which block the key transition until the checkpoint is happy (no more distress signals coming in)
-b/c this is a cycle, cells with problems can and will arrest (accumulate) at the next checkpoint to block their entry into the next phase
–> if the problem isn’t solvable, decision is made to go to cell death

Question 20

1st major checkpoint: G1-to-S transition or the go/no go

Answer 20

decision is made when it looks like the cells are still in G1

Question 21

what is the 2nd checkpoint?

Answer 21

G2/M transition

Question 22

what is a weak checkpoint?

Answer 22

metaphase-to-anaphase- cancer promoting event

Question 23

G1-to-S “restriction point”

Answer 23

-biochemical transition in S phase
-if it’s a divide, cells will get past transition point and enter S phase

Question 24

entire system of checkpoints is controlled by cyclin-dependent kinases (CDKs)

Answer 24

-controlling the transitions
-in the case of S phase, there’s a CDK riding the polymerase complex- maintaining ability to say stop the locomotive here b/c we have DNA damage ahead
-you enter the next phase of the cell cycle if you activate the right CDKs and how they do the entry is phosphorylating targets

Question 25

what are kinases?

Answer 25

very potent enzymes --> if you can't kill them, you'll stay in that next state forever

Question 26

what activates the next phase of transition?

Answer 26

triggered by the activation of the cyclin dependent kinases (CDKs) and need to be inactivated by destroying their cyclin (ubiquitin dependent proteolysis)

Question 27

overview of cell cycle biochemistry

Answer 27

-each stage is driven by one or more CDK proteins- CDK must bind a cyclin protein to be active -cyclins determine target specificity of the CDK -cyclins bound to a CDK can determine who that kinase phosphorylates -cyclins need to accumulate at appropriate times then be destroyed- they have to trigger their own assassins

Question 28

genome encodes multiple CDKs and cyclins, each expressed at specific times during the somatic cell cycle

Answer 28

-G1 CDK -G1/S CDK -S CDK -M CDK

Question 29

CDKs phosphorylate specific target proteins that achieve next stage

Answer 29

-for S phase, only one target: retinoblastoma (Rb)- one protein responsible for guarding entry to S phase- suppress going into a new round of cell division -for mitosis, CDKs target many different proteins that have to be phosphorylated to achieve the change in shape of entire cell, letting go of most of the adhesion complex -for anaphase, CDKs target M-phase cyclin and securin, which holds sister chromosomes together

Question 30

G1/S checkpoint or restriction site

Answer 30

-signaling proteins go into the nucleus and turn on genes --> cells start dividing -first genes that get turned on in downstream pathway include G1 cyclin, G1 CDK, CDK inhibitor p27, and early transcription factors --> they help activate later genes like later phase G1/S cyclin and G1/S CDK -slow b/c you have to turn on G1 phase genes to help turn on G1/S phase genes -entire army of CDKs is needed to overcome Rb protein and block going into S phase -those that overcome Rb are irreversibly committed to a new division cycle --> happens at G1/S phase transition site

Question 31

two waves of CDKs combine to hyper-phosphorylate Rb and overcome G1/S checkpoint

Answer 31

-1st wave in mid-G1: several G1 CDKS- can't phosphorylate Rb enough -2nd wave in late G1: G1/S CDK -8-10 phosophorylations that each Rb protein needs to finally let go of its target -both waves of CDKs can make Rb let go of its target

Question 32

why is Rb so important?

Answer 32

-E2F class transcription factors, which obligate heterodimers with Dp class transcription factors -2 waves together turn on all the genes that help you replicate DNA -E2F-dependent transcription factors sitting on their gene and another with Rb bound (Rb binds chromatin regulators to keep gene OFF) -when Rb gets phosphorylated, the Rb folds up and goes out of business temporarily --> lets go of transcription factors to turn genes ON -turning on all of the E2FB genes and you have all the genes you need to be in S phase

Question 33

E2F/DP-dependent gene products also reinforce S phase

Answer 33

-inactivated RB lets go and starts making other genes -another S phase CDK that's being turned on amongest the replication genes is special b/c it goes back and is activated and acquires a cyclin and maintains being in S phase by sitting on DNA replication complexes --> this is how the cell can sense DNA damage- CDK complex on replication engines and feedback to them to stop CDK activity and stop the engine

Question 34

Retinoblastoma- homozygous loss of Rb

Answer 34

-cells have no functional Rb --> never slow/pause or wait for the right signals to go into S phase and cells keep overproliferating -causes numerous tumors in the eyes -Rb is important tumor suppressor- 30% of human tumors lack Rb function

Question 35

who do Rb null children have tumors ONLY in their eyes?

Answer 35

-many important genes, including Rb, were duplicated during evolution -humans have 3 genes: Rb, p130, and p107 -cells that get tumors due to an Rb mutation, it will only be the cell types that rely exclusively on the Rb for this control

Question 36

the G1-to-S transition MOST resembles which event?

Answer 36

ski jump- once the cells decide to go into S phae it is irreversible- go/no go event

Question 37

G2/M checkpoint

Answer 37

-not satisfied if there's unreplicated DNA, cell is too small, or DNA damage -this transition to that entry to mitosis was so obvious

Question 38

cell division cycle genes, proteins, and mechanisms are highly conserved in eukaryotes

Answer 38

-baker's and brewer's yeast- cell division cycle (cdc) mutants and checkpoint control -surf clams- cyclin proteins -clawed toads- mitosis-promting factor (cyclin-dependent kinases)

Question 39

cyclins were discovered as proteins that first accumulated in 'interphase' then disappeared during mitosis

Answer 39

-put S35 methionine and let them watch newly synthesized proteins -when they were looking at the samples under the microscope, they could see when the nucleus was gone and when it was rebuilding again -took samples from embryos at different stages and ran them out on a blot -they found a band accumulating then disappeared then accumulated then disappeared -accumulation correlated with being in mitosis and the control protein was not fluctuating -called protein cyclin since it was cycling with accumulating then disappearing

Question 40

why is cyclin needed to activate the kinase?

Answer 40

-CDK has cyclin bound and opens the CDK arm (T loop gets moved up) and when the arm is up, the ATP binding site is exposed for the kinase -kinase works by binding ATP and using third phosphate to stick it on the target protein and lets ADP out -if it cannot bind ATP, it is not active as a kinase -kinases trigger conformational change in CDK subunit that opens the armpit and allows ATP to bind --> active kinase

Question 41

CDK activating kinase (CAK)

Answer 41

-armpit gets exposed with T loop up and CAK comes in to phosphorylate the up position of arm to keep site open --> kinase is now fully active and stabilized by CAK -you don't want to just jump into mitosis --> has to be controlled

Question 42

CDK is immediately MUZZLED by wee1

Answer 42

-wee1 phosphorylates and blocks the enzyme's active site -M phase kinase with the elbow up and ATP site open and stabilized but wee1 kinase phosphorylates and blocks ATP from binding --> stays there until checkpoint is satisfied

Question 43

when G2/M checkpoint is satisfied cdc25 is fully active and starts de-phosphorylating the M-CDK

Answer 43

-cdc25- takes off the phosphate group and reveals the CDK cyclin complex -M phase CDK cyclin binds inactive --> CAK stabilizes the ON position --> wee1 says no --> inactive cdc25 becomes activated and they rip off the first few phosphate groups with phosphatases -becomes M phase CDK -G2/M checkpoints mainly target (inhibit) cdc25

Question 44

feedback loops then explosively activate all the cdc25s and M-CDKs

Answer 44

-first few mitotic CDKs are feedback positive --> phosphorylates the inactive CDKs and turns them ON -activates its activator and inhibits wee1 -explosive activation of all ready-to-go M phase CDKS --> this is why mitosis happens massively and synchronously in a cell

Question 45

once M phase CDK is turned ON, it can phosphorylate its many targets

Answer 45

-condensin proteins and histone H3- contribute to chromosome condensation -lamins, NPCs, emerin- disassembles lamin filaments, nucleoskeleton, pore complexes, break apart as functional subunits -microtubule-associated proteins- enhances 'dynamic instability' of microtubules --> microtubules become dynamically unstable to capture chromosomes that are condensing -cyclin-specific Ub-ligase- complicated multi-protein complex that is a cyclin-specific Ubiquitin ligase- not instant since you don't want to destroy M-phase cyclin until you've reached metaphase arrest point and made sure all of the chromosomes are captured -metaphase is the end of biochemical mitosis b/c after you've undone all of the phosphorylations --> go back to building the nucleus

Question 46

anaphase promoting complex (APC) is a ubiquitin ligase E3 complex

Answer 46

when cells reach metaphase and all of the chromosomes have been captured, spindle checkpoint is satisfied and APC is allowed to target the M-phase cyclin to be degraded

Question 47

APC triggers anaphase by getting rid of M-phase cyclins and securins --> causes them to be degraded by proteosomes

Answer 47

-cohesin makes gigantic wishbone-shaped structures with ATPase activity- take 2 strands of DNA and encircle them -condensin is creating loops and holding loops together --> helps chromosomes condense -cohesin holds 2 chromosomes and loops around them --> gluing two sister chromosomes together

Question 48

securin stops pacman (protease) from eating the cohesin

Answer 48

by getting rid of securin, pacman chews up cohesin and chromosomes can be pulled apart

Question 49

APC stays active for awhile

Answer 49

-G1 CDK activity --> add in G1/S CDK activity --> S phase CDK activity --> G2 --> turn on M CDK activity and goes away at metaphase-anaphase transition b/c APC is turned on and you can exchange a subunit with another --> APC as a whole stays on for the rest of G1 -needs to stay on G1 to enforce no cyclins since we don't want any CDKs active in G1 until purposefully making them -that is why it is the one time in a cell's life that it is able to reassemble origins of replication --> can only assemble if there's no CDK activity

Question 50

what does the SCF-E3 ligase complex do?

Answer 50

-helps facilitate transitions by getting rid of cyclins or other proteins but only respond when proteins are doomed by being phosphorylated --> SCF ubiquitinates them and makes them be degraded -promotes exit from G1 and entry into S phase by getting rid of CDK inhibitors

Question 51

when CDKs are already active, what if something bad happens during G1, G2, or S?

Answer 51

FULLY ACTIVE cyclin-CDKs can be ARRESTED by CDK Inhibitors (CKIs)

Question 52

existence of CDK inhibitor p27- class of proteins important for cancer

Answer 52

-CDK inhibiors, including p27 -if there's a fully active CDK, CDKIs can bind to it (many to one) and stop it from being active -cell-type specific CDK inhibitors like p53

Question 53

TF p53 helps in times of trouble

Answer 53

-if it gets turned on, p21 can go in and stop any CDK complex and give cell chance to repair tissue -not a required part of the cell cycle --> there in times of trouble- sophisticated TF that makes tetramers and usually kept in the cytoplasm and destroyed -damage of various kinds cause it to be stabilized --> enter the nucleus --> turn on the right genes -leads to stopping cell cycle progression and gives cells time to repair -p53 can trigger a whole different set of genes that can lead cells to apoptosis

Question 54

what does p53 do for a cell with an issue?

Answer 54

decides to rescue or kill it

Question 55

why does loss of p53 promote cancer?

Answer 55

cells can't efficiently pause to repair especially DNA damage leading to new mutations --> why not having p53 is first in cascade of events that allow incidental mutations to accumulate

Question 56

positive and negative control of cyclin-dependent kinases

Answer 56

-kinase gets activated and can be inhibited -INK4- blocks cyclin binding and interferes with ATP hydrolysis -p27- blocks ATP binding to the active site

Question 57

cell cycle regulators

Answer 57

-when wee1 is missing, the cells just finished S phase and are in G2 --> the first one CDK gets there and mitosis begins when cells are still too small -if cdc25 is missing, you can never inhibit wee1 and cannot trigger entry into mitosis --> cell keeps growing

Question 58

checkpoint summary

Answer 58

-restriction checkpoint (G1-to-S)- checkpoint is enforced by Rb, which blocks activation of S phase genes and Rb is inactivated by G1/S-CDK-dependent hyperphosphorylation -G2/M checkpoint- controlled by pathways that monitor cell size, completion of DNA replication, and potential DNA damage prior to activating cdc25 (different cdc25 proteins work in G1 phase) -spindle checkpoint (metaphase-to-anaphase)- 'uncaptured' kinetochores arrest cell in metaphase and transition is triggered by activating Anaphase Promotion Complex -DNA damage is monitored throughout interphase- several mechanisms: DNA damage causes p53, an important tumor suppressor, to activate genes encoding CDK inhibitors to arrest progression while damage is repaired