W23

Question 1

Describe the general interaction between apoptosis and autophagy.

Answer 1

Autophagy slows apoptosis and is therefore cytoprotective. If apoptotic caspase activity breaks a threshold, then autophagy becomes inactivated. Some autophagy related genes (ATGs) take on pro-apoptotic roles when cleaved by caspases, as their function in autophagy is inhibited and some proteins adopt a new role involved in apoptosis

Question 2

What are the three major forms of autophagy?

Answer 2

Chaperone-mediated autophagy (CMA) - proteins with a particular sorting motif are pumped into the lysosome via the lysosomal receptor LAMP-2A.
Microautophagy - the lysosome engulfs a portion of the cytoplasm and degrades material.
Macroautophagy - a membrane surrounds cytoplasmic material and forms a double membrane vacuole that transports to the lysosome and fuses for material to be degraded. Can be nonspecific or specific for organelles, pathogens or misfolded protein

Question 3

What are the three stages of macroautophagy?

Answer 3

Initiation & nucleation, elongation & closing, maturation fusion & degradation

Question 4

Describe initiation and nucleation of macroautophagy.

Answer 4

Nutrients, growth factors and energy levels stimulate the protein kinase mTORC1 which promotes cell growth and inhibits the ULK1 complex involved in autophagy. In the absence of these factors, mTORC1 is inactive so cannot phosphorylate the ULK1 complex to inactivate it. The ULK1 complex is a protein kinase that translocates to the site the autophagosome is going to be generated, typically the ER. The ULK1 complex phosphorylates the lipid kinase VPS34 complex which produces phosphoinositides, mainly PI3P. PI3P forms a patch on the ER membrane to recruit soluble effector proteins

Question 5

Describe elongation and closing of macroautophagy.

Answer 5

The effectors bound to PI3P recruit the LC3 lipidation machinery. LC3 is one of several ATG8 proteins that decorate the autophagosome membrane surface, acting as a recruitment site for all the cargo to be degraded. LC3 and the other ATG8 proteins are covalently bonded to the lipid via a Gly residue at the C-terminus. This produces LC3II from ATG8 and allows the autophagosome to close and engulf its cargo for degradation

Question 6

Describe maturation, fusion and degradation of macroautophagy.

Answer 6

The autophagosome finds a lysosome, fuses with it to form an autolysosome and cargo is degraded

Question 7

Name six factors that cause apoptosis.

Answer 7

Damage to organelles, matrix detachment, cell surface death ligands, cytotoxic drugs, or growth factor withdrawal

Question 8

Describe caspases.

Answer 8

Aspartate-directed cysteine proteases, synthesised as zymogens with low intrinsic activity that, when activated, are processed into heterotetramers with two large subunits and two small subunits. Caspases cleave target proteins on the carboxyl-side of aspartic acid residues, the classical caspase site is the DEVD motif

Question 9

Describe extrinsic apoptosis.

Answer 9

Death receptors bind to death ligands which cause recruitment of adaptor proteins to the intracellular region of the receptor. The adaptor proteins bind to the receptor via death domains and also contain death effector domains. The death effector domains attach to death effector domains found within initiator caspases. With the recruitment of initiator caspases, the death-inducing signalling complex (DISC) has been formed. Initiator caspases become activated and cleave effector caspases, separating domains and removing the N terminus, which activates them to cleave target proteins in the cell. FLIP is a protein that has death effector domains but no caspase enzyme domains hence if the cell expresses enough FLIP then it can outcompete initiator caspases and extrinsic apoptosis can be prevented

Question 10

Describe intrinsic apoptosis.

Answer 10

Arises due to damage to organelles within the cell, particularly the mitochondria, and is controlled by BCL-2 family proteins. The protein Bak binds to the surface of the mitochondria and recruits the soluble cytosolic protein Bax to the mitochondrial surface. Some BH3-only proteins target Bax/Bak and cause a conformational change that forms a pore in the mitochondrial outer membrane, allowing substances to leak out form the intermembrane space. Cytochrome c is released from the mitochondria and associates with Apaf-1 via its WD40 repeats. dATP binds Apaf-1 triggering assembly of a 700-1400kDa apoptosome that recruits procaspase-9, which acts as an initiator caspase similar to caspase 8 and 10 in extrinsic apoptosis. The initiator caspase procaspase-9 activates downstream caspases that in turn cause apoptosis

Question 11

Describe how intrinsic apoptosis is regulated.

Answer 11

Bcl-2 and Bcl-xL are anti-apoptotic BCL-2 family proteins that inhibit the interaction between BH3-only proteins and Bax/Bak. Some BH3-only proteins target Bcl-2/Bcl-xL (derepression) to lower the threshold for mitochondrial outer membrane permeabilisation (MOMP)

Question 12

Describe how the intrinsic and extrinsic apoptosis pathways are linked.

Answer 12

The number of DISCs formed may be insufficient to bring about apoptosis alone, so as well as cleaving effector caspases the initiator caspases of DISC also cleaves/truncates the BH3-only protein Bid (inert) to form tBid (active). tBid is very potent in activating Bax to bring about MOMP

Question 13

Describe the difference between apoptosis, necrosis, and necroptosis.

Answer 13

Apoptosis is controlled for safe clearance by the release of anti-inflammatory cytokines by macrophages to minimise damage to surrounding tissue. Necrosis causes the cell to burst and release its toxic contents due to pro-inflammatory cytokines. Necroptosis causes the cell to burst and release toxic contents due to death receptor ligation in the absence of caspases

Question 14

Describe the necroptosis pathway.

Answer 14

When caspase-8 is inhibited, RIPK1 does not bring about apoptosis so RIPK3 activates MLKL that oligomerises and forms pores at the plasma membrane. RIPK3 and MLKL also activate the NLRP3 inflammasome, triggering caspase-1 processing and interleukin-1beta maturation. RIPK3 also activates caspase-8, which in turn contributes to NLRP3 activation and/or direct processing of IL-1beta. In some contexts, caspase-8 can suppress RIPK3-MLKL-mediated inflammasome activation.

Question 15

Describe the two steps in activation of the NLRP3 inflammasome.

Answer 15

Priming sees receptors activate NF-kB, which leads to upregulation of both NLRP3 and pro-IL-1beta. Activation involves external stimuli leading to assembly of the NLRP3 inflammasome, activating caspase-1 to cleave pro-IL-1beta to active IL-1beta. The pores created by MLKL allow IL-1beta to exit the cell and induce inflammation

Question 16

What are the three levels of control in regulation of CDK/cyclin activity?

Answer 16

Inhibitory/stimulatory phosphorylations on the CDK, CDK inhibitors, and synthesis/destruction of the cyclin subunit

Question 17

Describe CDK1 activation.

Answer 17

Upon CDK1 binding cyclin B, the T-loop within the CDK1 active site changes conformation to become partially active. It is only considered fully active when a phosphorylation event occurs on T161 of the T-loop, mediated by CDK-activating kinase (CAK). CDK1 is further regulated by the separate phosphorylation event (Y15) of the inhibitory kinase Wee1 and the activating phosphatase Cdc25. Once CDK1 is activated, it has a negative feedback loop on Wee1 and a positive feedback loop on Cdc25, encouraging more CDK1 activation

Question 18

Describe cyclin degradation via APC.

Answer 18

Cyclin ubiquitination is controlled by the anaphase promoting complex (APC). K48-linked polyubiquitin is involved in degradation by the proteasome. APC is an E3 complex with two regulatory subunits (cdh1 and cdc20). Cdc20 is active during anaphase of mitosis and targets mitotic cyclins for degradation. Cdh1 is active during G1 phase, ensuring mitotic cyclin levels remain low as not to bring about premature chromosome separation and mitosis. Cdh1 also targets Cdc20, degrading it so APC is active at the correct points of the cycle. CDK1/cyclin B phosphorylates APC during anaphase to activate it and phosphorylates the cdh1 subunit to inhibit it. Cdc14 removes the phosphate group from cdh1 during G1 to activate it

Question 19

Describe how the cell cycle is driven by cyclins.

Answer 19

Growth factors activate cyclin D/CDK4(6) to allow the progression of G1. Cyclin D/CDK4(6) phosphorylates retinablastoma protein, causing it to release the transcription factor E2F. E2F drives transcription of cyclin E, allowing the cell to move through the restriction point and commit to cell division. Late in G2, Wee1, cdc25, and CAK all interact with CDK1 eventually bringing about activation with cyclin B, causing mitosis. Cyclin B/CDK1 causes activation of APC/cdc25 which causes the destruction of cyclin B and the cell exits mitosis. APC/cdh1 then assembles and maintains low cyclin B as well as degrading cdc20

Question 20

Describe the three types of microtubule.

Answer 20

Kinetochore microtubules span from the centrosome poles to the kinetochores of sister chromatids, overlap/interpolar microtubules span from one pole and associate with microtubules of the opposing pole at the spindle equator, astral microtubules associate with the periphery of the cell

Question 21

Describe the role of kinesin-5 in mitosis.

Answer 21

Slides anti-parallel microtubules apart, playing a role in prophase and anaphase B

Question 22

Describe the role of kinesin-6 in mitosis.

Answer 22

Functions during cytokinesis to organise midzone microtubules and interacts with the cell cortex at the cleavage furrow

Question 23

Describe the role of kinesin-7 in mitosis.

Answer 23

Moves chromatids towards poles during anaphase by coupling the capture of microtubules at the kinetochore to the spindle assembly checkpoint

Question 24

Describe the role of kinesin-10 in mitosis.

Answer 24

Binds chromatid arms and pushes the chromatids towards the spindle equator (polar ejection force)

Question 25

Describe the role of kinesin-13 in mitosis.

Answer 25

Couples ATP hydrolysis to microtubule depolymerisation during anaphase A

Question 26

Describe prophase.

Answer 26

Anti-parallel microtubule sliding drives centrosome separation coordinated by BimC (kinesin-5) regulated by CDK1/cyclin B. Motor domains are bound to anti-parallel microtubules and translocate to the respective plus ends, sliding the two microtubules apart and forcing the centrosomes to either side of the nucleus. Binding of BimC to cytoplasmic dynein/dynactin at the minus end focuses microtubules together at the centrosome

Question 27

Describe prometaphase.

Answer 27

Nuclear envelope degrades and CENP-E supports bipolar attachment of microtubules to kinetochores as well as recruiting BubR1 in the spindle-assembly checkpoint pathway. Kid (kinesin-10) binds to the chromosome arms and moves towards the plus end in the direction of the spindle equator meanwhile the kinetochore microtubules depolymerise, pulling the kinetochore via CENP-E toward the spindle pole. Most sister chromatids are attached to only one spindle pole, making the process unstable. These opposing forces acting on the sister chromatids create the polar ejection force, providing tension that is detected by the spindle-assembly checkpoint machinery

Question 28

Describe metaphase.

Answer 28

The polar ejection force and chromatid cohesion is balanced as the kinetochore is bound by microtubules at both spindle poles, resulting in no net movement. Correct chromosome alignment on the spindle equator relieves the spindle-assembly checkpoint and activates APC/Cdc20. Cohesins are proteins that assemble on DNA when it is replicated and hold the identical strands of DNA together, effectively attaching sister chromatids to one another during S-phase. The presence of CDK1 stimulates APC/Cdc20 for the degradation of securin which binds separase in the inactive state, proteolysis of securin leads to loss of chromosome cohesion. Separase is activated and free to cleave cohesins, separating sister chromatids. Proteolysis of Kid via APC/Cdc20 removes the opposing centripetal force of polar ejection and facilitates anaphase onset

Question 29

Describe anaphase.

Answer 29

In anaphase A, sister chromatids are pulled towards opposing spindle poles. This is performed by dynein moving towards the microtubule minus end and MCAK (kinesin-13) depolymerises kinetochore microtubules at the plus end as well as microtubule flux contributing to the net poleward movement. In anaphase B, cortically-anchored cytoplasmic dynein pulls astral microtubules toward the minus end, thereby pulling the centrosomes apart. BimC crosslinks overlap microtubules in the midzone, sliding them apart

Question 30

Describe cytokinesis.

Answer 30

MKLP (kinesin-6) binds the remaining microtubules at the central spindle and forms the centralspindilin complex by targeting RacGAP50C/CYK-4 which is associated to Pebble/ECT2 at the cell cortex. These proteins are GEFs and GAPs for RhoA that activate the actin cytoskeleton. Plk1 phosphorylates Mklp2 and binds the phosphorylated site before phosphorylating other signalling molecules at the cleavage furrow to drive cytokinesis. These proteins accumulate in the midbody to drive cellular abscission, narrowing either side of the midbody until one side is determined the constriction zone that separates the cells