Cytoskeleton
What are the 3 main components of the cytoskeleton and what are their functions?
Intermediate filaments: maintain shape (allow cells to withstand mechanical stress), organelle organization and cell:cell adhesions
Microtubules: crucial role in organization, transmit information, regulate cell division and allow cilia and flagella movement
Actin filaments: essential for maintaining shape & movement; control non cilia/flagella cell movement
What is the Sliding Filament Theory?
During contraction, the sarcomere shortens as the filaments slide past one another (i.e. during contraction).
What are the 3 types of microtubules present during M-phase and how do they differ?
1) Kinetochore/spindle microtubules: attach @ kinetochore
2) Astral microtubules: short, radiate outwards towards cell periphery
3) Interpolar microtubules: long, project towards center of cell, not attached to kinetochores
How and when (i.e. stages) is the cell cycle regulated?
By cyclin-dependent kinases (CDKs), which are regulated by cyclins.
Cyclin/CDK complexes drive & regulate cell cycle at G1, G1 -> S transition, S, and G2 -> M transition stages.
Compare and contrast necrosis & apoptosis.
Necrosis: cell swells, membrane ruptures, cytosol spills out, triggers inflammatory response
Apoptosis: membrane remains intact, cytosol is contained, no inflammatory response; engulfed by macrophages
What kind of actin-associated proteins PROMOTE vs PREVENT polymerization?
Promote: nucleating proteins
Prevent: monomer sequestering proteins, capping proteins, and severing proteins
What 2 compounds are required by sarcomere proteins in order for crossbridge cycling to occur? Why?
1) ATP: hydrolysis of ATP -> ADP + Pi allows myosin to bind to actin + binding of ATP to myosin releases actin; calcium pumping (SERCA)
2) Ca2+: binds to troponin, exposing myosin binding sites on actin filament
What are the 3 regulatory/check points in the cell cycle? When do they occur?
1) Cycle initiation by nutrients or growth factors/hormones (mitogens). Occurs between G1 phase, before entry into S phase.
2) DNA damage checkpoints. Occurs between G2 phase, before entry into M phase.
3) Mitotic spindle assembly checkpoints. Occurs in late M phase.
Describe the 5-step process of initiating DNA replication.
1) Cdc6 is recruited to origin of replication by ORC;
2) DNA helicase hexamers are recruited to join & form pre-replication complex.
3) Cyclin E/CDK2 activates DNA helicase & promotes recruitment of other proteins.
4) DNA helicases move outward, unwinding DNA
5) DNA polymerase binds & begins replicating.
What are the 5 targets of caspase? What is the result of targeting each?
1) Inhibitor DNase = DNA fragmentation
2) Nuclear lamins = nucleus fragmentation
3) Cytosolic cytoskeletal proteins = cytoskeletal disruptions, cell fragmentation, membrane "blebbing"
4) Golgi membrane proteins = Golgi fragmentation
5) Scramblase = translocation of phosphatidylserine (eat me signal) to cell surface
Briefly describe the process of rescue vs catastrophe in microtubules.
RESCUE (growth): tubulin dimers w/ GTP bound add to the growing (+) end of the microtubule and forms a GTP cap; addition of tubulins proceeds faster than GTP hydrolysis by the dimers
CATASTROPHE (shrinkage): GTP hydrolysis proceeds faster than addition of new GTP-tubulin dimers (GTP cap is lost); protofilaments containing GDP-tubulin pull away from microtubule wall & GDP-tubulin is released into cytosol
Briefly describe the process of Ca2+ release from sarcoplasmic reticulum.
1) Upon stimulation, motor neuron releases acetylcholine (ACh) into neuromuscular junction, causing ACh receptors to open (& cell to depolarize).
2) T-tubule depolarization causes its voltage-sensitive DHP receptors to unblock Ca2+ release channels (RyR) on SR; Ca2+ rapidly released into cytosol.
3) Ca2+ actively pumped back into SR by SERCA.
What is the role of the central spindle and how is it formed?
Keeps structures of the two daughter cells separated & helps with final separation of the two cells' plasma membranes.
Formed by reorganization of mitotic spindle microtubules & interpolar microtubules.
Briefly state the roles of each type of cyclin.
1) Cyclin D: drives progression of cycle through G1 in response to extracellular signals
2) Cyclin E: drives G1 -> S phase transition - also restriction point (i.e. commits to progression to S phase)
3) Cyclin A: needed to progress through S phase & prevents re-replication (CDK1 & 2); needed to start mitosis (CDK1)
4) Cyclin B: needed to start mitosis (drives progression of M phase)
What are the 2 ways Pro-apoptotic Regulatory Proteins (PRPs) indirectly activate caspases?
1) Form complex with ARPs, blocking them from inhibiting caspase activity (i.e. negative control)
2) Activate PEPs, which in turn directly activate caspases
Briefly describe the steps of cell crawling.
1) Actin polymerization: extension of lamellipodia & filipodia at leading edge of cell
2) Attachment: integrins adhere to molecules in extracellular matrix & actin filaments in cell cortex
3) Contraction: myosin motor proteins slide along stress fibers (actin filaments) to drag cell body forward
How are intermediate filaments involved in muscle contraction? Give a specific example.
Maintain cell structure; stabilization/anchoring (via accessory proteins).
Desmin intermediate filaments anchor the Z-disc to nucleus, mitochondria, and plasma membrane.
How and when are intermediate filaments involved in the cell cycle?
Nuclear lamins below nuclear envelope are fragmented in prometaphase; reform with nuclear membranes in telophase.
What are the 4 processes regulated by cyclin B/CDK1? Briefly describe what happens during each process and what stage of the cycle they occur in.
1) Chromosome condensation: phosphorylation of cohesins & condensins. Occurs in prophase
2) Breakdown of nuclear envelope: phosphorylation of nuclear lamina. Occurs in prometaphase
3) Mitotic spindle formation: phosphorylation of centrosomes, kinetochores and microtubule associated. Occurs in prometaphase
4) Anaphase Promoting Complex/Cyclosome ubiquitin ligase: tags cyclins (all remaining) and securins for degradation. Occurs in metaphase
How do Pro-apoptotic Effector Proteins (PEPs) activate caspases?
Bak & Bax oligomerize to form pores in the outer mitochondrial membrane, leading to the release of cytochrome C from inner membrane.
Cytochrome C appearing in cytosol activates caspases.
Describe & differentiate between lamellipodia, filipodia and stress fibers.
Lamellipodia: thin, flat extensions at leading edge of cell. Composed of branched actin filaments. No myosin (can't contract).
Filipodia: stiff, narrow protrusions at leading edge of cell. Sense & navigate extracellular environment. Composed of bundled actin filaments. No myosin (can't contract).
Stress fibers: contractile actin:myosin bundles found in cytoplasm of non-muscle cells. Composed of bundled actin filaments.
Briefly describe the steps of a cross-bridge cycle.
1) ADP-bound myosin head is COCKED & ready to bind
2) Ca2+ binds to troponin, exposing myosin binding sites
3) Bound myosin rotates head, producing a power stroke (releases ADP)
4) ATP binds to myosin head, causing actin & myosin to detach
5) ATP is hydrolyzed to ADP + Pi
6) Cycle repeats
What 3 types of cells do not (or do not usually) divide?
1) Neurons (don't divide)
2) Skin fibroblasts (response to injury)
3) Liver hepatocytes (response to injury)
What 3 mechanisms are used by cyclin A to prevent re-replication of DNA?
1) Displaces cyclin E (prevents initiation of replication)
2) Inhibits pro-replication gene
3) Inhibits DNA polymerase
What is the role of the signaling pathway initiated by PI-3 kinase? How does it fulfill this role?
Inhibits apoptosis in the presence of extracellular pro-survival signals via AKT (protein kinase B).
AKT phosphorylates (inhibits) BAD, preventing the activation of PEPs & the inhibition of ARPs.