GET MILLARED Flashcards
(1) Structure of Microtubule
- Dimers of A-tubulin and B-tubulin
- GTP bound dimer is added to MT
- Each photofilaments has alternative A-Tub and B-Tub
- 13 parallel protofilaments which form MTs (forms lumen)
(1) Gamma-tublulin waht do?
Major component of gamma-tubulin ring complex, recruited to MT organising centres (centrosome)
(1) Formation of MTs
- MTs only form when above threshold concentration (Critical concentration)
- Crit conc at +ve end is lower than -ve end, MTs grow at +ve end.
(1) WAT is treadmilling
- Negative end allowed to disassemble while the +ve end assembles.
- Rate of polyermisation and depolyerisation is the same, polymer stays the same length. (causes MT flux to occur)
(1) WAT does Taxol do?
Blocks mitosis by stabilising MTs, but doesn’t block other functions of MTs
(1) WAT does Colcemid do?
Binds to tubulin dimer and prevents polymerisation. Blocks mitosis by dissolving spindle
(1) WAT does Nocodazole do?
Binds tubulin dimer and prevents polymerisation
(1) Outline the animal cell MTOC
Centrosome
- MTs are nucleated by gamma-TURs
- Gamma-tubulin prevents poly from -ve end of MT
- Negative end is near MTOC and +ve end is near cell pheriphery
(1) What do MTs do during interphase
- Positive ends directed to cell cortex where they interact with plasma membrane
- Allows MTs to maintain cell shape
(1) Effect of GTP to GDP hydrolysis in B-tubulin
- Conf change that tenses lattice
- GTP dimer has 5 degree straight angle (MT assembly)
- GDP dimer has 12 degree bent heterodimer angle (MT disassembly)
- Cap stops depoly
(1) Outline process of dynamic instability
1) Rapid growth with GTP cap
2) Due to the slow hydrolysis of GTP to GDP in B-tubulin, the GTP cap is lost (catastrophe)
3) Rapid shrinkage
4) Regain of GTP-cap (rescue, modulated by MAPs)
5) Repeat
(1) What happens to MT during mitosis
- Spindle MTs undergo MT flux (-ve end can now undero deploy at spindle poles)
- Helps form bi-oritentation during pro-metaphase
- MT length remains constant
(1) 3 types of single MTs
1) Astral MTs: Link spindle pole to cell cortex
2) Interpolar MTs: Inter-digitate at centre of spindle
3) Keinetochore Mts (k-fiber): Connect spindle polesat chromosome at kinetochore
(2) 2 Examples of MAPs
1) EB-1: Only binds GTP-tubulin (Stabilises the MT seam and is present on growing end of MT)
2) DASH-ring complex: Binds poly and depoly (couples kinetochore movement to MT depoly)
(2) 2 examples of cross-linking, stabilising, bundling proteins
1) MAP2, Tau: Binds side and stabilises parallel MTs (via promoting poly or inhibiting MT catastrophe)
- Space between MTs is greater with MAP2 than Tau expressing cells
2) MAP65: Bundles and stabilises anti-parallel MTs (key in bi-polar spindle)
- MAP65 both MT binding and dimerisation domains
(2) 2 types of MT motor proteins
1) Kinesin
- These are plus end directed motors (kinesin-14 is ive end motor)
- Use ATP to generate force
- Direct exocytosis
2) Dynein
- Fast -ve end direct motor
- Uses ATP to generate force
- Direct endocytosis
(2) Process of kinesin movement
1) Forward motor binds B-tubulin, releasing ADP
2) Forward head binds ATP
3) Conformational change in neck linker causes rear head to swing forward
4) New forward head releases ADP, trailing head hydrolyses ATP and releases Pi
(2) Cargo of kinesin motors
1) Organelles: ER and Golgi
2) Secretory vesicles: Mediation of exocytosis
3) MTs: sliding kinesin (5+6) used in anaphase of mitosis
4) Chromosomes (mitosis)
(2) Cargo of dynein
1) Direct endocytosis
2) Late endosomes, lysosomes, golgi positioning
(2) Differences between the 3 MT structures
1) Singlet: Simple tube, 13 protofilaments, flexible
2) Doublet: A + B tubule, cilia/flagella, less flexible
3) Triplet: A, B, C tubules, basal bodies/ centrioles
(3) Where is ATP binding cleft on actin
ATP binds to opposite of adjoining actin molecule.
Gives filament polarity with actin binding cleft exposed at -ve end.
(3) How F-actin filaments form
- When conc of G-actin is larger than Cc, filament gets longer
- When conc of G-actin is smaller than Cc, filament will disassemble
(3) Process of G-actin addition
- Rate of additon of ATP G Actin is 10X faster at +ve end than -ve end, dissocation rate similar
- ATP hydrolyses to ADP-Pi, Pi released slowly. Filament has ATP, ADP-Pi and ADP actin
- Treadmilling effect caused by adding ATP actin at +ve end.
(3) Function of thymosin-B4
-Binds to G-actin, cannot be added to filaments. (As conc of G-actin is 1000X more conc than Cc)
(3) Function of Profilin
-Binds to G-actin more weakly than thymosin-B4
-Binds to G-actin more strongly than actin +ve ends
Allows ADP/ATP exchange and promote ATP replacement and actin incorporation into filaments
(3) Function of Cofilin
-Binds sides of ADP-actin in filament causing them to fragment
(Replenishes pool of free ADP-actin to reuse
(3) Process of actin filament treadmilling
- CapZ binds to +ve end (high affinity) and inhibits both subunit addition and loss
- Tropomodulin binds -ve ends of filaments and promotes filament growth
(3) How are actin filaments regulated
Formin (Rho GTPase binding domain, profilin ATP actin binding domain and filament nucleating domain.
- RBD inhibits FH2 if not bound to Rho
- If FH2 is activates formin to plasma membrane, conf shift which releases FH1/FH2 domains to trigger filament formation
(3) Function of WASp
Not bound to Rho, RBD binding to Arp2/Arp3
-Cdc42 GTPase activated, WASp recruited to PM and conf shift releases Arp2/Arp3 and triggers branch formation
(3) 3 Actin binding toxins
1) Cytochalasin B, D: Binds monomers and barbed ends, stops poly
2) Phalloidin: Binds and stablilises filaments
3) Botulinum toxin: ADP ribosylates the monomer, stabilises G actin.
(3) 2 different types of actin bundles
1) A-actinin: Dimers which are para or anti para, allows myosin to get to actin (large gaps)
2) Fimbrin: Bundles filaments of same polarity and packs tightly (microvilli)
(4) 3 types of myosin filaments
Myosin I: Binds membranes and endocytosis
Myosin II: Forms dimers
Myosin III: Dimerises but doesn’t form filaments, binds via adaptors to vesicles. MTs and RNA.
(4) Process of myosin movement
1) Binds ATP, head released from actin
2) Hydrolysis of ATP to ADP+Pi. Myosin head rotates into cocked state
3) Myosin head binds actin filament
4) POWER STROKE. Release P and elastic energy straightens myosin. Moves actin filament left.
5) ADP released, ATP bound, head released from actin
(4) Differences between Myosin and Kinesin
1) M tightly bounds to act without nucleotide rigor, K bounds to act with ATP bound.
2) M releases from actin when ATP binds, K releases when ATP is hydrolysed.
3) M ATP hydrolysis causes cocking and power stroke by loss of P, K ATP hydrolysis causes binding and binding of ATP causes power stroke that throws partner head forward
(4) 4 proteins in muscle ell actin filaments
1) CapZ: Binds +ve ends and allows bury in Z-disks
2) Tropomodulin: Binds -ve ends
3) Nebulin: Repeated actin binding sites and determines length of actin filaments
4) Titin: Elastic molecule that prevents over-stretching of sarcomere.
(4) Process of muscle contraction
1) Depolarisation of sarcolemma
2) T-tubules lie adjacent to sarcoplasmic reticulum
3) Depolarisation causes voltage-gated Ca2+ channel that allows small burst of calcium in cell
4) Ca2+ binds to channel in SR to massive release of Ca2+ in cytosol
5) Ca2+ binds to Troponin
6) Troponin conf change causes conf change in Tropomyosin
7) Myosin bind site exposed
(4) Calculating motor size
1) Myosin bound at low density to immbolised bead. Actin filament held by 2 laser light sources and lowered towards bead
2) Myosin touches actin filament and ATP added. ATPase stimulated, myosin moves filament
3) Distance and force calucated by computer
(4) Differences between Myosin II and V
II: 5-15nm steps, multiple single heads, 10% duty ratio
V: 72nm steps, one head always bound, 70% duty ratio
(4) Functions of Myosin V
1) Budding yeast, V transports cargoes like vacuoles and ER.
2) Mediate asymmetric segregation of ash1 in budding yeast.
(5) Process of chemotaxis (keratocytes)
1) Begin with extension of one lamellipodia from leading edge of cell
2) New focal adhesions are established
3) Bulk of cytoplasm is pushed forward
4) Trailing edge of cell detaches from substratum and retracts into cell body
(5) 3 GTPases required for directional clue of movement thingy
Rac
Cdc42
Rho GTPase
(5) Structure of cillia and flagella
Form axonemes that are a ring of 9 doublet MTs with 2 single MTs in the centre
(5) Features of IFs
1) 8-10nm diameter
2) Doesn’t bind nucleotide
3) Great tensile strength
4) Unpolarised
5) No motors
6) Used in tissue and cell integrity
(5) Role of type I and II IFs
Keratins
- Attach to PM through demosomes and extracellular matrix via hemi-demmosomes
- Provides mechanical strength to epithelial cells and their derivatives when cross-linked
(5) Role Type III IFs
1) Skeletal muscle: Desmin/Synemin provide mechanical scaffold to sarcomere. Skelemin binds M line to support
2) Smooth muscle: Desmin cross-linked at dense bodies to provide resistive force to streching
(5) Role TYPE IV IFs
Neurofilaments
- NF light/med/heavy
- Structural support in axons and glial cells bound to MTs
(5) Role TypE V IFs
Nuclear lamins
-Nuclear structure and organisation
