Cell Organisation And Movement

Question 1

Micro filament definition
Sickle cell
Metastasis

Answer 1

Actin polymers organised by actin binding proteins
Plasma membrane integrity and micro villi
Tracks for ATP myosin motor proteins -> muscle contraction
Facilitates endocytosis

Sickle cell- b-globulin haemoglobin chain, aggregation under low O2. Cytoskeleton more rigid

Metastasis- changes in actin regulation allows non motile cells to move.

Question 2

Micro tubules definition
Colchicine
Taxol

Answer 2

Formed by Tubulin, organised by micro tubule associated protein
Framework for organelles and support cilia and flagella
Structure of the mitotic spindle
Kinesins and dyneins transport

Colchicine- meadow saffron. Inhibits polymerisation of Tubulin. Relieve gout joint pain. Reduces micro tubule dynamics in WBCs so cannot migrate.

Taxol- plant alkaloid. Binds and stabilises micro tubules. Prevents cells from mitosis. Cancer treatment.

Question 3

Intermediate filaments definition

Answer 3

Support, integrity, barrier. Not transport. A tetra ear is formed by side to side aggregation to form protofibril.
Neurofilaments- axon organisation
Lamins- inner surface nuclear membrane. Mutations cause emery-dreifus muscular dystrophy. Hutchinson-Guildford progeria (accelerated ageing)

Keratin- strength to epithelial cells. Epidermolysis bullosa- skins blisters.

Desmin, GFAP, vimentin

Question 4

Coordinating the migrating cell cytoskeleton

Answer 4

Motility stimulus
Activation of signal cascade
cdc42 -> polymerisation of actin -> crawling
Also promotes micro tubules to direct secretory vesicles and adhesion molecules and nucleus movement

Question 5

Structure of actin

Answer 5

F-actin - constructed of G actin monomers. Each actin molecule contains an Mg2+ complexed to ADP or ATP. G actin is divided into 2 lobes and ATP binds to bottom of the central cleft.
Addition of cations promotes formation of filaments
+ end is the joining end
One unit is 28 G actins

Question 6

Actin polymerisation

Answer 6

Nucleation- ATP-G actin monomers form complexes. Rate limiting step. 3 monomers needed. Critical concentration required.
Elongation- stable nuclei elongate, addition to both ends
Steady state- equilibrium of addition/subtraction

Faster addition to + end as lower critical concentration.
When Pi is released, ATP at + and ADP at -
This then promotes dissociation from the - end

Question 7

The cofilin cycle (actin polymerisation)

Answer 7

Cofilin binds to f and g actin. Specifically binds to ADP actin at the - end.
Bridges 2 adjacent actin monomers and changes twist -> destabilises
Increases the ADP actin that can be used by Profilin

Question 8

The profilin cycle (actin polymerisation)

Answer 8

Profilin binds G actin on the opposite side to the ATP binding site
Binds and removes the ADP, monomer free to bind ATP
Profilin-ATP-G actin binds to + end and profilin dissociates

Question 9

The Thymosin-β4 cycle (actin polymerisation)

Answer 9

Binds to G-ATP actin and inhibits addition. Creates G-actin reserve that can be used in blood clotting
CapZ caps the + end and stabilises, regulated by lipid.
Tropomodulin caps -end, abundant in cells that are stable e.g. RBCs and muscle
Gelsolin- binds to + end, regulated by Ca. Allows binding to side of an actin filament, creating new - end.

Question 10

Mechanism of actin filament branching

Answer 10

Arp2/3 binds to side of filament (7 subunits)
WASp binds (NPF)-> conformational change allowing complex to bind actin
Filament formation
WASp has an RBD element that binds to Cdc42. Releases the ve terminal domain and activates.

Question 11

Wiskott Aldrich syndrome

Answer 11

WASp defect. Not enough IgM produced, lack of platelets. X linked.

Question 12

Listeria motility

Answer 12

Polymerises actin into a comet like tail. ActA is a Arp2/3 activating protein. Leads to actin nucleation of branched filaments -> force

Question 13

Formins

Answer 13

Assemble unbranched filaments, FH1 FH2 adjacent domains with RBD. The FH1 contains Pro -> profilin binding. Increases concentration of ATP-G actin bound to profilin. Protects + from capping
FH2 inactive when associated to RBD. When bound to GTPase Rho, both domains exposed -> actin nucleation

Question 14

Actin based cellular structures
Cross linkers
Spectrin

Answer 14

Cross linkers- 2 actin binding sites within polypeptide. Fimbrin is an example, arranges filament with polarity in bundles.
Also formed when 1 actin site dimerises. These are more spaced out, a-actinin is example.

Spectrin- tetramer with 2 sites, forms networks under the plasma membrane. Cross linkers such as filamin serve as springs at leading edges.

Question 15

Diseases of the cytoskeleton

Answer 15

Arthritis
Osteoporosis- bone loses calcium and becomes thinner
Fibrodysplasia ossificans progressive- fibrous tissue ossified when damaged. Joints become frozen.

Question 16

How are actin filaments linked to membranes in RBCs

Answer 16

2x14 subunit filaments. Stabilised by tropomyosin and tropomodulin
Linked together by Spectrin
Spectrin links to Ankyrin -> transmembrane protein band 3
Spectrin links to band 4.1 to transmembrane protein glycophorin C

Question 17

Genetic disorders of actin linking to membrane

Answer 17

Spherocyic anaemia- mutations in Spectrin, band 4.1 and Ankyrin. Cells are rounder and rupture.

Sickle cell- mutation in B globulin. Low oxygen causes vessel occlusion and ischaemia. Actin oxidation stabilises abnormal cell type and exacerbates phenotype.

Question 18

Structures of cell migration

Answer 18

Filopodia- protrude from leading edge, tight actin bundles formed by formins
Lamellipodium- complex of branched actin formed by Arp2/3
Cortical actin cytoskeleton gives stability to membrane
Stress fibres attached to substratum of cell through focal adhesions

Question 19

Membrane extension

Answer 19

Leading edge actin is nucleated by Arp2/3 complex, filament grow from + end.
Actin tread milling mediated by cofilin and profilin
Formins and bundling proteins form leading edge

Question 20

Focal adhesions (migration)

Answer 20

After membrane extension, plasma membrane attached to substratum. Actin bundles anchored to focal adhesions, stops lamella retracting.
As moves forward, adhesions migrate to the back of the cell and are deassembled

Question 21

Cell body translocation

Answer 21

Bulk contents trans located forward

Organelles squeezed forward by cortical contraction of cytoskeleton

Question 22

De adhesion and endocytic recycling

Answer 22

Focal adhesions at rear are broken
Integrins are recycled
Freed tail brought forward
Snap forward by stress fibres contracting

Question 23

Control of actin organisation- injury

Answer 23

TGF-β released from injury sites and activates Rho

Activated platelets release LPA which activates Rho
RBCs release PDGF (platelet derived growth factor) at site of injury, which activates cdc42 and Rac

Question 24

Activation of Rho

Answer 24

GDP form complexed to GDI in inactive form
Activated by GEF which changes GDP to GTP
Causes localisation to membrane
Rho-GTP then binds effectors that cause changes
Binds to RBD of Formin, profilin binds to FH1 allowing addition at FH2
GAP returns it to the cytoplasm

Question 25

Activation of Rac

Answer 25

Activated by CD42 | WAVE -> Arp2/3 -> actin polymerisation -> lamellipodia

Question 26

Activation of cdc42

Answer 26

Activated par6 and WASP Par6-> polarity WASP -> Arp2/3 -> polymerisation -> filopodia formation

Question 27

Wound healing assay

Answer 27

Cells on plate scratched to simulate wound Neighbouring cells sense gap and move Dom negative Rac- needed to activate Arp2/3, lamellipodia not formed well Dom negative Cdc42- do not orientate in right direction as cdc42 needed for polarity Dom negative Rho- failure to establish contractile fibres

Question 28

Role of cdc42 in cancer

Answer 28

Stimulates protease release and invasion by invadopodia Increased cdc42 gene copy number common Leads to active Rac inducing leading edge and high Rho in rear to assemble contractile structures -> myosin II activation

Question 29

3 functional regions of myosin

Answer 29

Head- actin binding region, ATP binding region Neck- a-helical region, regulates head domain Tail- binding sites which determine specificity

Question 30

Myosin superfamily 3 classes

Answer 30

I- membrane association and endocytosis, 1 head II- contraction, 2 heads V- organelle transport, 2 heads and 2 feet

Question 31

Type V myosin in transport

Answer 31

One head binds while the other swings Binds site 72nm ahead Exact distance as helical repeats of the filament Used to segregate organelles

Question 32

Clathrin mediated endocytosis

Answer 32

Assembly of hemispherical clathrin coat Arp2/3 dependent formation of an actin network on the surface of the endocytic coat induced by Las17, Pan1 in yeast Elongation of incipient invagination by Myo-5 actin polymerisation Formation of 2 acto myosin structures which cooperate which yeast amphiphysins in the fission event by generating tension

Question 33

Recycling of endosomes

Answer 33

NMDAR requires myosin Vb which binds RAB11 NMDAR activation -> Ca influx -> myosin Vb activation Translocation of recycling endosomes and cargoes into the spine Transport from endosomes to spine surface mediated by RAB8

Question 34

Myosin in the cell

Answer 34

``` SW2- level arm recovery stroke SW1- cleft closing and actin binding II cell division V- transport vesicles V1- moves backwards along actin X- moves over actin bundles I- organises actin cytoskeleton As neck length increases, velocity increases ```

Question 35

The structure of Tubulin

Answer 35

α-β heterodimer, forms a protofilaments with β at the - end GTP bound to the α subunit is trapped by the β subunit, no exchange or hydrolysis. The GTP in β can be hydrolysed. 13 protofilaments -> sheet -> tube Stabilised cap from GTP bound units. Fast addition at + end.

Question 36

Tubulin in cell division

Answer 36

``` Assembled from MTOCs + ends grow outwards from centrosome Astral point into cytoplasm Kinetochore pull chromatids apart Polar contact each other and slide apart ```