invasion - regulation of cell cycle mobility

Question 1

what are the molecular mechanisms that regulate motility *

Answer 1

microfilaments

regulation of actin dynamics

cytoskeletal proteins

signalling proteins

Question 2

What are the steps in tumour progression *

Answer 2

homeostastis

genetic alterations

hyper-proliferation

de-differentation - disassemble cell-cell contacts, lose polarity

can form a tumour mass and invade BM

invasion - increased motility and cleavage of ECM proteins

(from hyperproliferation to invasion is the change from benign to malignant tumour)

Question 3

what is de-differentiation in tumour progression *

Answer 3

losing the knowledge that the cells are epiuthelial cells

normally tightly cohesive and highly polarised (ie nucleus at abse of cell)

normally cells are separated from stroma by the basement membrane which provides support for the epithelial cells

Question 4

describe invasion in tumour progression *

Answer 4

increased motility

cleave ECM proteins so cells find canals to migrate through the ECM

Question 5

describe the sequence of events in metastasis *

Answer 5

epi cells in primary tumours are tightly bound together - but not as organised as neighbouring normal cells

they cleave the BM

metastatic tomour cells become mobile mesenchyme type cells, enter the stroma, and then enter the bloodstream

metastatic cells then travel through the bloodstream to a new location in the body

they then exit the circulation through the endothelial cells and invade a new organ

the cells lose their mesenchymal characteristics and form a new tumour

sometimes the cells reverse and reacquire the neighbour-neighbour contacts

Question 6

describe the types of tumour cell migration *

Answer 6

tumours can travel alone or as a collective (cluster of cells - they maintain their attachment ti each other and have a higher metastatic potential)

Question 7

what are the different migration strategies *

Answer 7

ameoboid - individual tumour type - usually migrate as round structure but depends on the type - tumour tyoes are lymphoma, leukaemia, SCLC

mesenchymal - either single cell/chain - found in fibrosarcoma, glioblastoma, anaplastic tumour

cluster/cohorts - travel as a group through the ECM - found in epi cancer and melanomas

multicellular strands/sheets - larger number of cells - in epi cancer or vascular tumours

Question 8

describe the signalling involved in different tumour cell migration strategies *

Answer 8

different types need different signalling processes

all involve integrins and proteases - proteases digest the ECM, integrins receptor of ECM proteins (like foot of cell so cell can migrate)

for cluster migration - coordination is fone by the gap junctions between epi cells (they allow communication between the cells), also cadherins induce differentation and hold the cells together so they can drag each other along (they are different to the cadherins in normal tissue)

Question 9

describe how tumour cell metastasis mimics morphogenetic events *

Answer 9

2d sheet - cells migrate and drag cells behind

vascualr sprouting - need to regrow bv - tip cell drive newly formed vessel to a site where it needs to reconnect with the vasculature

branching morphogenesis - in mammary gland need spur of growth - need to proliferate and differentiate the epithelium - this is done by collective migration - form terminal end bud that brings cells together to invade and form the lactating gland

multicellular 3d invasion strands - also uses tip cells

detached cluster - have cells at the front and bring other cells along

border cells - in fly ovary cells - 1 side of ovary have cluster of nurse cells that feed the ovary - these celsl need to migrate through the egg so that they do their business at the front - migrate as a cluster - similar regulation in malignancies

Question 10

explain the migration of primary glial cells in a scratch assay *

Answer 10

scratch the assay of cells

teh cells recognise there is a gap so proliferate to fill it

when the cells reach each other, they stop growing

this takes 16hours

Question 11

describe the migration of a glial tumour cell line on a scratch assay *

Answer 11

the cells are not compact - loose cells close together

when they sense the space the cells migrate randomly and alone

when cells reach each other they continue to grow

this is much faster than in normal cells

Question 12

compare the expression profile of invasive cells v primary tumours *

Answer 12

in invasive cells there is an upregulation of genes involved in cytoskeleton regulation and motility machinery

to test - put EGFL in injection (potent mitogenic and induces motility) - so the tumour cells grow up into needles

collect teh cells from the heterogenous tumour mass that are highly likely to metastisise because could sense EGF and leave primary tumour

compare the expression of these cells to that of the primary tumour - measure the RNA - higher expression of EGFR etc

this upregulation is surprising because the level of oncogenes was similar

Question 13

what are stimulants for cell movement *

Answer 13

organogeneisis and morphogenesis

wounding

GF/chemoattractants - chemoattractants stimulate immune cells to move

dedifferentiation in tumour cells

Question 14

what happens to cells when they move *

Answer 14

they have to change their shape - become polarised - so they get a front/leading edge

teh organelles are directed to dorection of motility

Question 15

how do cells know where to go *

Answer 15

directionality - towards chemoattractant/space available

can either be to a specific place or with no purpose

Question 16

how do epithelial cells know when to stop *

Answer 16

contact-inhibition motility

tehy recognise their neighnours

Question 17

how do cells move *

Answer 17

they have specialised structures - focal adhesions, lamellae and filopodium

Question 18

describe the attachment of cells to the substratum - ECM proteins *

Answer 18

when moving the cells form focal adhesions - very close contact with the RCM

the actin filaments are organised as bundles - they finish at focal adhesions

the cytoskeleton (actin bundles) are coordinated to go to the focal adhesions - this provides a hook to make traction forces for the cells to move

Question 19

describe the complex joining the ECM to the cells *

Answer 19

the receptor is integrin - there are dimers of an a and b subunit, different combinations of the monomers will form different types of integrins

at cytoplasmic site - very short tail with no enzymatic activity so to do function it organises plaque of cytoskeletal proteins

the plaque acts as a signalling port and connects to the cytoskeleton

Question 20

describe filopodia *

Answer 20

tehy are finger like prtusions rich in actin filaments

vinculin is a protein that binds to filamentous actin - one of the main components of the integrin plaque - there is a high concentration of vinculin forming the filopodia

filopodia are used to sense the surroundings of the cells

Question 21

describe lamellipodia *

Answer 21

sheet like protusions rich in actin filaments

cells form contacts from large areas of membrane

the membrane attaches then comes back to the cell so that the cell can move

Question 22

what control is needed in cell movement and why *

Answer 22

• within a cell- to know what is happening in differnet parts of the cell
• regulate adhesion/release of ECM receptors
• control froom outside to respond to external influences - ie sensors (know where the GF and nutrients are) and cues for directionality

needed because if cant coordinate different parts, the cells will move in opposite directions and split

Question 23

what are the different types of motility *

Answer 23

hapoptatic - no purpose

chemotatic - purpose

Question 24

describe the steps in cell motility *

Answer 24

1 - extension - extend the lamellipodium

2 - adhesion - form a new focal adhesion - this is like a step

3 - translocation - contract the back of the cell and move the cell body forward

4 - de-adhesion - de-attach the last focal adhesion so you can move forward

Question 25

describe the relationship between G-actin and F-actin *

Answer 25

G-actin are small soluble subunits

F-actin are large filamentous polymers with polarity - have positive and negative ends so different ends have differnet functions

the small monomers can polymerase quickly

for example - a signal eg a nutrient causes disassembly of F-actin filaments quickly and rapid diffusion of subunits to side where nutrients are; then rapid reassembly of the filaments at a new site

Question 26

describe actin filament organisation ion differnt structures *

Answer 26

actin is a filamentous cabel - exerts force so cells can move

in filopodium the fibres are in parallel bundles - this allows movement

in lamellipodium - the filaments are short, branched and crosslinked forming filaments

behind the lamellipodium are arched filaments where the stress fibres are - they are antiparallel contractile structures - focal adhesion structure is at end

Question 27

describe the different processes involved in remodelling actin *

Answer 27

sequestering - maintains G-actin

bindling

severing - cut the filaments

nucleating

cross linking

capping

side binding

motor proteins - allows contraction

in order to react to a stimulus and disassemble then reassemble everything cells have to engage all these processes and proteins

Question 28

steps in actib remodelling *

Answer 28

1. nucleation
2. elongation
3. capping
4. severing
5. cross-linking and bundling
6. branching
7. gel-sol transition by actin filament severing

Question 29

describe nucleation *

Answer 29

this is the limiting step in actin dynamics

need 3 actin monomers together to start remodelling - not stable

so instead use nucleators (Arp3 and 2) that resemble actin - they bind to actin and the cell thinks that 3 actin monomers are bound together = begin polymerisation

G-actin monomers are added to the plus end of actin chain

Question 30

describe elongation *

Answer 30

when have actin filament - the arp complex can come off

actin-profilin complex fascilitates the monomer bidning

thymosin binds to G-actin and keeps it so it cannot bind to actin chain - restrict proliferation

prolifin competes for actin monomers with thymosine

molecules that inhibit actin binding are B4-thymosin, ADF/Cofilin

Question 31

describe capping of actin *

Answer 31

it tells actin to stop growing - hooks onto the end of filaments to stop monomers being added

then filament becomes depolymerised from the otehr end, unless it is blocked

caps on +end - Cap Z, gelsolin, fragmin/severin

-end - tropomodulin, Arp complex

Question 32

describe severing *

Answer 32

in unsevered population, actin filaments grow and shrink relatively slowly

in severed population actin filaements grow and shrink more rapidly

severing proteijns - gelsolin, ADF/cofilin, fragmin/severin

Question 33

describe the cooperation of actin function to generate filaments *

Answer 33

profilin-actin complex cause elongation at +ve end (barbed end)

filament severing at opposite end - barbed end capped and depolarisation and monomer recycling at otehr end

annealing - join together 2 short fragments

can have growth from pre-existing ends

Question 34

describe cross-linking and bundling *

Answer 34

group filaments in different states

fascin (mutated in melanomas)

a-actinin - dimer that cross links filaments

spectrin (stabalise cortical structure) and filamin - bring filaments at an angle - form a mesh

dystrophin - join actin to plasma mem, mutated in muscle wastage diseases

other proteins involved - fimbrin, villin, vinculin

this occurs when the actin filament is not severed so it is stabalised in the cells

filaments that are joined by myosin can be buckled if there is a contraction when the orientation of filaments is in opposite directions - opp polarity allows the filaments to slide over each other and cells contract

Question 35

describe branching *

Answer 35

filaments branch at 70degrees

up to 3 filaments bind and then polymerise a new branch

protein involved is the Arp complex

Question 36

describe gel-sol transition by actin filament severing *

Answer 36

network maintain rigidity of membrane in cytosol

but cell needs to protude so the network needs to open up - so cleave the filaments at certain sites = cross linked filaments that are not connected to each other

there is a transition from the gel phase to the sol phase

the protein that does this is gelsolin

Question 37

what phases of actin remodelling are at different stages of cell movement *

Answer 37

extension - polymerisation (disassembly, nucleation, branching, severing, capping, bundling)

adhesion - gel-sol transition, attachment to the ECM

translocation - contraction

de-adhesion - deattachment

Question 38

describe the processes involved in lamellae protusion *

Answer 38

branched filaments at 70degrees

there is capping so the filaments are short

polymerisation brings the membrane forward

back needs to disassemble and be clipped = form G-actin

G-actin goes to front of lamellae so it can be reassembled

this involves spatial, temporal, and molecular regulation

Question 39

describe actin regulation in filopodia *

Answer 39

in parallel bundles

comprehensive actin assembly at tip - need protein to bundle them

retrograde flow - so get deassembled at back

when filopodia form focal adhesions, need to stop growing so are capped - also disassembled at end so retract beck to the cell body

Question 40

what are the differnt cell shape and actin organisations *

Answer 40

if you have bristles/hairs - have fascin/forked heads

microvilli - cross linked filopodia proteins are villin, fimbrin and esprin

stereocilia in ears - haev a progressive height - esprin and fimbrin

filopodia - fascin, a-actin

lamellipodia - arp2/3, filamin, capping protein, cofiolin, gelsolin

Question 41

what are the signalling mechanisms that regulate teh actin cytoskeleton *

Answer 41

ion flux changes - intracellular ca - some cytoskeletal proteins bind ca = upregulated func

phosphonositide signalling - phospholipid binding

kinases/phosphtases - phosphophorylation cytoskeleton proteins

signalling cascades via small GTPases

Question 42

describe signalling by small G proteins *

Answer 42

Rho family of GTPases belong to the Ras superfamily - family members Rac Rho and Cdc42 afre best known

they participate in a variety of cytoskeletal processes, are activaetd by TK, adhesion receptors and signal transduction pathways

expression levels are upregulated in different tumours - this activates the pathways indirectly

there are some point mutations on rho

Question 43

what happens when rho family is activated 8

Answer 43

drive specific shapes

cdc42 = very long filopodia

rac = spread large lamellae in any direction

rho = 2 rho proteins - form stress fibres which are very pwerful contractors

Question 44

mechanism of rho proteins *

Answer 44

they are the top of the chain

layer of effectors that are specific from Rho gtpases - Pak, PI5K (this is a lipid kinase), formin, IQGAP

the actin binding proteins that are regulated by Rac/cdc42 are WAVE, arp2/3, cofilin, profilin, WASP, arp2/3

WASP is downstream of Cdc42 and activates Arp2/3

WAVE is a partner of the Arp2/3 complex and helps polymerisation

after this actin polymerisation and organisation into filaments, Rho can come in and bundle them

Question 45

where do GTPases fit in with the cell migration *

Answer 45

Rac activation restricted to one area of cell - lamellipodium

focal adhesion is rac/rho

assembly of stress fibres, control of myosin motor that gives tension to fibres, and contraction by myosin is contorlled by rho

rho is also de-adhesion

cdc42 - tell cell where to go by generating the filopodia and involved in polarised motility - receptors that sense chemoattractants need cdc42 to transduce the signal into the cell, important for actin pol because with raq controls the arp2/3 complexes