Invasion - Regulation of cell migration Flashcards
Summarise the steps of tumour progression
Steps of tumour progression:
o Homeostasis- polarity of cell, cell-cell contacts intact, functions apropiately.
o Genetic alterations.
o Hyper-proliferation.- formation of benign tumour
o De-differentiation:
Disassembly of cell-cell contacts.
Loss of cell polarity.
o Invasion:
Increased motility.
Cleavage of ECM proteins.- formation of malignant tumour
Describe how metastasis requires distinct and sequential events
Epithelial cells in primary tumours are tightly bound together Metastatic tumour cells become mobile mesenchyme-type cells and enter the bloodstream. Metastatic tumour cells then travel through the bloodstream to a new location in the body. Metastatic cells exit the circulation and invade a new organ. Cancer cells (for unknown reasons) lose their mesenchymal characteristics and form a new tumour.
Describe the different types of migration
Individual cell migration:
o Amoeboid – e.g. lymphomas, leukaemia, SCLC
o Mesenchymal (single) – e.g. Fibrosarcoma, glioblastoma, anapaestic tumours
Collective cell migration:
o Mesenchymal (chains) – same as mesenchymal single
o Cluster/cohorts – e.g. Epithelial cancers, melanoma
o Multicellular strands/sheets – e.g. Epithelial cancers, vascular tumours
Collective cell migration requires more coordination to metastasise and so still has some cell-cell junctions, and to cleave more of the BM.
What do all metastasising tumours require
requires integrins for movement and proteases to digest ECM
What does collective cell migration require
Cadherins and gap junctions- to keep the cells as a cohort.
Cadherins drag neighbours along- taste regulator for differentiation (not usually expressed in invasive tissues- but needed for these cells to migrate together)
Describe how metastatic tumour cells mimic morphogenetic events
Cell growth in 2D sheet moving along ECM
Vascular sprouting in angiogenesis- with tip cells leading growth
Branching morphogenesis- maxillary gland - towards terminal end bud- for lactation- migration and differentiation of epithelium- maybe stromal signals
multicellular 3D strands and detached cluster -led by tip cells
border cells- nurse cells migrating as cluster to front of ovum to provide nutrients.
What is important to remember about tumour cell migration
Tumour cell metastasis/migration MIMICS PHYSIOLOGICAL morphogenic events.
o E.G. Branching morphogenesis in the mammary glands.
o E.G. Migration of primary glial cells to repair a scratch wound (the cells stop migrating when the contact is made) – conversely, tumour cells will have no clear migration front and no sense of direction.
They also lose contact inhibiton of locomotion, this is normally found in normal tissues so that they know when to stop growing
Describe an experiment which showed the the in vivo expression of invasive cell profile vs primary tumours using cDNA microarray profiling-gene expression
Needle collection of invasive cells (stimulated with EGF to direct growth)
FACS carcinoma cells from primary tumour- sequence these too
Invasive cells showed Upregulation of genes involved in:
Cytoskeleton regulation
Motility machinery
compared to the tumour cell
these cells were more likely to metastasise
remember- tumours are heterogenous.
State some stimuli for cell movement
organogenesis and morphogenesis
wounding
growth factors/chemoattractants
dedifferentiation (tumours)
How do the cells know where to go
directionality (polarity)
will change shape to grow towards stimulus- organelles directed towards direction of movement
How do migrating cells know when to stop
contact-inhibition motility
How do cells move
specialized structures (focal adhesion, lamellae, filopodium)
Describe the importance of regulation in this process
Regulation: required to coordinate stages, control adhesion/release of receptors and to respond to external influences
- What are the attachments between the cell and the surface that it is moving along called?
Focal adhesions
Integrins provide Attachment to substratum
(ECM proteins)
Cytoplasmic tails of interns have no enzymatic activity- so need to recruit a plaque of proteins to attach to filamentous actin- which will contract to move the cell towards the site of attachment. to the ECM at the focal adhesion- works like a hook.
Describe the importance of attachment to the substratum
Attachment to the substratum: cells require attachment to the ECM to allow response to growth factors and movement across tissue - filamentous actins terminate at focal adhesions to the ECM substratum - allowing to provide traction forces for movement
Describe filopodia
Finger-like protrusions rich in actin filaments
Used for motility
Vincluin important for formation
Describe lamellopodia
Sheet-like protrusions rich in actin filaments
Important for motility- throw membrane off to attach- then re-cycle membrane
Describe the importance of control of cell movement
within a cell to coordinate what is happening in different parts
regulate adhesion/release of cell-extracellular matrix receptors- adhesion and de-adhesion to grow towards stimulus- if they grew in all directions- cell would split apart.
from outside to respond to external influences –
sensors
directionality
Describe the two different types of movement
Motility: hapoptatic (random) versus chemotatic (direction and purpose)
Ultimately, what does cell movement require
Cell movement = changing cell shape
Describe the different stages of cell motility
Extension: lamellipodium extends from cell in direction of movement
Adhesion: tip of lamellipodium attaches to ECM, forming a new adhesion
Translocation: posterior region of cell contracts, allowing the cell body to move forward
De-adhesion: most posterior cellular attachment is broken
What are the different types of actin
G-actin- monomers- small-soluble subunits
F-actin - large filamentous polymers
Describe F-actin polarity
plus end
minus end
different properties of proteins at each end
The monomers preferentially get added on at the plus end
What happens when cells are stimulated to grow towards a given stimulus (e.g a nutrient source)
disassembly of filaments and rapid diffusion of subunits
reassembly of filaments (F-actin) at a new site (towards direction of growth).
Describe the filament organisation and structure
Lamellopodium- branched and cross-linked filaments
Filopodium- bundle of parallel filaments
stress fibres- anti-parallel contractile structures- just being lamellopodium
Summarise the remodelling of actin filaments
G-actin can be sequestered or nucleated and polymerised to form F-actin
F-actin can be severed, cross-linked, capped, side-binding, motor proteins and bundling
Describe nucleation of G-actin
Limiting step in actin dynamics – formation of trimers to initiate polymerization- as G-actin trimers are unstable
Nucleation: actin monomer joins with Arp2 and Arp3 (ARP complex) to form a nucleated actin filament with the ARP complex at the minus end - limiting step, as require trimers to initiate polymerisation
Describe elongation
Elongation: profilin joins with free actin monomers to promote assembly of filaments (thymosin inhibits and competes with profilin to sequester actin monomers)
added to + end
Sequestering proteins:
b4-thymosin
ADF/cofilin (do not inhibit polymerization
Profilin competes with hymosin for binding to actin monomers and promotes assembly
Describe capping
Capping: ends of actin filaments capped to prevent further polymerisation
Positive end: Cap Z/gelsolin/fragmin
Negative end: ARP complex/tropomodulin
Describe severing
Severing: proteins such as gelsolin/ADF/fragmin promote severing of strands, causing filaments to grow and shrink more rapidly (shorter fragments can be depolymerised quicker - need capping to prevent reassembly- otherwise they will polymerise quickly).
in unsevered populations, actin populations grown and shrink relatively slowly.
Describe the cooperation of actin functions to generate filaments
Filament severing:
Barbed-end (+end) capping to prevent growth
This will allow for depolymerisation and monomer recycling
can anneal severed end- where it is joined to another protein (ADP-Pi actin)
can then grow from prexisting end- (addition of ATP-bound actin to prolfilin)
or can have barbed end proflin-actin elongation
Describe cross-linking and bundling
a-actinin (dimers) fimbrin filamin spectrin- imported for formation of meshwork villin vinculin fascin- mutated in melanomas
filamin- meshowrk
dystrophin- link to plasma membrane
Describe the cooperation of actin functions to organise filaments
Sever- barbed end capping
No severing- allows for stabilisation and bundling
If two filaments are organised in opposite directions- myosin can cross-link and two strands can slide over each other to contract cell
Describe branching
Branching protein: Arp complex
70 degrees
Describe 7 - Gel-sol transition by actin filament severing
Gel-sol transition: actin filament severing allows for transition from rigid “gel” state to “sol” state that can flow and allow for protrusions of membrane
gelsolin important for this
Describe the role of cytoskeletal dysregulation in disease
High blood pressure
Wiskott-Aldrich Syndrome – WAS (immunodeficiency, eczma, autoimmunity) - WASP- important in directing cell towards chemoattractant
Duchenne Muscular Dystrophy (muscle wasting)
Bullous Pemphigoid (autoimmune disease)- Abs against BM proteins
Alzheimer (neurodegenerative)
Describe the participation of different actin activities during cell movement
Extension: Disassembly Nucleation Branching Severing Capping Bundling
Lamellopodium important in polymerisation.
Adhesion- gel/sol transition and attachment ECM
Translocation- contraction of posterior regions
de-adhesion- de-adhesions of posterior focal adhesion
Describe actin activities in lamella protrusion
Polymerization, disassembly, branching, capping
Net filament assembly at leading edge
net filament disassembly behind leading edge- this release G-actin for polymerisation at the leading edge.
Describe the role of actin activities in filopodia protrusion
Actin polymerization
Bundling
Crosslinking
Initiation
Protrusion - elongation and polymerisation of actin at top, bundling and cross-linking by fascin at base
retrograde flow- where the base is degraded
Retraction- filament is capped to stop growth, continuous retrograde flow shortens protrusion
Describe the relationship between cell shape and actin organisation
Bristels
Fascin, forked
Microvilli
villin, fimbrin, espin
stereocilia
espin, fimbrin
fliopodia
fascin, a-actinin
lamellopodia
arp2/3, filamin
Summarise the signalling mechanisms that regulate the actin cytoskeleton
1 - ion flux changes (i.e. intracellular calcium)
2 – Phosphoinositide signalling (phospholipid binding)
3 – Kinases/phosphatases (phosphorylation cytoskeletal proteins)
4 - Signalling cascades via small GTPases
Describe the Rho family
Rho subfamily of small GTPases belongs to the Ras super-family
Family members: Rac, Rho, Cdc42 best known
Participate in a variety of cytoskeletal processes.
These proteins are activated by receptor tyrosine kinase, adhesion receptors and signal transduction pathways to trigger motility- filopodia
Expression levels up-regulated in different human tumours.
Describe the activation of Rho
Inactive- bound to GDP
Active- bound to GTP
Transiently activated- Pi released by GTPase to return GDP
90-95% inactive
Whereas Ras gets mutated, Rho is unregulated and levels increase- leading to more activation of pathways- point mutations rarer in Rho compared to Ras.
Describe some roles of the Rho family
Cdc42: filopodia
Rac: lamellipodia
Rho: stress fibres
Describe signalling through Rac GTP
IRSp53
Rac binds to and activates WAVE
WAVE then activates Arp2/3, which is important in actin organisation and polymerisation
Can also activate cdc42GTP and related pathways
Describe signalling through cdc42 GTP
Cdc42 binds to WASP
WASP also activates Arp2/3
Can also bind to formin to activate profilin- actin polymerisation and organisation
can bind to Bak- activate LIM kinase to activate cofinin- actin organisation and polymerisation, also activate Raf TO activate MAPK
Describe the participation of small GTPases on cell migration
Extension- Rac- actin polymerisation and branching (lamellopodium)
Adhesion- Rac and Rho in focal adhesion assembly
Transloction- Rho- stress fibers
tension (myosin)
contraction
de-adhesion - Rho
Cdc42
filopodia
polarized motility
actin polymerization
Describe phosphorylation of actin
Remodelling of actin filaments: growing end of filament comprised of ATP-actin, with dephosphorylation occurring behind leading edge to form ADP-actin
