Invasion - Regulation of Cell Migration

Question 1

What is the sequence of steps that lead to tumour formation and spread

Answer 1

1. Homeostasis
2. Genetic Alterations
3. Hyper-proliferation
4. De-differentiation
5. Invasion
6. Metastasis

Question 2

Describe the epithelial tissue in the homeostasis stage of tumour progression

Answer 2

In homeostatic epithelial tissue there is a layer of cells tightly attached to each other lying on a basement membrane
Beneath is a stroma consisting of blood vessels and supportive tissues

Question 3

What is the consequence of genetic alterations in tumour progression

Answer 3

If there are a cumulation of genetic alterations, there may be hyperproliferation of some cells – this leads to the formation of a benign tumour

Question 4

Explain the de-differentiation stage of tumour progression

Answer 4

Progression from a benign to malignant tumour involves the cells de-differentiating (losing their identity and function as epithelial cells)

disassembling cell-cell contacts
losing polarity

Question 5

Explain the invasion stage of tumour progression

Answer 5

Malignant cells invade the basement membrane, requires:
Cleavage of ECM proteins
Increase motility

Question 6

Describe metastatic cells under the microscope

Answer 6

Cell migrate much faster
No attachment to neighbours
Movement in every direction, over each other
Do not stop by contact inhibition

Question 7

Describe the sequential events of metastasis

Answer 7

1. Epithelial cells in primary tumours are tightly bound together
2. Metastatic tumour cells become mobile mesenchyme-type cells and enter the bloodstream
3. Metastatic blood cells then travel through the bloodstream to a new location in the body
4. Metastatic cells exit the circulation and invade a new organ
5. Cancer cells lose their mesenchymal characteristics and form a new tumour

Question 8

How are tumour cells profiled

Answer 8

Tumour cells are inoculated in a mouse, and they form a primary tumour. Growth factors can then be used to stimulate cell migration

Question 9

How is the extraction of both the primary tumour cells, and the more invasive cells compared with regards to their expression profile ≈

Answer 9

By measuring mRNA levels

It is found that there is upregulation of genes involved

Question 10

Which genes are upregulated in comparison of expression profile of invasive cells vs primary tumours

Answer 10

Cytoskeleton regulation

Motility machinery

Question 11

What are the stimuli for cell movement

Answer 11

organogenesis and morphogenesis
Wounding
Growth factors/chemoattractants
Dedifferentiation (tumours)

Question 12

What are the factors involved in regulation of cell movement

Answer 12

Direction (polarity)
Contact-inhibition motility
Specialised structure formation (focal adhesion, lamellae, filopodium)

Question 13

What are focal adhesions

Answer 13

Focal adhesions are the sites at which the cell attaches to proteins which make the ECM

Question 14

Explain how ECM proteins cause cell attachment to the substratum

Answer 14

Filamentous actin lies beneath the membrane, and hooks the focal adhesions to the
cytoskeletons via integrins
On the intracellular side, integrins interact with various cytoskeletal proteins to form a plaque

Question 15

What are filopodia and what do they do

Answer 15

Finger-like protrusions rich in actin

Protrude from the cell to sense where they want to attach, and direction of movement

Question 16

Which structures in actin filaments attach to the substratum and what is the name given to them when they move back

Answer 16

Lamellipodia

Ruffles

Question 17

What is vinuncilin

Answer 17

Proteins that overlie the protrutions of filopodia

Question 18

Control is needed:

Answer 18

Within a cell to coordinate what is happening in different parts
To regulate adhesion/release of cell-ECM receptors
From outside to respond to external influences

Question 19

What are the 2 types of cell motility

Answer 19

Hapoptatic motility - movement with no direction

Chemotactic movement - movement in which the cell senses a stimulus and goes towards it

Both involve changes in cell shape

Question 20

How do cells regulate their shape changes

Answer 20

Regulation of actin cytoskeleton

1. Signal (e.g. nutrient source) at one end
2. F-actin at the other end disassembles
3. Subunits diffuse to the signal side
4. Reassembly of the subunits at the new site
5. Movement facilitation

Question 21

What structure can actin be found as in the cytoskeleton

Answer 21

Can be found as small soluble globular monomers (G-actin)
Or
in a large polymerized filamentous polymer (F-actin)

Question 22

How does actin facilitate movement

Answer 22

1. Signal (e.g. nutrient source) at one end
2. F-actin at the other end disassembles
3. Subunits diffuse to the signal side
4. Reassembly of the subunits at the new site
5. Movement facilitation

Question 23

Describe the actin filament arrangement in the cell cortex

Answer 23

random gel-like arrangement to provide support to the plasma membrane

Question 24

Describe the actin filament arrangement in the stress fibres

Answer 24

anti-parallel arrangement, forming contractile bundles

Question 25

Describe the actin filament arrangement in the filopodium

Answer 25

tight parallel bundles

Question 26

What is remodelling of actin filaments driven by and what does actin dynamics refer to

Answer 26

Different actin binding proteins The cycle between G-actin monomers

Question 27

Describe the G-actin monomers in solution and what is the solution to the problem it poses

Answer 27

Polymerises very quickly, because the lowest energetic state is as a filament (but you can’t just have loads of filaments in the cell) The solution to this is to have sequestering proteins, which store the G-actin monomers until they are required.

Question 28

What are capping and severing proteins

Answer 28

Stop signals which prevent further growth. | Severing proteins act like scissors, and various proteins regulate how the filaments are arranged

Question 29

Draw a diagram that illustrates the remodelling of actin filaments

Answer 29

(refer to notes)

Question 30

What is nucleation in actin filament remodelling

Answer 30

Rate limiting step in actin dynamics | Formation of trimers to initate polymerisation

Question 31

Explain what occurs in nucleation during actin filament remodelling

Answer 31

Arp 2 + 3 (actin-related proteins) are similar to actin, and form a complex on which trimers of actin can attach (on the minus end) Elongation of the filament can then occur from the Arp2/3 complex

Question 32

Explain what occurs in the elongation stage of actin filament remodelling

Answer 32

Profilin binds to the monomers and helps to elongate the filament. Thymosin is a sequestering protein which prevents polymerisation. There is a balance between these two proteins to regulate filament growth, as profilin competes with thymosin for binding to actin monomers and promote assembly.

Question 33

Explain what occurs in the branching stage of actin filament remodelling

Answer 33

branched filaments form a precise 70 degree angle. Arp complex binds to the ide of the filament, providing nucleation at a different site

Question 34

What do capping proteins do

Answer 34

Prevents further growth of the filament | Can be used to regulate directionality of growth

Question 35

Which proteins are in the plus end of capping proteins

Answer 35

Cap Z Gelsolin Fragmin Severin

Question 36

Which proteins are in the minus end of capping proteins

Answer 36

Tropomodulin | Arp complex

Question 37

What are the two types of cross-linking and bundling

Answer 37

1. Actin filaments and alpha-actinin, which forms a contractile bundle, which a loose packing allowing myosin-II to enter the bundle 2. Actin filaments and fimbrin, which forms parallel bundling with tight packing

Question 38

What is the role of severing during action filament remodelling

Answer 38

Prevents low rate of growth/shrinking of very long filaments

Question 39

Give examples of severing proteins

Answer 39

Gelsolin AF/Cofilin Fragmin/severin

Question 40

What do membrane connections allow the cell to do

Answer 40

Sense surfaces and suitable environments for protrusions

Question 41

What do membrane connections involve

Answer 41

Transmembrane protein, and a intracellular plaque which is attached to the cytoskeleton Connections are then mediated by cell-cell and cell-ECM receptors

Question 42

Give examples of proteins that membrane connections involve

Answer 42

``` Talin Alpha-catenin Spectrin Ezrin All of these proteins are perturbed (Changed, or truncated) in cancer ```

Question 43

Which actin activities occur in cell movement

Answer 43

``` Disassembly Nucleation Branching Severing Capping Bundling ```

Question 44

Give an example of participation of action in cell movement and what actin activities does it involve

Answer 44

Lamellae protrusion | Involves polymerisation, disassembly, branching and capping.

Question 45

What are the signalling mechanisms that regulate the actin cytoskeleton

Answer 45

Ion flux changes (i.e. intracellular calcium) Phosphoinositide signalling (phospholipid binding) Kinases/phosphatases (phosphorylation cytoskeletal proteins) Signalling cascades via small GTPases

Question 46

Explain how ion flux changes regulate the actin cytoskeleton

Answer 46

Gelsolin severing is Ca2+ dependent, as this exposes the fast depolymerising end Alpha-actinin crosslinks actin filaments, and this is decreased in high Ca2+ levels

Question 47

Explain how signalling cascades via small GTPases regulates the actin cytoskeleton

Answer 47

``` Uses Rho (subfamily from Ras) to participate in cytoskeletal processes Activated by receptor tyrosine kinase, adhesion receptors and signal transduction pathways ```

Question 48

What are filopodia, lamellae and stress fibres activated by

Answer 48

Filopodia - Cdc42 Lamellae - Rac Stress fibres - Rho

Question 49

What do the G-proteins )GTP-ases) of the Ras super family cdc42, Rac, and Rho activate

Answer 49

Filopodia - Cdc42 Lamellae - Rac Stress fibres - Rho

Question 50

What occurs during protrusion in lamellar protrusion

Answer 50

Net filament assembly at the leading edge, and net filament disassembly behind the leading edge.

Question 51

What do filopodia involve

Answer 51

Polymerisation / growth Bundling Fimbrin cross-linking