ECM and Cell Motility

ECM

Network of macromolecules organized in a tissue specific manner to bind cells together and regulate cell growth, differentiation, adhesion and migration

Chemokinesis

factor regulates cell movement but direction

Chemotaxis

Factor determines direction

What do chemokines do?

Secreted molecules that promote chemotaxis

How does the wound healing/scratch assay work?

On a 2D culture you make a scratch and see how long it takes to grow back

What does microfluidic migration devices do?

It introduces two different cehmoattractants at a gradient and observes the speed at which the cells will move towards it, or if it will just cause it to randomly move around

Transwell Assay/Boyden Chamber Assay

Cell suspenstion is placed in upper chamber

Migratory cells pass through polycarbonate membrane and cling to the bottom side.

After removal of non-migratory cells, migratory cells are stained and quantified

What method of cell movement do we really care about?

Amoeboid movement, extending the plasma memrbane to cause movement toward a specific location

How can the speed at which the cell moves be changed?

It can be changed in physical variables like rigidity, confinement, adhesion, topology. These all influence cell migration!

Structural elements of migrating cell

They’ve got the protrusion!

Cell migration

Different cell types on different ECMs will move around differently.

How does a cell pull foward?

With cell adhesion and traction! It has integrin-mediated attachement at leading edge at focal adhesions.

Cells then generate traction to pull cell foward

Why are focal adhesion important?

It is where ECM, integrins, and the cytoskeleton interact

How fast is adhesion assembly and disassembly?

Very rapid!

How is does the cell activate intracellular signaling?

With integrins connecting to fibronectin! Which then activate the focal adhesion complex!

What are the main components of ECM?

protiens, glycosaminoglycans, and glycoconjugates

Functions of ECM

Functions as adhesive substrate

Provides structure

Presents growth factors to their receptors

Sequesters and stores growth factors

Sense and transduces mechanical signals

Key functional properties of ECM

Factor administration, network structure, adhesiveness, shape, degradability

Integrin signaling variables

ECM components, stiffness, subunits

FAK

Focal adhesion Kinase

What does FAK do?

It is activated by integrin and growth factor receptor signaling, regulates focal contact assembly and disassembly. changes actin and microtubules structures

What are the domains of FAK?

FERM

Kinase

FAT

What does the FERM domain of FAK do?

localizes protiens to PM. Mediates interaction with growth factor receptors

What does the FAT domain of FAK do?

targets to focal contacts. Mediates interactions with integrin-associated proteins

What happens if FAK is changed?

It leads to failure of proper focal adhesion formation

It can also inihibt cell migration

What can molecules that block FAK do?

They can decrease cancer growth and reduce signalling!

What does invasion require?

cell movement through the ECM

What are a class of proteins that degrade ECM?

Active Matrix Metalloproteinases, proteolytic ezymes degrade ECM including collagen, gelatin, elastin

what happens if MMPs (Matrix Metalloproteinases) are targeted?

It prevents invasion but not cell growth

What does 3D invasion Assay measure?

They measure the amount of cells growing in matrix

Invasion assay/boyden chamber

Cell suspension is placed in upper chamber

Invasive cells degrade the matrix and pass through porse in the membrane, non-invasive cells stay in the upper chamber

After removal of matrix and non-invasive cells, invasive cells tained and quanitified

What are some important controls in the invasion transwell assay

Look at the ratio of invading and migrating cells

Measure MMP activity??

Fluorescnce based approaches

FRET-based protease substrates

How can we monitor invasion in vivo?

Imaging?

Also being very specific with the models used for the experiment we want to do (duh)