MCDB 165A Measuring/Manipulating Mechanical Forces Flashcards

Luciferase Assay

Used to measure the activity of specific regulatory elements (enhancers and promoters). It is really a measure of activity of transcription factors.

Your favorite gene (UFG) is replaced by the Luciferase protein gene (under control of enhancers/promoters). If luciferin is present, luciferase catalyzes a change in this molecule, creating a small molecule and emitting light.

Bioluminescence

Utilizes a chemical reaction to create light.

The amount of light emitted reflects the amount of luciferase protein, which indicates…

how active the enhancer/promoter is.

Differences in the mechanical properties of tissues can...

have consequences on the behavior of cells (e.x mechanical properties of tissues can affect cell differentiation).

Durotaxis

Cells tend to crawl towards stiffer substrates. One group tested this by making a gel soft on one side and stiff on the other side.

Tissues and cells are…

viscoelastic substances (characteristics of solids and liquids). Depends on time scale: with slow deformation cells often flow but with rapid deformation they can store stress like solids.

How do you measure viscoelasticity?

Apply a defined force to a tissue/cell and measure the resulting deformation. “Young’s modulus”, measured in pascals, describes the elasticity of a material.

Atomic Force Microscopy (AFM)

A point is pressed into a cell. If the cell is stiff, the cantilever will deflect, but if the cell is soft the cantilever will sink in. This deflection is measured, allowing you to obtain the elasticity value.

Actin

Exists in two forms, G-actin (monomers) that polymerize into F-actin (filaments). These are the main components of stress fibers.

Myosin

Myosin II relevant to stress fibers. Dimers of myosin can form bipolar arrangements (minifilaments) that bind to actin. This arrangement allows both sides to move actin to the center. This process is ATP-dependent.

One mechanism for cells to sense the state of the ECM:

Myosin-based stress fiber contraction pulls on focal adhesions. If integrins are bound to a rigid substrate, pulling will deform intracellular focal adhesion components, like Talin and Vinculin. The altered conformation of these proteins allows it to signal to downstream factors.

Methods to Change Cell Tension

1) Change the stiffness of the ECM/gel the cells are plated on.

2) Crowding of cells

3) Put cells on lawns of different sizes (allowing them to stretch more or less).

Micropillars

Can be made stiff/nondeformable or soft/deformable. The tension changes, as a stiff substrate generates a harder pulling force. Biochemical environment does not vary much.

Artificial Gels (Varying Stiffness)

Altering cross-linking in artificial gels alters the stiffness of the environment. The biochemical conditions are kept the same, while changing the mechanical properties. Fewer cross-links makes the gel more deformable.

Lysyl oxidase (LOX) enzymes cross-link…

collagens to stiffen the ECM.

You can plate cells on a dish that has an empty chamber that is filled with fluid (creating a dome)…

causing the cells on top to be stretched without changing the number of cell-cell adhesions.

Microfluidic Channels

Cells move through narrowings in a channel, creating mechanical tension/challenges.

How can we measure cell tension?

1) Cells under tension have more stress fibers. 2) Cells under tension activate YAP/TAZ.

Severing Assay

Use a laser to burn a junction, and monitor the two ends and see the rate of separation, which indicates the tension at that junction.

Tension Biosensors

Measures the conformational change of the focal adhesion proteins. For instance, you can take Vinculin and put two FRET fluorophores in between. When the protein is not pulled, the fluorophores are close and FRET can occur. When the cells are pulled, you lose FRET (higher tension).

Traction Force Microscopy

Cells are placed on a gel impregnated with fluorescent beads. As the cell moves, it displaces the beads. Bead displacement can be used to calculate direction and magnitude of cellular forces.