2. Cytoskeleton • Gives the cell mechanical strength, controls its shape, drives and guides its movement • Four classes • Muscle thick filament • Microfilaments • Intermediate filaments • Microtubules

3. Changeable cytoskeleton • The cytoskeleton changes in response to its environment • Are there chemicals the cell likes to stick to? • Is the surface flat or bumpy? • Is the surface stiff or soft? • Are the cells near other cells?

4. Where is actin in a cell? Contractile ring In dividing cells Microvilli cores And terminal web Leading edge of Motile cell Stress fibers And cell cortex

5. Lamellipodia, filipodia and blebs Pinocytotic cup lamellipodia • Membrane protrusions under the control of actin • Can view these as cells move across a surface Bridge still connecting recently divided cells filipodia bleb

6. Solutions and Equipment • PBS • Used to wash the cells • 4% Formaldehyde • Preserves the cells • Glycine • Will prevent other chemicals in the cells from glowing • 0.4% Triton X-100 • Pokes holes in the membrane so the stain can go in

7. Rhodamine Phalloidin • Phalloidin • From the poisonous mushroom Amanita phalloides (Death Cap) • Binds specifically to actin • Rhodamine • Fluorescent marker stuck to phalloidin • When we shine green light on the sample (l = 520-560 nm) • The sample will glow red (l = 650-720nm)

8. Mounting Media • Helps the coverslip stick to the slide

9. Fluorescence Microscopy • Green light photons “promote” rhodamine electron to a higher energy level. When they return to original level, they emit a lower energy photon. • What you can expect to see • Black background, with red stress fibers visible

10. Photobleaching • Keep sample protected from light after adding rhodamine phalloidin • If any fluorescent sample is left exposed too long to light, the fluorescence will fade • Results will not be as good

11. Fluorescence Microscope Diagram

12. Multi colored fluorescence images require changing excitation sources and layering the images • Blue is the nucleus • Red is actin • Green is golgi • Pink is mitochondria