1. INTRODUCTION
Unit 8 - Cytoskeleton

2. Microtubules Actin Microfilaments
3 types of fibers make up the cytoskeleton
                        
                                                    
Intermediate Filaments
Microtubules
Actin Microfilaments
Microtubules interact with motor systems: dyneins and kinesins
Microfilaments interact with myosins
Intermediate filaments do not interact with motor proteins

3. “provide a supporting framework within the cell”
Intermediate Filaments
FUNCTION:
“provide a supporting framework within the cell”
These supportive fibers provide for mechanical reinforcement of tissues and range in diameter from 8-10 nm

4. Intermediate Filaments - STRUCTURE
monomer
dimer
Have great tensile strength!
Strengthens cells against mechanical stress!
tetramer
2 tetramers coiled together
8 tetramers twisted into a ropelike filament
10nm

5. Types of intermediate filaments
nuclear lamins form a meshwork that stabilizes the inner membrane of the nuclear envelope;
keratins – junctions in epithelial cells (desmosomes) and also form hair and nails;
neurofilaments - strengthen the long axons of neurons;

6. What cytoskeletal element is stained green in this immunofluorescence experiment? Why?
Intermediate filaments. They form a network that links to other cells at junctions on the plasma membrane
Cell boundaries stained in blue

7. Intermediate Filaments
Why can you use intermediate filaments to tell whether a cancer has spread in the body?

8. Microtubules
FUNCTION: These macromolecular assemblies are involved mainly in the movement and positioning of cell organelles.
- Minus end is attached to centrosome (or Microtubule organization Center)
- Plus end is free

9. Microtubules - STRUCTURE
Long, hollow cylinders made of 13 protofilaments and built by the assembly of dimers of alpha tubulin and beta tubulin.
Right. Electron Microscope Image of microtubules

10. MICROTUBULES Rapid Growth = plus end
- The growing end of the microtubule has subunits arranged with the beta-tubulin on the outside. The subunits in the microtubule all show a uniform polarity

11. Microtubule polymerization
Tubulin dimers + GTP (red) bind more tightly to one another than tubulin dimers + GDP (dark green) creating a GTP cap.
microtubules keep growing with freshly added tubulin dimers and GTP

12. Microtubule depolymerization
- Slow microtubule growth causes the "GTP cap“ to hydrolyze GTP to GDP The GTP cap is lost
the GDP-carrying subunits are less tightly bound in the polymer and readily released from the free end
The microtubule shrinks

13. Centrosomes
Organized array of microtubules that radiate outward from it through the cytoplasm.
Contains ring-shaped structures of g-tubulin.
Serves as starting point for growth of new microtubule

14. - - + + Motor Proteins Dyneins Kinesins Travel towards MINUS END
Travel towards PLUS END - - + +
Dyneins
Kinesins

15. Actin microfilaments
FUNCTION: support components of the cell and provide for the movement of cytoplasm and the cell surface
Microfilaments Interact with many types of molecules including its own class of motor proteins, the myosins

16. Bundles of Actin filaments in cells
Actin in RED:
Sheet-like and finger-like protrusions from the leading edge of a moving cell
Contractile ring during cell division
Microvilli
Contractile bundles in cytoplasm

17. Actin - STRUCTURE
Actin microfilaments are comprised of actin monomers that polymerize to form long, thin fibers. These are about 7nm in diameter.

18. Actin Polymerization Can occur at either end Faster rate at PLUS end
Triphosphate (ie ATP) hydrolyzed to diphosphate (ie ADP) soon after incorporation into actin filament.
Nucleotide hydrolysis promotes depolymerization

19. ATP Hydrolysis
Actin monomers in the cytosol carry ATP, which is hydrolyzed to ADP soon after assembly into growing filament.
ADP molecules remain trapped within actin filament, until the actin monomer that carries them dissociates from filament to form a monomer again.

20. Drug Treatments
Colchicine: Binds free tubulin and inhibits formation of microtubules by preventing polymerization
Taxol: Stabilizes microtubules by preventing depolymerization
Cytochalasin: inhibits formation of actin by preventing polymerization
Phalloidin: stabilizes actin filaments preventing depolymerization

21. Practice Questions:

22. Here we see Phalloidin linked to a green fluorescent dye
Here we see Phalloidin linked to a green fluorescent dye. What cytoskeletal element is being stained green? How do you know?
Nucleus stained blue
Golgi stained red
What technique was used?

23. What cytoskeletal element is indicated by the arrow
What cytoskeletal element is indicated by the arrow? What are TWO other functions of this element?

24. NEXT TUTORIAL
MITOSIS