INTRODUCTION Unit 8 - Cytoskeleton

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  

“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  

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

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;

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

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

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  

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

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

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

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

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

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

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  

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

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

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

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.

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

Practice Questions:

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?

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

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