Lecture 6 - Actin and Intermediate Filaments

Describe the structure, subunits, and function of Actin filaments (microfilaments)

Structure:

• Strands in double helix

Subunits

• Actin

Functions

• maintain cell shape by resisting tension (pull)

• move cells via muscle contraction or cell crawling

• divide animal cells in two

• move organelles and cytoplasm in plants, fungi, and animals

Describe the structure, subunits and functions of intermediate filaments

Structure:

• Fibers wound into thicker cables

Subunits:

• Keratin or vimentin or lamin or others

Functions

• maintian cell shape by resisting tension (pull)

• anchor nucleus and some other organelles

Describe the structure, subunits, and functions of microtubules

Structure:

• Hollow tube

Subunits

• a- and B-tubulin dimers

Functions:

• maintain cell shape by resisting compression (push)

• move cells via flagella or cilla

• move chromosomes during cell division

• assist formation of cell plate during plant cell division

• move organelles

• provide tracks for intracellular transport

What is the cortical cytoplasm?

• It is just beneath the plasma membrane and helps to maintian cell shape

• Dominated by an interconnected meshwork of microfilaments (acitn)

• Large membranous organelles and other bulky structures are mostly excluded form this region

What is the Subcortical Cytoplasm (aka Endoplasm)

• Major organelles of the cell reside in this region and are spatially organized and othen attached to cytoskeletal elements

What is the nuclear cytoskeleton?

• Important in mitosis, allows the nucelus to aintain and change its shape

• Nuclear lamins (intermediate filaments) give the nucleus form like a tennis ball

Describe the structure of actin (a microfilament). What is G-actin?

G actin: actin monomer

• Globular

• 42 kDa protein (375 a.a.)

Non-symetrical

• Pointed end (-)

• Barbed end (+)

Binds ADP or ATP in a cleft

• Binding ATP promotes polymerization

• ATP eventually hydrolyzes

How are microfilaments formed? Mention nucleation and F actin

Nucleation: the formation of trimers, followed by more rapid growth

F-actin: Actin polymers

• (-) ends only add to (+) ends, giving the chain polarity

• this is reversible and depends on subunit availability

What is the microfilament tread-mill?

• Occurs because the barbed end of a filament grows 5-10 times faster than the pointed end

• G actin bound to ATP favorably associates with the positive end of the microfilament (where the barbed aspect of actin is exposed)

• Binding of G actin will eventually lead to hydrolysis of ATP to ADP

• The presence of ADP on an exposed end promotes depolymerization

Actin binding proteins are regulated via cell signaling. What are the different actin binding proteins

1. Profilin - stimulates ADP exchange for ATP

2. Formin - catalyzes nucleation and extension of a microfilament

3. Arp 2/3 complex - initiates the formation of branches

4. Capping proteins and tropomyosin - stabilize filaments

5. Cross-linking proteins: cross-link filaments into bundles and networks

6. Cofilin - severs acting filaments

Where are actin filaments located? What do they form?

• Microfilaments (including actin filaments) are more concentrated in the periphery (creating the cell cortex)

• Cortical cytoskeleton is made of actin and spectrin (anchoroed by ankyrin and protein 4.1)

What are focal adhesions and stress fibers?

• Focal adhesions are areas of the cell that makes connections with the extracellular matrix

• These areas of the membrane also tend to have a lot of actin attachments (stress fibers)

Differentiate microvilli and stereocilia

Microvilli

• finger like extensions; abundant on cells involved in absorption formed by actin bundles

Stereocilia

• finger like extensions; not involved in absorbtion, but involved with dtecting extracellular changes (auditory hair cells) formed by actin bundles

What are ‘podia?

These are cell dependent and allow for different variations of movement. They are a more transient structure (unlike microvilli)

What are the proposed steps in locomotion (true for all ‘podia)

1. Protrusion: The leading edge usually shows filopodia and lamelipodia. These extensions probe forward in search of a new substatum contact site

2. Substrate Adhesion: If and when the leading edge ecnounters and adhesive region or the substratum it will attach, stabilizing the forward extension. The mechanism of attachment involves integral membrane receptors and their coupling to internal cytoskeletal elements

3. Traction/Cell Body movement: Once a new forward contact has been made a mechanism must exist to move the bulk of the cell cytoplasm in that direction. Several activities are believed to contribute

What does this movement look like in a cell?

• In most cases, cells move in response to cues from the environment

• Rho family proteins are coupled to receptors and respond to envrionmental cues by activating Acting Binding Proteins

  • This stimulates actin remodleing and cell movement

List the actin associated proteins

1. Profilin

2. Formin

3. Arp 2/3 complex

4. Capping proteins

5. Tropomyosin

6. Cross-linking protiens

7. Cofilin

8. Spectrin

9. Ankyrin

10. Protein 4.1

11. Rho

Describe the structure and assembly of Intermediate filament proteins

• All Intermediate filaments have a similar general structure

  • N and C terminal globular domains

• Step 1 - Two polypeptides will dimerize connected by central rod domain (coiled-coil)

  • This process is in parallel (N-N/C-C)

• Step 2 - Two dimers combine to form a tetramer

  • This process is in antiparallel (N-C/C-N)

• Step 3 - Tetramers will stack on one another end to end to form a protofilament

• Step 4 - 8 protofilaments will combine, twisting around each other to form a rope-like structure called a filament

Describe specifically the roles of intermediate filaments

• Stablize structure and support within the cell

  • Intermediate filaments are more resistant to strech and bending forces than microtubules, but less resistant than actin

  • Positioning of the nucleus and certain organelles

  • support of the plasma membrane

  • support of the nuclear membrane (nuclear lamina)

• Transmitting force between cells or to the extracellular matrix

  • Requires attachement to one or the other via peripheral and transmembrane proteins

  • Resisting and distributing externally generated stresses over multiple cell to prevent damage to individual cells

  • Desosomes - junctions between cells (cell to cell)

  • Hemidesmosomes - jucntions between cell and connective martrix (cell to substratum)