Cytoskeleton 2: Actin & Cell Motility Flashcards
- Describe the three types of cytoskeletal elements, their properties, their functional roles, and their protein composition.
Microfilaments/actin:
-essential for amoeboid motility,
-cell shape, and polarity
-7nm in diameter, with
-globular actin (G-actin) that assembles into 2-stranded, helical filaments (F-actin).
-Also polarized –> + end (hidden cleft, grows) and – end (exposed cleft = depolymerization).
-If ATP-bound, forms filaments; if ADP-bound, doesn’t form filaments.
Kd ratio describes the rate of dissociation at – end.
-Steps regulating formation of actin:
= nucleation, and extension/retraction.
-Actin needs 3 monomers to form a nucleation center.
-Doesn’t occur spontaneously; helped by formin and Arp2/3.
-ARP allows for polymerization at the minus-end, allowing rapid elongation at + end branched actin filaments (necessary for lamellipodia).
-Formin forms parallel actin bundles (like microvilli, filopodia, and actomyosin ring).
-Formin mimics actin and brings 2 actin monomers together to form the trimer –> makes an actin dimer.
-Arp2/3 works the same way in that it facilitates the process; if i am correct, it doesnt bring in 2 actin monomers like the other one, but it acts like 2 actins and binds a single monomer of actin .
-Regulated by RhoGTPase, which opens the formin up and activates it to help form nucleation center.
-Actin helps polarize epithelial cells (apical vs. basolateral side).
- The 2 domains are separated by tight junctions and adherens junctions.
-These junctions only work if they’re fixed to the cytoskeleton (decreased association of AJ proteins, like cadherin and catenin, with actin –>internalization and loss of adhesion –> EMT and cancer).
-Actin helps form apical microvilli.
-MF bundle forms the core of microvilli; actin + ends anchored in the protein cap and bundles held together by villin and fimbrin proteins.
-Bundles linked to plasma membrane by myosin-I.
-(Loss of microvilli = microvilli inclusion disease).
-MF helps stabilize plasma membrane –> determines shape and movement of cell surface.
Appendages = microvilli, filopodia, and lamellipodia.
-Filopodia and lemellipodia can adhere to or detach from the cell substratum using adhesion molecules.
-F-actin can form stress fibers (contractile bundles) that associate with focal adhesions.
-These things anchor cells to the substratum and exert tension across the cell –> low motility.
-Actin organization and cell shape controlled by Rho family (members of Ras GTPases).
-Actin + GTP = active.
-Actin + GDP = inactive.
-Rho causes formation of stress fibers and focal adhesions.
-Rac causes formation of veils.
-Cdc42 causes protrusion of filopodia.
- Discuss cytoskeletal dynamics and the role of certain proteins in actin filament formation, polymerization/depolymerization.
- Actin-binding motor proteins belong to myosin family (heavy chain) –> related to kinesins and contain a head region (with ATPase activity and actin binding sites) and tail region.
- ATPase activates when bound to actin and moves to the + end.
- Tail region binds other molecules.
- Myosin II in striated muscle –> 2 heavy and 2 light chains –> form thick filaments in muscle that use ATP to walk down actin and contract muscle.
- Non-conventional myosins like I and V associate with membrane –> F-actin mediated movement of organelles.
- Describe the role of actin cytoskeleton in epithelial cell polarity and discuss some diseases associated with that.
Loss of microvilli observed in microvilli inclusion disease.
- Explain the concept of molecular motion, and the mechanism of actin-based organelle movement and muscle contraction.
Myosin:
-Has two head domains and an alpha helical domain.
-Actin and myosin filaments are arranged into thick and thin filaments –> hydrolyze ATP –> change conformation –> causes a step, or power stroke that moves myosin along the actin and causes muscle contraction.
-Has a much slower hydrolysis cycle compared to dynein/kinesin –> only 5% bound to ATP at a time, but lots of heads are working –> allows for muscle relaxation.
Conventional myosin = skeletal muscle. Unconventional myosin involved in transport (associated with membranes and movement of organelles – MyosinV acts like kinesin).
- Discuss the concept and the key steps of cell movement.
- Actin aids with amoeboid movement –> crawling along a surface –> requires actin cytoskeleton and cell adhesion.
- Changes in actin cytoskeleton aid with movement –> involve Rho/Rac GTPases that regulate random migratory activity (chemokinesis) and directed migration (chemotaxis) –> cells can respond to attractants (positive chemotaxis) or repellents (negative chemotaxis) –> causes movement towards or away from the sources of these factors. (Ex: inflammation produces attractants so attract leukocytes).
1) Protrusion:
- Movement of filopodia and lamellipodia driven by polymerization of actin at the leading ends.
- The + end faces forwards, - ends linked to other MFs via ARPs (in a branched web) –> controlled by Rac and WASp –> stimulate Arp2/3.
- Actin assembly at leading edge push plasma membrane forward, while disassembly happens in the back.
2) Attachment and traction:
- protrusions must form attachment with substratum –> attachments sites are anchorage points that pull cell forward.
- Traction force generated by myosin.
3) Detachment:
- earlier formed adhesions in the back have to be released for movement.
* *Motility depends on adhesion in a biphasic manner.
- If cells adhere too tight, can’t migrate.
- As adhesion decreases, motility increases, to a certain point.
- If adhesion decreases too much, you also lose motility because there’s no traction.
- Eventually, cells detach completely.
- Goes to show that attachment and detachment must be coordinated for movement.
- Discuss cell motility in the context of developmental and disease processes.
-Phalloidin toxin stabilizes F-actin and causes net increase in actin polymerization (from death cap mushroom).
- In development, neural crest cells have to migrate to reach their destinations –> originate from ectoderm and give rise to pigment cells and PNS.
- Axons also depend on nerve growth cone (amoeoid) to grow over long distances.
- Leukocytes have to move to infection sites.
- Cancer becomes metastatic when they invade healthy tissues.
Wiskott-Aldrich syndrome:
- WAS = X-linked immunodeficiency disease resulting from WASp mutations.
- Results in thrombocytopenia and recurrent infection.
- Platelets get sloughed off lamellipodia –> thrombocytopenia results from defective lamellipodia/platelet formation.
Lissencephaly:
-brain has smooth cortical surface (no gyri) due to loss of function in n-cofilin, an actin depolymerizing factor –> severe mental retardation.
- Describe the role of actomyosin ring in cell division.
- Actin helps with pinching the two cells apart in cytokinesis.
- Formation of actomyosin ring = a contractile ring that pinches the cell in 2–> drives formation of the cleavage furrow and separation of daughter cells.
- Site of ring formation and timing of contraction = highly regulated because needs to happen AFTER chromosomes separate –>otherwise can get trisomy.
- Regulated by Rho-GTPase, which activates formin to help form contractile ring. Rho activates ROCK kinase as well –> turns on the myosin part of ring.
- Describe the mechanisms regulating the establishment and activation of the actomyosin ring and identify examples of asymmetric cell division.
-Sometimes need to have asymmetric cell division.
-In an enucleation process, division happens such that only the nucleus in pinches off to form a separate ‘cell’ that gets eaten by a macrophage.
-Resulting reticulocyte has no nucleus.
-In the formation of platelets, don’t get cytokinesis until you have up to a 128n polyploidy megakaryocyte –> then cytokinesis can occur and you get platelets (also without nuclei).
Spermatogonia also undergo incomplete cytokinesis–> delay in cell division.
