Cytoskeletal Filaments and Tubulins, Exams of Cell Biology

Download Cytoskeletal Filaments and Tubulins and more Exams Cell Biology in PDF only on Docsity! Exam 2 (CBIO 3400 Gaertig, Fall 2020) page 1 Name: 1) Describe the conditions under which cytoskeletal filaments undergo treadmilling. What is the role of treadmilling in the cell? (7). Treadmilling occurs at the concentrations of g-actin above the critical concentration for the plus end and below the critical concentration for the minus end. The plus end grows while the minus end shrinks. Treadmilling maintains the length of the filament. The growing plus end can produce force by pushing the membrane (e.g. plasma membrane or vesicle membrane). 2) Briefly compare the functions of a-, b- and -tubulin. (6). All these tubulins share amino acid sequence homology. a-tubulin and b-tubulin form dimers that are the building blocks of the microtubules. Within the protofilament, the a/b dimers are arranged head-to-tail which results in b-tubulin being exposed at the plus end and a-tubulin being exposed at the minus end. Both a- and b-tubulin bind GTP but only GTP on b-tubulin hydrolyzes and this promotes microtubule depolymerization. g-tubulin functions at the minus end of microtubules as part of the ring-shaped g-TuSC complex that nucleates microtubules. 3) List the mechanisms that mediate either the addition or loss of tubulin subunits in microtubules (7). addition loss Polymerization at the plus or minus end (can be enhanced by XMAP215 polymerase at the plus end) Internal incorporation following generation of “bites” by katanin/spastin Depolymerization at the plus or minus end (can be enhanced by kinesin-13) Severing or “biting” by spastin or katanin 4) Do intermediate filaments have polarity? Explain your answer based on the structural data (6). No. The basic unit of IFs is a dimer of two proteins that are connected by a coiled coil. The dimer is polarized because the N-and C-termini of the two subunits are located at the opposite ends. However, these dimers form antiparallel tetramers (by hydrophobic “lock and key” interactions). Next the tetramers associated side-by-side to form protofilaments. A completely assembled intermediate filament has identical ends and therefore is not polarized in the way microtubules or microfilaments are. 5) Give an example of a disease caused by a mutation in cytoskeletal protein. Explain how the mutation lead to the disease phenotype (6). Examples of good answers that link the protein/filament level, cell level and organism/disease level: 1) Epidermis bullosa simplex is caused by mutations in keratin. Mutant keratin fails to form proper intermediate filaments. The epithelial cells that express mutant keratin become more fragile due to reduction in the mechanical strength. 2) Wiscott-Aldrich syndrome is caused by mutations in Wasp. Wasp is an activator of Arp2/3. A mutation in Wasp reduces the formation of the branched network of microfilaments which in turn impairs cell motility and cell shape of blood cell types (lymphocytes and platelets). stereocilia to move which stretches the inter-row links. The stretching of the plasma membrane opens up the mechanosensitive cation channels which ultimately leads to depolarization and release of neurotransmitters from the hair cells. Exam 2 (CBIO 3400 Gaertig, Fall 2020) page 3 Name: 1) Explain the origin of bipolarity of the mitotic spindle (7). In interphase, the centrosome undergoes duplication. The two centrioles move away and each serves as a template for the formation of a daughter centriole. At the onset of mitosis, the two centrosomes move away from each other to establish the spindle poles. 2) Predict the consequences of treating mammalian cells with monastrol during anaphase. Explain how monastrol acts at the molecular level. (7) Monastrol is a specific inhibitor of kinesin-5, a bipolar plus end directed mitotic kinesin. During anaphase, kinesin-5 mediates sliding of the interpolar microtubules as part of anaphase B which promotes separation of the spindle poles. With monastrol, spindle would fail to elongate properly. 3) Describe the experiment which led to a model that microtubule depolymerization produces force during mitosis. (7) The plus ends of microtubules of marked polarity were allowed to be captured by chromosomes in vitro. Microtubule depolymerization was induced by dilution of tubulin concentration below the Cc+. as the microtubule plus ends depolymerized, the chromosomes remained bound. No ATP was included so motor proteins were not active. Thus, microtubule depolymerization can produce force for chromosome movement that mimics the movement that occurs during anaphase A. 4) Propose an approach to rapidly inactivate formin during the late phase of cell division (after metaphase) and predict the likely results. (7) Develop a plasma membrane penetrating inhibitor. Alternatively, inject anti-formin antibodies (that block its activity) or identify a temperature-sensitive mutation in the formin gene that at the permissive temperature leads to a loss of function. One of these treatments will block cytokinesis due to inability to assemble microfilaments of proper length. This is caused by blocking the activity of formin as a plus end polymerase. 5) The organization of microfilaments changes in a migrating cell in response to external signals. Explain the underlying mechanism (in broad terms). (6) An external signal binds to a plasma membrane-based receptor and activates a signaling response that ultimately leads to activation of GEFs, also at the plasma membrane. These GEFs then mediate loading of G-proteins (CDC42, Rac and Rho) with GTPs. Active G-proteins bind to their respective effectors that function as regulators of microfilament organization. For example, in the front of the motile cell, activated Rac activates Wasp which in turn activates Arp2/3 to form the branched network of microfilaments that pushes the plasma membrane forward.