CMM 577 EXAM 2 2024 WITH 100% CORRECT ANSWERS

CMM 577 EXAM 2 2024 WITH 100% CORRECT ANSWERSWhat are the three different cytoskeletal elements? Describe them. - correct answer Actin: Responsible for cell protrusions Concentrated at the cell cortex Organize into bundles/networks Microtubules: Long, larger, Made of tubulin Give rise to cilia, mitotic spindles and interphase array Intermediate Filaments: Ropelike fibers made of IF proteins Mechanical strength and support for the entire cell and nuclear lamina Describe the classification system of IF's? - correct answer 5 Main Types: Toward the N terminus there is a head domain, form a dimer through coiled coil interaction. C terminal has C terminal tails. Tripartite structure: alpha helical central rod flacked by non helical head and tail domains of variable length and sequence. Thought to regulate intxns b/w Ifs and other proteins. Head domains are essential for IF assembly. Tail domains involved in lateral IF intxns and organization of IF networks. Describe the conserved structural motifs of IF protein subunits. - correct answer Coiled coil is a structural motif where 2-7 alpha helices are coiled together like strands of a rope (typically dimers and trimers). Tertiary structure-protein protein interaction motif, happens in regions that are encoded by alpha helices and that are elongated structures. Coiled coils are composed of heptad repeats, repeating pattern of 7 AAS stabilized by hydrophobic intxns b/w residues in the a and d positions. G position is charged. Describe the assembly of intermediate filament polymers. How are they elongated? How are subunits added/removed? What experiments were used to test this? - correct answer IF assembly occur in stepwise manner. 2 alpha subunits come together to form a coiled coil dimer then 2 dimers come together and form a staggered tetramer of 2 coiled -coil dimers (anti-parallel stagerred tetramer) then 8 of these tetramers come together to form a unit-length filament (cylinder shaped, 16 dimers or 32 coiled-coils). (Monomer, dimer and tetramer are ALL soluble subunits of IFs) Can self-assemble in vitro and does not req energy!!! Describe the mechanical behaviors of IFs. What experiments were used to test this? - correct answer 1. Atomic force microscopy was used to show the mechanical behavior of IFs in vitro. IF can stretch unlike actin and MTs. Fxn as security belt in tissues exposed to mechanical stress like epithelial cells IF assembly: In vitro they assemble in step wise manner. Tagged using fluorescent proteins and visualize their assembly. Then the cells were induced to fuse and if you give them enough time the cells came together end to end. 2. IF elongate by end to end annealing and exchange subunits along their length in cells. Photoactivation and bleaching strategy: Took Mcherry and tagged it to neurofilament protein and used photoactivating GFP. Certain wavelength will activate the GFP on the cell. Take the cell, photobleach one wavelength to bleach out the mcherry signal and then shine a different light the green. In a single cell half should be green and half should be red. 3. IFs incorporate subunits along their lengths Bleach entire cell. Photoactivate ½ the cell to see green. Overtime, the red fluorescent is always being expressed so then both the green and red is visualized together. Describe the keratin cycle. - correct answer 1. Nucleation: Soluble keratin oligomers assemble into particles into the cell periphery near the focal adhesion center 2. Elongation: Particles grow 3. Transport: As they grow, they move toward cell center in an actin dependent process. KF particles are integrated + bundled during transport 4. Maturation: KF bundles are stabilized forming the perinuclear cage or disassembled into soluble oligomers which diffuse and become recycled. How is IF disassembly mediated? What happens to the nuclear lamina during mitosis? How so? - correct answer -Lamina is a biological polymer which can undergo reversible disassembly during mitosis -Lamins head and tail domains are phosphorylated (causes them to become more acidic) by Cdk1--which blocks the ability to form networks--during mitosis to promote their disassembly.