Exam 3 Flashcards
What are the major features of the 3 cytoskeletal systems and what cellular processes
do they participate in? (Note that mitosis involves all 3!)
The three cytoskeletal systems are microfilaments, microtubules, and intermediate filaments. MF are made of actin, which along with myosin is responsible for muscle contraction, cellular movement (filopodia, leading edge), cell cortex, and microvilli. MT are made of alpha-beta tubulin dimers, and are responsible for axons, cilia, cellular movements (determine direction of migration). In mitosis, MF are used for the contractile ring, MT all come from the centrosomes and pull apart chromosomes, and intermediate filaments are important for permanent structure and change the nuclear membrane prior to formation of the mitotic spindle.
What are the primary structural and biochemical properties of G-Actin and F-Actin? Why do the (+) and (-) ends have different critical concentrations (Cc)? How do these
differences govern microfilament polymerization and depolymerization?
The monomer form is G-actin, the polymer form is F-actin. Actin is an ATPase, meaning it can hydrolyse ATP into ADP on its own. G-actin has four domains and a cleft that ATP/ADP and magnesium binds to. Binding of both ATP and ADP causes a conformational change in G-actin. It is the presence of ATP on the G-actin, not ATP hydrolysis, that is required for polymerization into F-actin. At the plus end of actin, the ATP cleft is hidden. At the minus end of actin, the ATP cleft is exposed. Myosin points towards the minus end due to its tilt. Since G-actin undergoes a conformational change between ATP and ADP binding, the Cc is different at the plus and minus ends. When G-actin initially joins the MF on the plus end, there is a small section where it is still bound to ATP. After time passes, ATP is hydrolysed. The middle part of the MF is ADP-P-Actin and the minus end is ADP-Actin. Treadmilling occurs because of dissociation at the minus end, leading to little to no net growth. Cc is the lowest concentration of G-actin needed to form F-actin; nucleation is the rate limiting step.
How do cells regulate preferential assembly or disassembly at either the (+) or (-) ends
of microfilaments independently of [G-Actin]?
Capping proteins: CapZ prevents assembly at the plus end, tropomodulin prevents disassembly at the minus end. Cofilin fragments ADP-actin at the minus end: small fragments of ADP-actin disassemble quickly. Profilin accelerates the exchange of ADP for ATP and only binds to the plus end: accelerates growth on the plus end only. Thymosin-[beta]4 is a G-actin sequestering protein: it inhibits addition of G-actin to either end.
How can toxins that target the polymerization cycle of actin and are therefore toxic to
cells, be used to study actin dynamics?
Cytochalasin D and latrunculin both promote depolymerization of actin via different methods. The first binds to the plus end and prevents assembly, the second is a G-actin sequestering toxin. These toxins were able to show how a loss of actin assembly affects cell motility, as well as the rate of actin structure turnover. Other toxins that prevent disassembly show the importance of actin turnover.
How do extracellular signals activate small, monomeric G proteins to mediate and
coordinate microfilament dynamics at the leading edge of a migrating cell?
Rac, Rho, and Cdc42 are the three G proteins in the Rho family involved in cell movement. All three are inactive when GDP bound and active when GTP bound. The growth factor binds to a receptor on the cell, which acts as a GEF and activates Cdc42, Rac, and Rho respectively. Cdc42: WASP, Rac: WAVE. These two create branched microfilaments for filling in the parts near the leading edge. Arp2 and Arp3 act as nuclei for branches to form off of. Rho activates formin, which facilitates linear MF formation. Responsible for pseudopods on the leading edge. Rho also can activate myosin to contract stress fibers.
How do dominant negative and dominant active alleles of Rac, Cdc42 & Rho affect cell
migration and morphology?
Dominant negative for all G proteins: wound fails to close if even 1 has a mutant form that can’t exchange GDP for GTP. Dominant active actin: pushing out in all directions, no directed migration. Dominant active Rho: lots of long shards of MF. Dominant active Rac: forms excessive branching near the membrane. Dominant active Cdc42: branched MF go across the entire cell.
