Projects for Research Methodology

Submitted by margaret on Thu, 10/27/2011 - 20:50

Goals: determine the contribution of one (or more) spindle-­-associated protein(s) to mitosis in drosophila S2 cells (controls) and S2 cells in which the centrosome pathway is inhibited.

A. Culture S2 cells (untreated controls) and cells depleted of SAS-­-4 to block the centrosome pathway for spindle formation. Examine spindle formation for both conditions.

B. Select one or more Spindle associated proteins (list below), and deplete the protein from control cells and cells lacking SAS-­-4. Examine spindle formation for both conditions.

C. As an alternative to depletion of SAS-­-4, examine spindle formation in cells treated to disassemble microtubules and then allowed to reassemble microtubules (an assay to examine microtubule formation at chromosomes and centrosomes separately).

D. generate model(s) to explain your observations and test using additional depletions or manipulations.

Rationale: Many proteins have been shown to have a role in spindle formation, but how they contribute to spindle formation when the centrosome pathway is inhibited has not been examined.

Reading: Goshima et al Science 2007. Genes needed for mitosis in drosophila cells; also download the supplemental material for this paper. Goshima et al, JCB Mitotic motors paper Proteins.

  • 1. minus-­-end directed motor proteins (i.e. dynein) link the centrosome to the kinetochore fibers. Proteins to examine: Dynein Heavy chain (DHC); Lis1, a regulator of dynein; dynactin components (another regulator of dynein). Proteins that target dynein/dynactin to kinetochores (Spindly, zest white, rod, etc).

  • 2. kinesins. KLP61F, a crosslinking kinesin. This protein is needed when centrosomes are present, but not when they are absent (in mammals); has not been specifically tested in drosophila. Other kinesins can also be examined (see Goshima, JCB paper).

  • 3. Short spindle phenotype, see Goshima. These proteins were identified in his screen of S2 cells, and have not been examined for a role in spindle formation in cells lacking centrosomes.

  • 4. kinetochore proteins. There are too many to list. We tried Nuf2 and found it has no effect on microtubule assembly in cells released from nocodazole (Tulu, et al). Others have shown that the Ran pathway components (which is required for microtubule formation near chromosomes) localize to kinetochores.

  • 5. Unknowns. Check the list from Goshima. Most of the mitocheck genes that I lookediinto were not present in Goshima’s list, suggesting that they were mammalian specific.