Cytoskeleton and Cell Motility

V.1 Coordinators
V.2 Participants
V.3 Introduction
V.4 Specific Research Objectives
V.5 Background and Significance
V.6 Research Plan

• V.6.ii Subproject 2 - Microtubule Interactions with the Cortex
• V.6.ii.a Advantages of C. elegans Model
• V.6.ii.b Aims
• V.6.ii.c Methodology
  • V.6.ii.c.1 Identification of regulators of astral microtubule dynamics and interactions
  • V.6.ii.c.2 Develop approaches for quantifying astral microtubule plus-end behavior and force generation; and measure changes in those parameters as a function of position in the embryo, stage of mitosis, and presence vs. absence of selected proteins
    • V.6.ii.c.2.i Mutant embryos
    • V.6.ii.c.2.ii Analyzing microtubule ends
    • V.6.ii.c.2.iii Cortical interaction sites
    • V.6.ii.c.2.iv Localizing and analyzing local cortical forces
  • V.6.ii.c.3 Mathematical models of rotational alignment, spindle asymmetry, and spindle rocking, based on measurements and predictions

V.7 Relation with Organogenesis (project 3) and Biological Networks (project 1)
V.8 Timeline

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V.6.ii.c.2.iii Cortical interaction sites:
1. Rotational alignment: By analogy with findings in the P₁ cell of the two-cell embryo (Hyman, 1989), an anterior cortical site, which may form around the remnant of polar body extrusion appears to control rotational alignment of the spindle in the one-cell embryo (Skop and White, 1998). At that site, a tethered, minus-end-directed motor may capture astral microtubules of the nascent spindle. Preferential attachment to and reeling in of microtubules from one spindle pole rotate the spindle. Dynein is probably the motor; it is minus-end-directed and dynein/dynactin inactivation, prevents rotational alignment (Skop and White, 1998; D. Schmidt et al., in preparation).

2. Spindle asymmetry: Grill et al. (Grill et al., 2001) elegantly demonstrated that differential pulling forces on the two asters of the spindle contribute to spindle asymmetry. They observed that after severing the two half-spindles, the posterior cortex exerted a stronger pulling force on the posterior aster than the anterior cortex did on the anterior aster. The cortical PAR proteins at least in part control this asymmetry: in par-2 mutants, both asters behave like the anterior aster in wild type (both asters are pulled weakly), while in par-3 mutant embryos, both asters behave like the posterior aster in wild type (both are pulled strongly). PAR-2 and PAR-3 localize in the posterior and anterior cortex, respectively; they help restrict the other to its proper domain. Thus, PAR-regulated posterior and anterior cortical domains exert differential pulling forces on astral microtubules.

3. Spindle rocking: We have demonstrated that rocking is dispensable. Under permissive conditions, our ts dynein mutants display little or no rocking and yet show proper spindle asymmetry and develop normally (D. Schmidt et al., in preparation). This result also suggests that dynein is involved in rocking and that rocking is especially sensitive to any perturbation in dynein function. Based on our analysis of GFP::microtubules during rocking in wild type, we previously speculated that the force for rocking stems from microtubule capture by a small number of lateral-posterior cortical sites containing minus-end-directed microtubule motors; those tethered motors (probably dynein) generate force by binding to and pulling on a few astral microtubules (Strome et al., 2001).

VI.6.ii.c.2.iv Localizing and analyzing local cortical forces:

We need to understand the forces that cortical sites exert on nearby astral microtubules. We will perform experiments that will complement those being done by Tony Hyman's lab (sever spindles and analyze the movements of each aster) and Bob Goldstein's lab (laser ablate one centrosome and analyze the movements of the remaining aster). In addition to using their approaches to analyze individual asters, we will use a complementary approach. The activity of MEI-1, which is a homologue of the microtubule severing protein katanin, is required to generate a small meiotic spindle; it must be destroyed to allow assembly of a large mitotic spindle with elaborated asters (Srayko et al., 2000). Mutations that prevent destruction of MEI-1 cause small spindles to form, usually in the posterior of the embryo (Mains et al., 1990; Mains et al., 1990). We can cause them to form in the anterior of the embryo by fertilizing mei-1 oocytes with sperm from males (instead of by the hermaphrodite's own sperm) (Goldstein and Hird, 1996). Our goal is to generate stunted and GFP::tagged spindles in various positions in the embryo, and then measure the rates and extents of movement of the spindles toward the cortex as a function of spindle position in the embryo and the stage of the cell cycle. Some predictions are that a stunted spindle in the anterior of the embryo will be pulled to the anterior cortex during rotational alignment, and that a stunted spindle in the posterior of the embryo will be pulled to the posterior cortex during spindle asymmetry or to a posterior-lateral position during rocking. The effects of depleting MCAK, dynein, gamma-tubulin, and interacting proteins will be analyzed in our mei-1(gf) stunted-spindle assay and also in the aster assays developed by the Hyman and Goldstein labs.