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

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V.6.ii.b Aims:
1. Identify proteins that control astral microtubule plus-end dynamics in the embryo.
2. Develop better methods to quantify astral microtubule distributions, dynamics, and residence times of plus ends at the cortex. Develop methods to identify locations and directions of forces exerted on astral microtubules.
3. Measure changes in those parameters as a function of position in the embryo, stage of mitosis, and presence vs. absence of specific plus-end interacting proteins.
4. Use our data and relevant insights from our plus-end dynamics colleagues (Section V.6.i) to generate mathematical models of rotational alignment, spindle asymmetry, and spindle rocking.

V.6.ii.c Methodology:
V.6.ii.c.1 Identification of regulators of astral microtubule dynamics and interactions:

The Strome and Saxton labs have collaborated to determine which of the 18 kinesin-related and 2 dynein microtubule motors encoded by the C. elegans genome are required in the early embryo, and what roles they serve. In a further collaboration with the White lab, we have investigated the role of γ-tubulin in microtubule nucleation and behavior. These studies have identified three proteins that influence astral microtubules. C. elegans MCAK, like MCAK and its homologue XKCM1 in other organisms, promotes depolymerization of microtubules. C. elegans requires MCAK to orient and position the spindle and to maintain connections between the two half-spindles during anaphase (D. Rose et al., in preparation). C. elegans cytoplasmic dynein is necessary for the correct orientation and positioning of the spindle and for anaphase separation of the spindle poles and chromosomes (D. Schmidt et al., in preparation). C. elegans γ-tubulin, concentrated at centrosomes, is required for correct positioning of the spindle and for formation of a functional spindle. These and other results led to the surprising conclusion that γ-tubulin influences the behavior of spindle microtubule plus ends (Strome et al., 2001). We will investigate how these 3 microtubule-interacting proteins contribute to astral microtubule dynamics.

The motor proteins and γ-tubulin are important, but each represents only a part of a more complex machine. Our objective is to identify all the component parts of these cytoskeletal machines, so we can model their functions accurately. Our initial experimental effort will define the C. elegans microtubule motor "interactome." We have planned a high-throughput yeast two-hybrid screen, using the Gateway system for fast DNA manipulations to identify proteins that interact with the twenty microtubule motors and with γ-tubulin (Walhout and Vidal, 2001). Most of the intended bait cDNAs are already in the Gateway entry vector. After moving the remainder into entry vectors, we will send members of our group to the Vidal laboratory (Harvard), which is actively engaged in C. elegans interactome screens (Walhout et al., 2000). Verification of novel interactions will use GST pull-down assays, which the Strome lab does routinely (Amiri et al., 2001) and which will be facilitated by virtue of the fact that the prey libraries are also constructed in the Gateway cloning system. We will focus subsequent analyses on the interacting proteins for MCAK, dynein, and γ-tubulin.

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:

Interactions of the distal "plus" ends of astral microtubules with the embryo cortex seem to induce rotational realignment of the spindle, anaphase posterior shift, and rocking. Therefore, we will focus initially on the microtubule plus ends at the cortex: the density of microtubule plus ends at the cortex, their residence time at the cortex, and the forces the cortex exerts upon them. We will make these measurements at different positions in the embryo (i.e., anterior, posterior, and equatorial) at various stages of the first cell cycle. After determining values in wild-type embryos, we will determine values in embryos depleted of functional MCAK, dynein, or γ-tubulin. We will also deplete any newly identified interactors to assess their influence on microtubule dynamics and spindle movements.

V.6.ii.c.2.i Mutant embryos:

RNAi can effectively deplete MCAK and γ tubulin (Strome et al., 2001; D. Rose, et al., in preparation). PCR-based screens for deletion mutations are in progress, to provide genetic alternatives to RNAi. We can inactivate dynein by shifting embryos carrying temperature-sensitive alleles of the dynein heavy chain (encoded by let-354) from 16°C to 25°C. We have four dominant and four recessive temperature-sensitive alleles of let-354. For several alleles, dynein inactivation occurs within one minute of the temperature shift and is reversible on a similar time scale, allowing us to disrupt function with excellent time resolution (D. Schmidt et al., in preparation). We will use RNAi to disrupt interacting proteins, identified in the interactome screens.

V.6.ii.c.2.ii Analyzing microtubule ends:

We will measure the density of microtubule plus ends (average number of ends per unit area of cortex) and their residence time (average number of seconds an end stays visible in a cortical optical section) in embryos expressing GFP::tagged β-tubulin (Strome et al., 2001) using an imaging methodology from the Goldstein and Salmon labs (University of North Carolina) (Labbe et al., submitted). We will use a Perkin Elmer spinning disk confocal microscope to image ends of GFP::microtubules in optical sections of cortex. We can determine plus-end residence times for wild-type embryos and for those lacking particular motors or motor associated proteins and compare them with the data from the Goldstein group, which is testing embryos lacking cortical polarity components (e.g., the PAR proteins).