Research

Figure 1: Bipolar spindles formed in C. elegans embryos during mitosis (top) and oocyte meiosis (bottom). Each image shows an entire embryo, so the spindle sizes are to scale. The poles of mitotic spindles contain centrosomes, which nucleate and organize microtubules, leading to large astral arrays of microtubules. Acentrosomal oocyte spindles are much smaller and lack these astral microtubules.

Background and Relevance

Organisms that reproduce sexually utilize a specialized cell division program called meiosis to reduce their chromosome number by half to generate haploid gametes (sperm and eggs). Proper execution of this process is crucial, since errors in meiotic chromosome segregation result in aneuploidy, a leading cause of miscarriages and birth defects in humans. Female meiosis in particular is highly error prone and this vulnerability has a profound impact on human health; it is estimated that as many as 10-25% of human embryos are chromosomally abnormal, and the vast majority of these defects arise from problems with the oocytes. However, despite the importance of female meiosis for human fertility, much of the research done on cell division to date has focused on mitosis, leaving many unanswered questions about how genomic integrity is maintained during the meiotic divisions.

While some mechanisms are likely shared between mitotic and meiotic cell division, oocytes have some key distinguishing features. Perhaps most significantly, oocytes lack centrosomes, which nucleate microtubules and act as structural cues to define and organize the spindle poles during mitosis. Consequently, oocyte spindles are morphologically distinct (Figure 1) and it is therefore important to study oocytes directly to understand how these acentrosomal spindles form and mediate accurate chromosome partitioning. Conversely, studies of oocytes will also inform our view of mitotic cell division; centrosome-independent mechanisms are known to play roles in mitotically-dividing cells but these mechanisms are often masked when centrosomes are present. Therefore, studies of acentrosomal spindles will not only shed light on the special divisions of oocyte meiosis but may also reveal previously hidden mechanisms that operate during mitosis.

 

RESEARCH INTERESTS
In the Wignall lab, we study the molecular mechanisms that contribute to acentrosomal spindle assembly and function. Specifically, we are addressing the following questions:

Figure 2: Imaging of oocytes in live worms expressing GFP::tubulin and GFP::histone to visualize microtubules and chromosomes, respectively. A normal oocyte spindle is shown (top left), in addition to the three phenotypic classes identified in an RNAi screen. Class 1 has reduced microtubules, Class 2 has disorganized microtubules, and Class 3 has monopolar spindles.

1) How are microtubules nucleated?
2) How are spindle poles formed?
3) How do chromosomes align and segregate?

To tackle these questions we use a combination of genetic, genomic, biochemical, and cell biological approaches in the nematode C. elegans.

One major research area in the lab focuses on proteins identified in an RNAi screen. This screen identified new factors involved in multiple steps of acentrosomal spindle formation; the first class plays a role in microtubule nucleation and/or stabilization, the second in organizing microtubules, and the third in establishing and/or maintaining spindle bipolarity (Figure 2 shows the phenotype associated with each class). Projects are now available examining the functions of these proteins, as a means of dissecting the underlying mechanisms of acentrosomal spindle formation.

Another area of research expands upon published discoveries regarding acentrosomal spindle organization (Wignall and Villeneuve, 2009). Specifically, homologous chromosome pairs in oocytes (Figure 3, blue) are ensheathed by microtubule bundles that run along their sides, whereas microtubule density is low at chromosome ends. Further, the plus-end directed motor KLP-19 (Figure 3, red) is targeted to a ring around the center of each chromosome pair and provides a force required for metaphase plate formation.

Figure 3: Model of chromosome congression on acentrosomal spindles. Microtubule bundles (green) ensheath homologous chromosome pairs (blue). A ring of the chromokinesin KLP-19 (red) forms around the center of each chromosome pair, and provides a force to move chromosomes towards microtubule plus ends. Chromosomes therefore align in the center of the spindle, where microtubule plus ends overlap.
Figure 3: Model of chromosome congression on acentrosomal spindles. Microtubule bundles (green) ensheath homologous chromosome pairs (blue). A ring of the chromokinesin KLP-19 (red) forms around the center of each chromosome pair, and provides a force to move chromosomes towards microtubule plus ends. Chromosomes therefore align in the center of the spindle, where microtubule plus ends overlap

Going forward, we will extend these studies by investigating the forces, components, and mechanisms that drive chromosome movements on acentrosomal spindles during both congression and segregation, in the context of this remarkable organization. Specific areas of interest include the contribution of molecular motors and microtubule dynamics to chromosome movements, and the monitoring of the process by checkpoint proteins.

 

 

 

 

 

 

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