Biology Department

Research Overview

In eukaryotic cells, the mitotic spindle is the cytoskeletal apparatus that segregates the genetic information contained in chromosomes to each of the daughter cells. In artistic renditions of cell division, the placement of mitotic spindle is often depicted exactly in the center of the two dividing cells. But for a large number of cell types, the mitotic spindle is actually found off-center. Asymmetric positioning of the mitotic spindle is an active process that allows cells to generate asymmetric cell divisions. This permits the asymmetric distribution of cell-specificity determinants and the establishment of different cell fates, a critical aspect of many types of cell differentiation. When spindle positioning does not occur correctly, chromosomal mis-segregations can occur, resulting in genetic instability. This is an important component of many cancers.

The work in my lab is focused on elucidating the molecular mechanisms that control spindle positioning. For this, the Miller laboratory uses the model organism S. cerevisiae. In this yeast, the cell divides by budding. Thus, the plane of cell division is specified by the position of the emerging bud, yielding an asymmetric cell division. This type of cell division requires that the mitotic spindle be placed not at the middle of the cell, but instead across the mother-bud neck of the dividing cell. In S. cerevisiae, the spindle is contained entirely within the nucleus. The minus ends of cytoplasmic microtubules are attached to the nucleus at the cell’s microtubule-organizing center, the spindle pole body (SPB), which is embedded within the nuclear envelope.

In yeast, spindle positioning requires two separate but partially redundant systems. The first, referred to as the Kar9p pathway, involves the movement of the nucleus up to the bud neck and the alignment of the mitotic spindle along the mother-bud axis. A defining feature of the Kar9p pathway is that it provides orientation to the cytoplasmic microtubule. It does this by connecting the cytoplasmic microtubules (that are attached to the mitotic spindle) to the polarized actin cytoskeleton within the cell. The second process, referred to as the dynein pathway, is responsible for the translocation of the elongating nucleus across the bud neck. Dynein is a well-characterized minus-end directed microtubule motor involved in several important processes within the cell, including vesicle transport, kinetochore functions, and spindle assembly in mammalian cells. Dynein is complexed with and activated by the multi-subunit dynactin complex. After anaphase, the nucleus continues to elongate until the nuclear masses are separated at the distal ends of the mother and bud. Finally, cytokinesis ensues and the cells enter G1.

Spindle positioning is a process that integrates many facets of cell biology. For many cell types, it utilizes both the actin and microtubule networks, two of the major cytoskeletal systems in the cell. Accurate spindle positioning also requires that the spindle recognize the polarity cues within the cell. Further, placement of the spindle must also be integrated within the proper context of the cell cycle, occurring neither too early nor too late. Many of the components utilized in positioning the mitotic spindle are conserved across many species, including mammals.

The Miller lab is particularly interested in the mechanisms that regulate spindle positioning. We focus on two post-translational modifications, phosphorylation and sumoylation. Phosphorylation is the process by which a phosphate group is added to a protein, usually through a residue containing a hydroxyl group. This occurs through the coordinated action of a kinase. Sumoylation adds the small ubiquitin-like protein SUMO to target proteins through a covalent attachment on lysine residues. We use a variety of approaches to answer questions on these topics, including genetics, biochemistry, and cell biology with an emphasis on microscopy.