The primary research focus of our lab is the structure and function of the centrosome. The animal cell centrosome is a tiny non-membrane-bound organelle (its volume is ~1 µm3) consisting of a pair of centrioles sourrounded by the ‘pericentriolar material’. Because it is the primary microtubule organizing center and also serves as a cellular ‘meeting point’, the centrosome is essential for a plethora of fundamental intracellular processes that involve microtubules, including (but not limited to) vesicular trafficking, mRNA localization, cell polarity, morphogenesis, cell migration, and, perhaps most importantly, cell division. Not surprisingly, defects in centrosomal protein function lead to a number of human diseases, including developmental defects, cancer, infertility, mental retardation, and obesity. We would like to understand how this important organelle carries out its functions, and how these functions are regulated during the cell cycle. We currently concentrate our research efforts on proteins required for microtubule nucleation, stabilization, and organization. We combine a variety of modern cell biological approaches for our work including light and electron microscopy, molecular genetics, biochemistry, protein crystallography, and molecular modeling. We collaborate with various labs in the Biochemistry Department, across campus, across the country, and in Europe to achieve our experimental goals.
Microtubule organization in the cell – the function of the centrosome
Centrosomes require a specialized tubulin named γ-tubulin for their function. One major focus of the lab is in understanding how γ-tubulin contributes to microtubule formation and anchoring. γ-Tubulin associates with at least two additional proteins, GCP2 and GCP3, in all cell types examined. In metazoans, the core tetramer composed of two molecules of γ-tubulin and one molecule each of GCPs 2 and 3, associates with 4-5 additional proteins to form a 2.2 MDa structure that appears as an open spiral when isolated from Xenopus eggs or Drosophila embryos. This structure is named the γ-tubulin ring complex (γTuRC) and its components the gamma complex proteins (GCPs).
We are interested in understanding, in molecular detail, how the γTuRC functions in microtubule formation and organization. Using a combination of experimental tools including molecular genetics, molecular biology, high-resolution fluorescence and time-lapse microscopy, and in vitro analysis of purified proteins, we are probing the mechanisms of the centrosome that are responsible for much of the overall structural organization within a living cell.
The role of importin-β in regulating mitotic spindle assembly
The mitotic spindle is an elaborate macromolecular assembly that is responsible for the segregation of chromosomes during cell division. Assembly of the mitotic spindle is tightly regulated and requires precise spatial and temporal coordination of many cellular proteins and processes. In most animal cells, the centrosome plays an important role during spindle assembly by nucleating and organizing many of the microtubules that make up the spindle. However, microtubules are also nucleated in the vicinity of chromosomes and are organized into a bipolar array by the action of molecular motors. The chromosome-dependent pathway of spindle assembly is particularly important in female meiosis, which occurs naturally without centrosomes. Chromosome-dependent microtubule assembly is mediated by the small GTPase, Ran, and its downstream effector, importin β. Importin β acts like a brake on the system; binding of RanGTP to importin β inactivates the brake, thus allowing microtubule assembly to proceed. Our current models postulate that importin b regulates spindle assembly by inhibiting proteins that are required to stabilize and organize microtubules. We have developed assays to identify candidate proteins that may be regulated by importin β in mitosis. Our goal is to determine how these candidate proteins participate in spindle assembly, and how importin β regulates
Nuclear envelope assembly
The defining feature of eukaryotic cells is the separation of the chromosomes from the cytoplasm, which is achieved by the nuclear envelope. The nuclear envelope is highly selectively permeable and is composed of a double lipid bilayer (inner and outer nuclear membranes, respectively), nuclear pore complexes, and the nuclear lamina. The enclosure of the genetic information within the nucleus generates a unique environment that separates DNA replication, transcription, and RNA processing from protein synthesis. In most metazoan cells, the nuclear envelope disassembles at the beginning of mitosis and reforms following segregation of the duplicated chromosomes. We have recently begun to study the roles of spindle assembly factors in post-mitotic nuclear envelope assembly.