Our lab has two primary areas of interest: (1) the structure, function and assembly of the vertebrate nucleus, and (2) the mechanism by which the cell regulates where and how to assemble major mitotic structures, such as the mitotic spindle, the nuclear membranes and nuclear pore complexes.
A focus of the research in my laboratory is the eukaryotic nucleus: its structure and function. We work both in vivo and in vitro. For in vivo studies, we use mammalian cultured cell lines to examine nuclear transport or assembly using RNAi technology to knock down expression of genes of interest to determine their effect on function. For in vitro studies, we use a nuclear reconstitution system which efficiently assembles nuclei in the test tube from soluble and membrane components. This system makes use of an extract of Xenopus eggs, which naturally store abundant amounts of disassembled nuclear components in preparation for the rapid cleavages of early division. Upon addition of DNA or chromatin to the in vitro interphase extract, nuclei containing nuclear membranes, a nuclear lamina, and nuclear pores quickly assemble. The in vitro reconstituted nuclei are capable of nuclear import, DNA replication, pol III transcription, and even mitotic disassembly. In parallel, we use a mitotic egg extract to study spindle and kinetochore assembly.
A long-standing interest of the laboratory has been the nuclear pore, a large macromolecular complex of 120 million daltons that spans the nuclear membranes. All communication between the nucleus and cytoplasm occurs through the nuclear pore. In vivo, the pore complex imports nuclear proteins and exports mRNA, tRNA, snRNAs, and ribosomal precursors. We study: (1) assembly of the nucleus itself, (2) assembly of nuclear pores, (3) the regulation of these processes, and (4) the mechanism of nuclear transport, including mRNA export, protein export, and protein import. Using antibodies we can immunodeplete a nuclear assembly extract of individual nuclear pore proteins, then reconstitute nuclei which lack that protein. Such "designer" nuclei can be tested for alterations in nuclear pore structure, pore function, or pore assembly. In this way we discovered the protein responsible for initiating pore assembly at the nucleus (Rasala et al, 2006, 2008). This protein, ELYS, acts by binding to chromatin at AT-rich sequences and recruiting key structural subunits of the nuclear pore to these sites to initiate nuclear pore assembly. With experiments of this type, we hope to molecularly dissect and elucidate the gates that "guard the fortress" of the genome.
The second major interest of the laboratory involves understanding the regulation of assembly of the major mitotic structures. Specifically, we are focused on how assembly is regulated such that the mitotic spindle, and later the nuclear membranes and nuclear pores, form only around chromatin, i.e., in a spatially precise manner. We have shown that an abundant cellular protein, Importin beta, acts in mitosis to negatively regulate both nuclear membrane fusion and nuclear pore assembly everywhere except in the vicinity of the chromosomes (Harel et al, M.B.C, 2003). The small GTPase Ran, produced in its GTP-bound form only near chromatin, acts as the counteracting positive regulator, a “GPS” to tell where assembly should occur. We have extended this work to Transportin, a second karyopherin. We believe that Importin beta and Transportin act as global regulators of cellular events involving the genome, including those controlling nuclear import, spindle formation, correct nuclear membrane assembly, and nuclear pore assembly.
Douglass Forbes received her Ph.D. from the University of Oregon and was an American Cancer Society postdoctoral fellow at UCSF in the Dept. of Biochemistry and Biophysics. She was a PEW Fellow in the Biomedical Sciences, and an elected member of the Governing Council of the American Society of Cell Biology. Dr. Forbes served as the Vice Chair of Cell and Developmental Biology from 2000-2005 and 2007-2009. She has served on multiple editorial boards, both past and present.