Research focus:  Inner nuclear membrane protein localization and role in chromosome positioning and segregation

In most eukaryotic cells, chromosomes assume a non-random distribution within the nucleus, with each chromosome occupying its own unique position or territory. How the spatial arrangement of chromosomes within the nucleus is established and maintained is largely unknown, but changes in chromosome position have been shown to have dramatic effects on gene expression and genomic stability. This is illustrated by the connection between a number of inherited human diseases and certain types of cancers and mutations in inner nuclear membrane (INM) proteins, which play a critical role in chromosome positioning, gene expression and cellular signaling as well as maintain the structural integrity of the nucleus. Research in my lab uses biochemistry, cell biology and genetics in the yeast model system to study the regulation and function of INM proteins in
chromosome organization and genomic stability.

The following is a selection of ongoing projects in the lab:

SUN proteins
     The conserved SUN (for Sad1-UNC-84 homology) family of INM proteins has been shown by our lab and others to mediate chromosome positioning during meiosis and in mitosis. In Saccharomyces cerevisiae the sole SUN protein Mps3 has an essential function in chromosome segregation due to its role in duplication of the SPB. Mps3 also has non-essential functions in establishment of sister chromatid cohesion during S-phase, in certain DNA damage repair pathways, in telomeric silencing and in rapid prophase movements prior to chromosome synapsis and meiotic recombination. The N-terminal acidic domain of Mps3 extends into the nucleoplasm where it is able to interact with chromatin-associated proteins, such as the histone variant H2A.Z, the silent information regulator Sir4 and factors involved in telomere maintenance. To better understand these diverse nuclear functions of the SUN proteins, we are analyzing mutants in the Mps3 N-terminus that are defective in localization to the INM, are not able to bind to chromatin or are unable to be acetylated in vitro by Eco1, a conserved acetyltransferase required for cohesion that has previously been shown to interact with Mps3. Our preliminary results suggest that multiple pathways converge upon the Mps3 acidic domain to control chromosome positioning, cohesion and segregation.

Inner nuclear membrane localization
     A critical part of understanding how the three dimensional architecture of the nucleus is established and maintained is determining how proteins are targeted to the INM. Some INM proteins contain nuclear localization sequences (NLSs), and it appears that continual nuclear import is mediated by factors analogous to those used by soluble proteins and is primarily responsible for their INM localization. However, many INM proteins, including Mps3, lack a classical NLS sequence and it is unclear how they come to reside in the INM. The import of non-NLS-containing INM proteins may involve passive diffusion through nuclear pores or association with an actively transported protein. We are using avalanche photodiode imaging techniques to examine the effects of disruptions in both active transport and passive diffusion pathways on Mps3 localization to determine how proteins are localized to the INM. To identify Mps3 binding partners in the nuclear membrane, we are examining a subset of the yeast GFP library to screen for proteins which require Mps3 function for correct localization. These studies will elucidate our understanding of how SUN proteins are targeted and maintained at the INM.

Nuclear positioning
     Ashbya gossypii is a multinucleate filamentous fungus that shares a common ancestor with the unicellular budding yeast S. cerevisiae, and this system is amenable to molecular, genetic, biochemical and cytological analysis using many of the same tools we employ in our studies budding yeast. We became interested in the A. gossypii system due to the fact that its nuclei undergo a complex pattern of oscillations, by-passing and positioning that are not observed in budding yeast. Increasing evidence suggests that the most pronounced defect in animal cells lacking SUN function occurs in multinucleated cell types such as muscle and germline. Therefore, the A. gossypii as a model system is ideal to study the function of SUN proteins and their binding partners in the regulation of nuclear dynamics.

SPB duplication
      The budding yeast SPB is the functional equivalent of the centrosome and is the sole site of microtubule nucleation in S. cerevisiae. The SPB is embedded in the nuclear envelope throughout the yeast life cycle and must be inserted and anchored into the membrane following its duplication, similar to nuclear pore complexes. How a massive protein complex inserts into the double lipid bilayer of the nuclear envelope is poorly understood and is an ongoing area of study in the lab. We are also interested in factors that coordinate SPB duplication with cell cycle progression since this is critical for bipolar spindle formation and accurate chromosome segregation during mitosis.