Academic Background

Ph.D., 1985, Michigan State University

Nuclear Architecture and Gene Expression

Nucleic acid metabolism is architecturally organized in the eukaryotic nucleus. Nucleic acids themselves- as well as their metabolism in transcription, RNA processing, and RNA export- are structurally constrained to dynamic nuclear domains. Our larger goal is to understand the mechanisms that accomplish the self-assembly of these domains and achieve the spatial organization of gene expression. Our approach is an interdisciplinary one, combining biochemistry and molecular biology with confocal and electron microscopy.

One ongoing project studies the role of the nuclear matrix protein, SRm160, in RNA splicing and export. SRm160 is recruited to pre-mRNA during spliceosome formation, participates in splicing, and then is retained as part of an Exon Junction Complex (EJC) of proteins bound to the fully processed mRNA. This EJC facilitates the efficient export of mRNA from the nucleus to the cytoplasm. The EJC restructures at the periphery of the nucleus and SRm160 is retained in the nucleus and recycled.

One of our current approaches is to track the movement of EJC complexes and components in the nuclei of live cells by time-lapse confocal microscopy and fluorescence recovery after photobleaching (FRAP). New FRAP techniques we have developed have shown that the intranuclear mobility of the EJC components SRm160 and RNPS1 is ATP dependent, suggesting that their assembly into complexes and movement are regulated. Electron microscopy studies of the SRm160 distribution have shown tracks of the protein between sites of RNA splicing and the nuclear lamina. Our working model, combining these observations is that SRm160-containing complexes may move toward the cytoplasm by a docking-release mechanism regulated by ATP. Stringing binding sites along a track would tend to constrain diffusion to preferred paths within the nucleus.

Other ongoing studies projects study additional protein components of the EJC, study the SRm160-interacting splicing factor SRm300, and measure the binding and mobility of mRNA itself in the nucleus. One collaborative project studies the changes in nuclear and chromatin structure that accompany and facilitate the tissue formation of human breast epithelial cells. We continue to develop new techniques for FRAP experiments and for the analysis of FRAP results.

Director for Cell Biology Confocal Core - Three Dimensional Microscopy - www.umassmed.edu/3dml

Figure

Figure: Resinless section of a CaSki cell nuclear matrix. Soluble proteins and chromatin have been removed from this nucleus, uncovering the nuclear matrix which consists of two parts. The nuclear lamina is the outer shell of the matrix which lies just under the nuclear envelope and is primarily composed to the lamin proteins A, B, and C. Connected to the lamina and extending throughout the nuclear volume is the internal nuclear matrix, an intricate structure built on a scaffolding of 10 nm filaments whose molecular composition remains unknown. The largest masses remaining in the interior are remnants of nucleoli.