UC San Diego

The nucleus is the defining structure of eukaryotic cells. The nuclear envelope acts as a barrier between nucleus and cytoplasm. Nuclear pore complexes perforating the envelope control all traffic into and out of the nucleus, and thus act to regulate transcription, translation, and other essential cellular processes. During mitosis, the nuclear envelope from flies to mammals disassembles into its component parts, with the nuclear pore breaking into multiple subunits. The pore then reassembles in a step- wise process as the nuclear envelope reforms in late anaphase. The major focus of this thesis has been to better understand the assembly and function of the nuclear pore. It has resulted in three published papers and one paper in preparation. First, I participated, with postdoc Dr. Corine Lau, in a study of the novel vertebrate transmembrane nucleoporin, Ndc1. I resolved the topology of yeast Ndc1p and identified conserved amino acids to target for future functional studies (Chapter 1). In a second study, I participated in a collaboration with the laboratory of Dr. Pamela Silver at Harvard Medical School in identifying a new role for the vertebrate nuclear pore in the regulation of transcription. We found that in vertebrates, specific chromosomal regions move to the nuclear pore complexes during transcriptional activation (Chapter 2). Key work next centered on the mechanism of action of importin beta in negatively regulating nuclear membrane fusion and pore assembly. (The small GTPase, Ran, positively regulates both these processes.) A major unanswered question has been, which specific steps in nuclear pore assembly are regulated by importin beta or RanGTP. I determined, using Xenopus constructs, that importin beta is an authentic regulator of nuclear pore assembly and that, contrary to previously published results, Ran reverses this negative regulation (Chapter 3). Finally, a fourth study, done with Dr. Corine Lau, established that the distant importin beta relative, transportin, also negatively regulates nuclear membrane and pore assembly (Chapter 4). I showed that both transportin and importin beta act early to control the initial step of pore assembly: the binding of the pore- targeting protein ELYS to chromatin, which sets in motion the specific targeting of nuclear pores to the nuclear surface