The world is an ever-changing environment. The cell's ability to adapt to environmental changes and take advantage of new resources is crucial to its survival. The careful control of gene expression is the key to this flexibility, and nucleocytoplasmic trafficking lies at the heart of this process. Our aim is to understand at the molecular level the mechanism for highly selective, bidirectional exchange of macromolecules between the nucleus and cytoplasm. Our strategy is to attack at the border, the site of entry and exit, the NPC.
In all eukaryotes, from unicellular organisms to mammals, the NPC is the sole conduit for transfer of material between the nucleus and cytoplasmic compartments While molecules smaller than ~30kDa diffuse freely across the nuclear envelope (NE) through NPCs, large proteins and RNA move through the NPC in a directed manner by active transport. Messenger RNA (mRNA) must travel from the nucleus to the cytosol for translation, while import and export of proteins are essential to virtually every level of cellular function.
The NPC is a huge (>60 MDa) macromolecular assemblage of ~30 nucleoporin proteins (Nups) embedded in pores spanning the double lipid bilayer of the nuclear envelope (NE). There are three distinct NPC domains, the nuclear basket, the cytoplasmic fibrils, and the central core. On the nuclear face, the basket is composed of a protein ring suspended from filaments in eight fold symmetry. Likewise, at the cytoplasmic face, unstructured filaments also positioned in eight fold symmetry serve as attachment points for Nups and other regulatory proteins. The central NPC core proteins are also symmetrically organized, forming a structure often described as a series of concentric rings. The outer ring is composed of a structural scaffold by which more functional Nups are secured. Two major subcomplexes make up this scaffold: the Nup84 subcomplex and the Nic96 complex. The inner ring is populated with functional Nups whose unstructured phenylalanine-glycine (FG) rich protein domains extend into the pore’s central channel. These domains play important roles in the selectivity of nucleocytoplasmic trafficking.
We have three specific areas of interest:
1. How is the functional NPC architecture assembled from its parts, and how does NPC integrity at the nuclear periphery impact gene expression? We are using yeast genetic strategies coupled with fluorescent imaging and biochemical approaches to identify assembly factors and to monitor nuclear dynamics in vivo.
2. What are the mechanisms for regulated movement through NPCs in yeast and mammalian cells? To be transported through the NPC, a cargo must bind to its specific transport receptor, which interacts with phenylalanine-glycine (FG) repeats in a subset of nucleoporins (FG-Nups). Through genetic, molecular and biochemical means, we are investigating interactions between NPC proteins and essential import and export receptors. Studies are focused on the regulation of mRNA export and dynamics of mRNP remodeling.
3. How do NPC-associated factors contribute to other steps in gene expression and in pathology? NPC-associated proteins at both the nuclear and cytoplasmic face are dynamic, functioning apart from the pore at steps all along the gene expression pathway. Thus, there is a vast potential in higher eukaryotes for unidentified tissue-specific roles of Nups and misregulation leading to disease pathologies. For instance, mutations in the gene encoding the mRNA export modulator Gle1 are causatively linked to lethal congenital contracture syndrome 1 (LCCS1). LCCS1 is an autosomal recessive disease resulting in embryonic lethality as a result of severe motoneuron defects. We have determined that mRNA export defects underlie the pathogenesis of LCCS1.