Nuclear pore complexes (NPCs) are large proteinaceous assemblies that provide the only known portals for the exchange of macromolecules between the nucleus and cytoplasm. Revealing how the transport apparatus is assembled will be critical to understanding the mechanism of nucleocytoplasmic communication. The goal of our work is to investigate the pathway of NPC biogenesis at the molecular level. Our experimental method is designed to benefit from the strength of combining molecular genetic, biochemical, and cell biological approaches. Our first specific aim is to identify genes that regulate NPC formation with a fluorescence-based screen in the yeast Saccharomyces cervisiae. This approach is based on the functional tagging of NPC proteins with the green-fluorescent protein (GFP), and the hypothesis that NPC assembly mutants will have distinct GFP signals as compared to wild type cells. These GFP signal differences will allow mutants to be isolated by fluorescence activated cell sorting and direct microscopic screening. Three classes of mutants are expected: 1) NPC clustering, 2) fewer total NPCs per nucleus, and 3) wild type NPC number, but each NPC has a decreased amount of GFP-protein incorporated. In the second aim, we will analyze the mechanism of action of potential assembly factors by assaying the mutants in situ for perturbations of NPC assembly. NPC density and general morphological structure analysis will be assessed by electron microscopy. Rate of NPC assembly will be determined by two complementary approaches: monitoring incorporation of GFP-proteins in live cell assays, and pulse chase analysis. NPC dynamics will be monitored with a live cell assay measuring NPC movement rates. This analysis will include both novel mutants and a nup57 mutant identified in our preliminary studies.
We aim to reveal whether the proteins are required for the incorporation of a nearest neighbor interacting protein(s) or if they are global assembly factors required for multiple aspects of NPC structure. The third specific aim is focused on characterizing a novel integral membrane protein with connections to NPC function. We will determine the membrane topology of Sn11p, and test for genetic and biochemical interactions with NPC components. Finally, we propose to isolate vertebrate homologues of yeast NPC assembly factors, and conduct direct tests for their roles in NPC assembly with the established Xenopus nuclear assembly assay. Together these studies are expected to significantly advance our understanding of how the nuclear transport apparatus is assembled and functions.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM057438-01
Application #
2599007
Study Section
Molecular Cytology Study Section (CTY)
Project Start
1998-07-01
Project End
2002-06-30
Budget Start
1998-07-01
Budget End
1999-06-30
Support Year
1
Fiscal Year
1998
Total Cost
Indirect Cost
Name
Washington University
Department
Physiology
Type
Schools of Medicine
DUNS #
062761671
City
Saint Louis
State
MO
Country
United States
Zip Code
63130
Casey, Amanda K; Chen, Shuliang; Novick, Peter et al. (2015) Nuclear pore complex integrity requires Lnp1, a regulator of cortical endoplasmic reticulum. Mol Biol Cell 26:2833-44
Adams, Rebecca L; Wente, Susan R (2013) Uncovering nuclear pore complexity with innovation. Cell 152:1218-21
Vargas, Jesse D; Hatch, Emily M; Anderson, Daniel J et al. (2012) Transient nuclear envelope rupturing during interphase in human cancer cells. Nucleus 3:88-100
Casey, Amanda K; Wente, Susan R (2012) Nuclear transport: shifting gears in fungal nuclear and cytoplasmic organization. Curr Biol 22:R846-8
Burns, Laura T; Wente, Susan R (2012) Trafficking to uncharted territory of the nuclear envelope. Curr Opin Cell Biol 24:341-9
Casey, Amanda K; Dawson, T Renee; Chen, Jingjing et al. (2012) Integrity and function of the Saccharomyces cerevisiae spindle pole body depends on connections between the membrane proteins Ndc1, Rtn1, and Yop1. Genetics 192:441-55
Burns, Laura T; Wente, Susan R (2012) Nuclear GPS for interchromosomal clustering. Dev Cell 22:1119-20
D'Angelo, Maximiliano A; Gomez-Cavazos, J Sebastian; Mei, Arianna et al. (2012) A change in nuclear pore complex composition regulates cell differentiation. Dev Cell 22:446-58
Vargas, Jesse D; Hatch, Emily M; Anderson, Daniel J et al. (2012) Transient nuclear envelope rupturing during interphase in human cancer cells. Nucleus 3:
Talamas, Jessica A; Hetzer, Martin W (2011) POM121 and Sun1 play a role in early steps of interphase NPC assembly. J Cell Biol 194:27-37

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