Reformation of the nuclear envelope (NE) around the segregated chromosomes is a key event at the end of mitosis. Defects in this key process may result in alteration of gene expression patterns and genomic instability. We have generated new information about the two major regulators of nuclear assembly, the GTPase Ran and the AAA-ATPase p97.
Our specific aims are: 1. To understand how Importin b, a major RanGTP binding protein, regulates NE fusion and to identify the molecular targets with which it interacts. We will use a 2-color fusion assay and transmission electron microscopy (TEM) to characterize how Importin b inhibits the fusion of chromatin bound vesicles. The dynamics of NE tubule formation and reorganization will be visualized by live imaging on chromatin-coated glass slides. We will use biochemical fractionation methods and affinity chromatography to identify the binding partner(s) of Importin b. 2. To characterize NE membrane sealing and to identify the Ufd1/Np14 regulated NE fusion machinery. To understand p97/Ufd1/Np14-dependent formation of a closed NE, we will use TEM, a novel nuclear exclusion assay, and real time microscopy. To identify additional proteins that interact with Ufd1/Np14 and participate in NE sealing, we will use a recombinant form of Ufd1/Np14 complex as matrix for affinity chromatography. 3. To characterize a second GTPgS-sensitive step in NE formation. We will use the 2-color fusion assay and TEM to characterize this membrane fusion event. We will analyze a membrane-associated GTPase activity on chromatin-bound vesicles. To identify the nature of the additional GTPase(s) we will perform an RNAi screen in C. elegans to test if GTPases known to mediate intracellular membrane fusion (e.g. Rab GTPases) are involved in NE formation. As an alternative approach we will use photo-affinity methods, overlay assays and proteomic approaches to identify the second GTPase.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM073994-02
Application #
7220556
Study Section
Nuclear Dynamics and Transport (NDT)
Program Officer
Shapiro, Bert I
Project Start
2006-05-01
Project End
2011-04-30
Budget Start
2007-05-01
Budget End
2008-04-30
Support Year
2
Fiscal Year
2007
Total Cost
$353,298
Indirect Cost
Name
Salk Institute for Biological Studies
Department
Type
DUNS #
078731668
City
La Jolla
State
CA
Country
United States
Zip Code
92037
Hetzer, Martin W (2010) The nuclear envelope. Cold Spring Harb Perspect Biol 2:a000539
Anderson, Daniel J; Vargas, Jesse D; Hsiao, Joshua P et al. (2009) Recruitment of functionally distinct membrane proteins to chromatin mediates nuclear envelope formation in vivo. J Cell Biol 186:183-91
Anderson, Daniel J; Hetzer, Martin W (2008) The life cycle of the metazoan nuclear envelope. Curr Opin Cell Biol 20:386-92
Schulte, Roberta; Talamas, Jessica; Doucet, Christine et al. (2008) Single bead affinity detection (SINBAD) for the analysis of protein-protein interactions. PLoS One 3:e2061
Kutay, Ulrike; Hetzer, Martin W (2008) Reorganization of the nuclear envelope during open mitosis. Curr Opin Cell Biol 20:669-77
Anderson, Daniel J; Hetzer, Martin W (2008) Reshaping of the endoplasmic reticulum limits the rate for nuclear envelope formation. J Cell Biol 182:911-24