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Research

The following is an overview of the types of questions the lab is interested in studying:

 

1.) Inner nuclear membrane composition
In order to understand how the nuclear envelope affects aging and other diseases, we first need to determine what it is made of.  To this end, we have developed an assay to analyze nuclear envelope composition in intact living cells, which will allow us to test if the proteins present at the nuclear envelope change during the cell’s life and affect nuclear function.

 

2.) Transport to and from the inner nuclear membrane
A critical part of understanding how cells build and maintain functional nuclei is to determine how integral and peripheral membrane proteins are targeted to the inner nuclear membrane. It is also important to determine how cells remove non-functional or damaged proteins from the inner nuclear membrane since their dysfunction can result in defects in chromosome organization, transcription, nuclear positioning and permeability.

 

3.) Regulation of spindle pole body duplication
The yeast spindle pole body (SPB) is the functional equivalent of the centrosome. In normal cells, duplication of the SPB/centrosome is coupled with DNA replication so that two microtubule-organizing centers are available to form the poles of the mitotic spindle. We are interested in mechanisms that regulate SPB duplication so that it occurs exactly once each cell division.

 

4.) Formation of nuclear envelope pores
Because yeast undergo a closed mitosis, the SPB(s) must be inserted into the nuclear envelope and separated prior to mitosis to facilitate bipolar spindle formation and accurate chromosome segregation. My lab has elucidated key components of involved in SPB insertion and shown that the SPB provides an excellent paradigm to study the mechanism by which large complexes are assembled into an intact nuclear envelope. Although all eukaryotes undergo de novo assembly of NPCs during interphase, the larger size of the SPB (~500 mDa compared to ~60 mDa NPC in yeast) and fixed timing and position of its insertion (late G1 adjacent to the existing SPB) make it an ideal model for analysis of nuclear envelope remodeling. 

 

Inner nuclear membrane composition

Given the key role the nuclear envelope plays in cellular architecture and in regulation of various biological processes, it is surprisingly how little we know about its composition in any system.  Because of the intricate connections of intracellular membranes, it is virtually impossible to isolate nuclei for proteomic analysis that are not contaminated with ER and mitochondrial proteins as well as many ribosomal and chromatin-associated proteins. Biochemical analysis is further complicated by the fact that many NE proteins are large, hydrophobic and low-abundance.  Because the inner and outer nuclear membranes are separated by only 10-50 nm, electron microscopy (EM) has been the only unequivocal method for determining inner nuclear membrane localization.

 

While precise, EM is time-consuming and not well suited for any type of large-scale study. Therefore, it was essential to develop methods that would allow us to determine the protein composition of the nuclear envelope under different conditions using live imaging in a genetically tractable organism.  Our strategy utilizes a variation of bimolecular complementation system to ‘mark’ proteins that localize in the same nuclear compartment, such as the inner nuclear membrane, perinuclear space or outer nuclear membrane. 

 

We are using this system to determine the composition of the inner nuclear membrane of budding yeast.  Its annotated genome, closed mitosis and established protocols to study aging and aneuploidy make it an ideal model eukaryote.


MYTH

 

The membrane based yeast two-hybrid system used to study inner nuclear membrane composition. Adapted from Chen et al. J Cell Biol. 2014.



Transport to and from the inner nuclear membrane

It has been widely assumed that the localization of inner nuclear membrane proteins would follow similar principles as that of soluble cargo.  The protein might diffuse or be trafficked to the inner nuclear membrane by an inner nuclear membrane protein-derived targeting sequence in an NPC-dependent pathway. This is particularly true in fungi that undergo a closed mitosis where NPC-mediated transport is the sole mechanism of macromolecular exchange between the nucleus and cytoplasm. However, studies of several conserved inner nuclear membrane proteins, including our work on the SUN protein Mps3, have failed to provide a simple paradigm for inner nuclear membrane localization.

 

At least three types of transport pathways have been proposed to account for the current data. It is unclear if the diversity in inner nuclear membrane transport reflects a true diversity of mechanism or if the discrepancies in data are experimental, perhaps based on the reporters and systems used to study inner nuclear membrane localization. We are in a unique position to address this question because of the powerful genetic features of yeast, its well-annotated genome and closed mitosis and our novel inner nuclear membrane localization assay that can be applied to a panel of panel of multiple inner nuclear membrane proteins.  The cis- and trans-acting factors for their localization to the inner nuclear membrane is being evaluated using simple and quantitative molecular genetic approaches.

 

INM transport

Proposed transport pathways of integral membrane proteins from the ER to the inner nuclear membrane.  Adapted from Katta et al. Trends Cell Biol. 2014


Regulation of spindle pole body duplication

In order to form a bipolar spindle during mitosis, two events must occur every cell cycle: DNA replication and duplication of the microtubule-organizing center, known as the SPB in fungi or the centrosome in higher eukaryotes. Defects in centrosome/SPB duplication, including both a failure to duplicate or over duplication, lead to errors in spindle assembly, chromosome segregation and aneuploidy.  Unlike DNA replication, the mechanisms that lead to a precise doubling of centrosomes/SPBs each cell cycle are poorly understood.

 

The SPB is an ideal model to study cell cycle-dependent control of centrosome duplication.  Although the SPB and centrosome are morphologically distinct, multiple components are conserved between SPBs and centrosomes. Many of the same regulatory molecules, including cyclin-dependent kinase, Mps1 kinase and the SCF ubiquitin ligase complex, regulate duplication of both SPBs and centrosomes.

 

We are interested in the role that phosphorylation plays in the control of SPB duplication and have a long-standing collaboration with Mark Winey’s lab at the University of Colorado-Boulder in which we have mappped and mutated phosphorylation sites on SPB components in budding yeast. While cell cycle dependent phosphorylation is important, we are also interested in other mechanisms that may regulate SPB assembly, such as recruitment of factors that remodel the nuclear envelope to allow for SPB insertion. A rate limiting factor appears to be the integral membrane protein Ndc1, which is also a component of NPCs. A nuclear membrane dependent regulator may explain how SPB duplication is controlled in multinucleated filamentous fungus Ashyba gossypii, which undergoes asynchronous nuclear cycles.

 

SPB duplication

 

Schematic of SPB duplication pathway.  Adapted from Jaspersen and Winey Annu. Rev. Cell Biol. 2004; Jaspersen and Ghosh Nucl 2012.



Formation of nuclear envelope pores

A wealth of structural and functional data made the S. cerevisiae SPB an ideal place for the lab to begin studying the proteins involved in forming a pore into which the soluble core SPB is anchored in the nuclear envelope. We term this SPB domain the pore membrane based on its similarity to the region at the NPC where the inner and outer nuclear membranes are contiguous. 

 

Our work on Ndc1, a conserved integral membrane protein found at NPCs and microtubule-organizing centers including the SPB, and the reticulons and Yop1/DP1, conserved ER membrane bending proteins, provides compelling evidence that the same molecular events are likely required to create pore membranes at both the NPC and SPB.  We are using advanced imaging methods to study interactions between Ndc1 and other putative pore components to learn how they are recruited to the pore membrane and interact with each other and the core SPB.

 

Unlike budding yeast, where the SPB is embedded in the nuclear envelope throughout cell division, in S. pombe, the SPB sits on the cytoplasmic face of the nuclear envelope during interphase where is duplicates in late G1 phase. Unlike budding yeast, the fission yeast SPB only enters into a limited fenestrated region of the nuclear envelope upon entry into mitosis, during which time it can assemble nuclear components, undergo separation and form the mitotic spindle.  During telophase, the SPBs are extruded out of the nuclear envelope into the cytoplasm. This cycle of insertion and extrusion is highly regulated and we are using it as a simple screening tool for nuclear envelope remodeling factors.

 

Figure 6-FCCS-final

 

Fluorescence cross-correlation spectroscopy as a method to probe interactions between proteins on the nuclear membrane. From Chen et al. J. Cell Biol. 2014.
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