Research in my lab is focused on understanding how molecules are targeted to particular locations inside cells. Most activities taking place inside cells are carried out by proteins, which are large molecules synthesized in the cytoplasm of eukaryotic cells. Most of these proteins perform their functions at specific sites within the cell, such as within a specific organelle or on a particular cellular membrane. In order for a protein to function at a particular location, it must be targeted to that location by a network of interacting molecules within the cell. Our research addresses how this “network” of targeting molecules functions. Specifically, we are interested in how large molecules, such as proteins and RNAs, are transported between the cytoplasm and nucleus of eukaryotic cells and how the regulation of this transport affects cell functions.
Gaining an understanding of the mechanism of nuclear transport is fundamental to our comprehension of important cellular events, including regulation of gene expression, response of cells to their environment, and even infection of cells by certain types of viruses, including HIV. In eukaryotic cells, proteins and RNAs must constantly be transported across the nuclear envelope separating the nucleus from the cytoplasm. The RNA molecules transcribed from genes in the nucleus must be exported to the cytoplasm before they can be translated into the proteins that carry out most cell functions. Specific proteins, synthesized in the cytoplasm, must be imported into the nucleus to carry out such functions as DNA replication, RNA transcription and processing, ribosome assembly, and regulation of nuclear structure. These RNAs and proteins cross the nuclear envelope through a large, multi-protein channel termed the nuclear pore complex (NPC) [See figure -Schematic diagram of nuclear pore complex structure. From M. Rout and J Aitchison. (2001) J. Biol. Chem., Vol. 276 (20): 16593-16596.
My lab is focused on understanding how the proteins comprising the NPC interact with each other and with soluble “nuclear transport factors” to mediate translocation of proteins and RNA into and out of the nucleus, and on how the regulation of this translocation affects other cellular events and activities.
The primary method we have used to examine nuclear transport is to take advantage of the outstanding genetic methods available in the yeast Saccharomyces cerevisiae. Yeast genes can be fairly easily manipulated, so we can alter specific genes and see how the changes in those genes affect nuclear transport. By studying yeast nuclear transport mutants, we can then begin to understand the function performed by the proteins encoded by these genes. Importantly, nuclear transport occurs by the same mechanism in yeast as it does in other eukaryotic cells
, so identifying these transport proteins and their functions in yeast tells us much about how nuclear transport occurs in our own cells.
Undergraduate students in my lab are working with yeast mutants on three different types of projects, each of which uses a different technique to ask how a particular protein (or set of proteins) might function in nuclear transport. These projects include:
Defects in nuclear transport can be detected by observing altered localization of proteins that normally travel between the nucleus and cytoplasm. We can observe the intracellular localization of a particular protein by expressing it as a fusion with “green fluorescent protein” (GFP) and then observing the location of the glowing GFP in the cell. We are currently looking at changes in localization of specific proteins in cells that are normal versus cells that contain mutations in specific nuclear pore complex proteins.
Cells undergo changes in function in response to signals from their environment. We have been investigating the role of regulated nuclear transport of several proteins in these functional changes. My students and I recently published work
that examined how the nuclear transport of a nuclear mediator of apoptosis (Nma111) is regulated and how this regulation affects programmed cell death (apoptosis) in yeast. We are currently examining the role of several other proteins in regulated cell functions, including the nuclear transport factor Kap108. We have used DNA microarrays to investigate how changes in Kap108 function affect the expression of every gene in the yeast genome, and how exposing cells to stress conditions alter these gene expression patterns.
Proteins are synthesized in the cytoplasm and then targeted to their ultimate location in the cell. This targeting usually is the result of specific amino acid sequences within the protein that function like a type of ‘zip code’ to get it to the correct location. However, the targeting sequence has not been identified for many proteins. We are examining how the nuclear pore complex protein Pom152 gets transported from ribosomes on the endoplasmic reticulum specifically to the nuclear pore complex.
We are performing this investigation of Pom152 targeting in both yeast and mammalian cells using molecular techniques and fluorescence microscopy.
If two proteins are involved in a cellular activity, they will often physically interact or “bind” to each other to carry out that activity. We are testing for interactions between specific nuclear pore complex proteins and soluble transport factors in order to determine if such interactions are important for the translocation of molecules across the NPC. We not only are utilizing these biochemical assays in our research lab, but also have incorporated an experiment examining interactions between nuclear transport factors into our Biology 212 (Molecules, Cells, and Genes) teaching laboratory (see “Teaching Interests” on my main directory page
In my research laboratory (like all labs at Colgate) the original, publishable research we perform is carried out by undergraduate students only. This means that students obtain hands-on experience in the lab, working closely with their research advisor to design and implement a research project in which they are intellectually invested and which utilizes laboratory skills they are interested in developing.