Kathryn Ryan

Research Interests

Nuclear Pore Complex Assembly

Eukaryotes require continual exchange of macromolecules between the nucleus and the cytoplasm. Nuclear pore complexes (NPCs) are large, proteinaceous structures that span the fused inner and outer nuclear membranes to form the passageway. Multiple copies of ~30 different proteins, collectively termed nucleoporins, comprise the mature NPC. Its overall structure is characterized by 8-fold rotational symmetry. A combination of spoke- and ring-like substructures forms a core to which cytoplasmic fibrils and a nuclear basket attach. Changes in growth conditions, developmental state, signaling cascades, cell cycle, and viral infection affect which proteins and RNAs are trafficked between the nucleus and the cytoplasm; however, all movement occurs through NPCs. Therefore, elucidating the molecular mechanism of NPC assembly and determining the interactions required for its structural integrity are the long-term research goals in the Ryan lab.

The lab takes advantage of the yeast Saccharomyces cerevisiae to focus on the mechanism of NPC assembly. As a postdoc, Dr. Ryan conducted a genetic screen to identify factors required for NPC assembly using GFP-reporters in live cells. Using this approach, a large number of n uclear p ore complex a ssembly (npa) mutant were isolated. Current research in the Ryan lab has two major objectives:

1. Delineate the function of the Ran cycle in NPC assembly
Ran is a small GTPase that cycles between a GTP and GDP bound form to regulate many nuclear processes. All 4 components of the Ran cycle were isolated in the npa screen. Characterization of these mutants revealed membrane defects and the accumulation of nucleoporin containing vesicles in the cytoplasm. The accumulation of such vesicles in these npa mutants suggests that NPC assembly involves a Ran-mediated vesicular fusion event at the outer nuclear envelope. In this model of NPC assembly, a subset of nucleoporins is first concentrated in vesicles (A). When the vesicles fuse with the outer nuclear membrane in a Ran-dependent manner (B), a critical, localized concentration of these nucleoporins triggers pore formation (C) and nucleates new NPC assembly (D and E).

To test the model, work is being done to characterize these vesicles. This includes biochemical approaches to purify vesicles and cell biological and genetic approaches to determine how vesicle-associated proteins contribute to NPC assembly. In addition, we are working to understand how Ran interacts with these vesicles to mediate vesicle fusion to the outer nuclear membrane.

2. Define additional steps in the NPC assembly pathway
There are events both upstream and downstream of the Ran cycle in the assembly pathway. Further cloning and characterization of mutants from the npa collection will continue to identify factors involved in other steps of NPC biogenesis and provide a platform from which to study these discrete events.

Laboratory Details

Laboratory Address:
Biological Sciences Building West
Room 435
979-458-1422