Professor
(Rockefeller University)
CIHR Scientist
AHFMR Scientist

Phone: 780-492-1384
Fax: 780-492-0450
Email: rick.wozniak@ualberta.ca

Summary of Research Plan

The Human Genome Project defines, for the first time, the blueprint for human life. The daunting challenge now facing the scientific community is to understand how genes are specifically and coordinately expressed to control cell growth and differentiation. The DNA in every eukaryotic cell is encapsulated in the nucleus by an impermeable membrane called the nuclear envelope and the proteins that regulate its expression are synthesized in the cytoplasm. Cells have made use of this physical separation to control the passage of molecules into the nucleus thereby governing the access of activators and repressors of gene expression to the DNA. To enter the nucleus and access the DNA all molecules must travel through elaborate macromolecular gateways termed nuclear pore complexes (NPCs) embedded in the nuclear envelope. Passage of a protein through this portal requires specific amino acid sequences that are recognized by a class of transporters we term karyopherins, which bind to these sequences and escort the proteins through the NPC. The main focus of my laboratory is to understand the mechanism by which the NPC and karyopherins control constitutive and regulated nuclear transport and how these processes mediate gene expression, chromosome segregation, and cell-cycle progression. Both yeast and mammalian cell model systems are being employed by our laboratory to address the following questions.

How do macromolecules move through the NPC?
Karyopherin/cargo complexes appear to move through the NPC by a series of docking and release steps along an ~200 nm pathway made up of multiple NPC proteins (termed nucleoporins). However, the molecular mechanism that underlies this movement remains undefined. We previously identified a subcomplex of the NPC that contains a receptor nucleoporin (Nup53p), a bound karyopherin (Kap121p), and two other nucleoporins. The dynamics of their interactions are being used to test our hypothesis that the release of a karyopherin/cargo complex from a receptor nucleoporin is driven by conformational changes induced by a separate group of nucleoporins that form the structural framework of the NPC. Release from proximal sites and subsequent binding to more distal sites would allow the import complex to progress through the NPC. Our approaches include a variety of genetic, in situ, and biochemical assays, including the in vitro reconstitution of the NPC subcomplexes.

Are nuclear transport pathways differentially regulated?
In yeast, 14 separate karyopherins have been identified, each of which is believed to transport separate, but overlapping, groups of cargoes. Thus altering the translocation of a specific karyopherin through the NPC could provide a mechanism for coordinately controlling the import of its cargoes. Such a mechanism could be employed in response to environmental cues or temporally controlled events such as cell cycle progression and development. We proposed this idea on the basis of our studies showing a specific interaction of Kap121p with Nup53p. We suggested that this binding step reflects a distinct intermediate in the movement of Kap121p through the NPC and thus a potential point of regulation. In this regard, we have made the observation that during mitosis Nup53p is phosphorylated and this is accompanied by molecular rearrangements within the NPC that enhance its binding to Kap121p. Coincident with these events, Kap121p-mediated transport is inhibited. These data, coupled with the observation that kap121 temperature-sensitive mutants arrest in mitosis, are consistent with a role for Kap121p in modulating the import of substrates critical for the execution of mitosis. On the basis of these observations, our objectives are to define the mechanism of Kap121p-mediated import inhibition and the functional basis for Kap121p's essential role in progression through mitosis.

What other functions do NPC play?
Recently a number of intriguing observations made by others and us have opened our eyes to other functions performed by the NPC. Our data have established a physical and functional link between the NPC and the spindle checkpoint machinery in the yeast. This machinery is responsible for the faithful separation of sister chromatids during mitosis. We have shown that two proteins required for the execution of the spindle checkpoint, Mad1p and Mad2p, reside predominantly at the NPC throughout the cell cycle until the checkpoint is activated. At this point Mad2p is released from the NPC and accumulates at kinetochores. These findings represent the first report that Mad1p and Mad2p may require specific nucleoporins as a scaffold for their function. Moreover, we have shown that mutations in specific nucleoporins produce a chromosome loss phenotype. Our future goals are to define the mechanistic role that the NPC plays in chromosome segregation - a process essential to life - that when it goes awry, as in cancer, has catastrophic consequences for the cell.

Selected Publications

Role of the nuclear envelope on genome organization and gene expression. Van de Vosse, D.W., Y. Wan., R.W. Wozniak, and J.D. Aitchison (2010) Wiley Interdisciplinary Reviews: Systems Biology and Medicine. In Press

Nuclear transport and the mitotic apparatus: an evolving relationship. Wozniak R, Burke B, Doye V. Cell Mol Life Sci. 2010 Apr 8.

A role for the karyopherin Kap123p in microtubule stability. Ptak C, Anderson AM, Scott RJ, Van de Vosse D, Rogers RS, Sydorskyy Y, Aitchison JD, Wozniak RW., Traffic. 2009 Nov;10 (11):1619-34.

The nucleoporins Nup170p and Nup157p are essential for nuclear pore complex assembly. Makio T, Stanton LH, Lin CC, Goldfarb DS, Weis K, Wozniak RW., J Cell Biol. 2009 May 4;185(3):459-73.

The nuclear export factor Xpo1p targets Mad1p to kinetochores in yeast. Scott RJ, Cairo LV, Van de Vosse DW, Wozniak RW., J Cell Biol. 2009 Jan 12;184(1):21-9.

Cyclin-like oscillations in levels of the nucleoporin Nup96 control G1/S progression. Wozniak RW, Goldfarb DS., Dev Cell. 2008 Nov;15(5):643-4.

Nup53 is required for nuclear envelope and nuclear pore complex assembly. Hawryluk-Gara LA, Platani M, Santarella R, Wozniak RW, Mattaj IW. Mol Biol Cell. 2008 Apr;19 (4):1753-62.

Cell biology: pore puzzle. Aitchison JD, Wozniak RW., Nature. 2007 Nov 29;450(7170):621-2.

Nup53p is a target of two mitotic kinases, Cdk1p and Hrr25p. Lusk CP, Waller DD, Makhnevych T, Dienemann A, Whiteway M, Thomas DY, Wozniak RW., Traffic. 2007 Jun;8(6):647-60.

The role of karyopherins in the regulated sumoylation of septins. Makhnevych T, Ptak C, Lusk CP, Aitchison JD, Wozniak RW., J Cell Biol. 2007 Apr 9;177(1):39-49.

Graduate Students

Luc Cairo

Juliana Capitanio

Diego Lapetina

Jana Mitchell

Chris Neufeldt

David Van de Vosse

Lab Assistants

Anh Dang

Barinder Minhas

Research Associates

Dr. Neil Adames

Dr. Tadashi Makio

Dr. Chris Ptak