Primary Goal The goal of this project is to unravel the nucleocytoplasmic transport mechanism and to advance our understanding of how transport maintains or through dysfunction fails to maintain in cells. The advanced knowledge obtained by these investigations provides the foundation for further disease diagnosis, treatment and prevention. Intellectual Merit Nuclear pore complexes (NPCs) span the nuclear envelope and enable bidirectional trafficking between the cytoplasm and nucleus in eukaryotic cells. Small molecules (<20-40 kDa) passively diffuse through the NPCs. Translocation of larger molecules (up to 50 MDa) is hindered by the phenylalanine- glycine (FG) nucleoporin (Nups) barrier inside the NPCs unless they are chaperoned by transport receptors. However, challenged by measuring the spatial structure of FG repeats and a series of transient interactions between the transport receptors and the FG repeats, the precise transport mechanism remains in dispute as to how different-sized molecules pave their pathways through the permeable barrier of the NPC. To unravel the transport mechanism, principal Investigator develops an innovative single molecule method to meet the major challenges. Research Approach The principal Investigator's lab develops a novel single-molecule approach, single-point edge-excitation sub-diffraction (SPEED) microscopy, to test nuclear transport models with a spatiotemporal resolution of 9 nm and 400 ms. SPEED microscopy enables us to capture transient interactions occurred in the NPC under real- time trafficking conditions that have escaped detection by previous single-molecule methods or electron microscopy. Principal Investigator proposes studies with the following SPECIFIC AIMS: (1) To determine the 3D spatial locations of transport pathways for small molecules in permeabilized cells. (2) To determine the 3D spatial locations of transport pathways for more transport receptors and their cargo complexes in permeabilized cells. (3) To characterize the 3D conformational change of FG-Nups at various transport receptor concentrations in permeabilized cells. (4) To determine the 3D pathways for small molecules, transport receptors and facilitated translocations in live cells.
Specific aim 1, 2 &4 will examine the hypothesis that small molecules diffuse through a central channel, and that small and large molecules spatially separate the pathways.
Specific aim 3 &4 will specifically test whether the transport receptors collapse the FG-repeats filaments or dissolve into filamentous meshwork. Broader Impacts Dysfunctions of the nuclear transport system are linked to numerous human diseases including leukemias, cancers, and primary biliary cirrhosis. Success of this project will significantly change the landscape of research on nuclear transport and also provide new approaches to biomedical imaging, drug development, and medical diagnostics and treatments of these human diseases.

Public Health Relevance

Dysfunctions of the nuclear transport system are linked to numerous human diseases including leukemias, cancers, and primary biliary cirrhosis. Understanding of nuclear transport mechanism will directly impact our understanding and development of therapeutics for a number of human diseases. In this proposal, we employ novel single molecule approaches to further unravel the nuclear transport mechanism.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM097037-03
Application #
8549263
Study Section
Nuclear and Cytoplasmic Structure/Function and Dynamics Study Section (NCSD)
Program Officer
Lewis, Catherine D
Project Start
2011-09-15
Project End
2016-08-31
Budget Start
2013-09-01
Budget End
2014-08-31
Support Year
3
Fiscal Year
2013
Total Cost
$280,526
Indirect Cost
$97,176
Name
Temple University
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
057123192
City
Philadelphia
State
PA
Country
United States
Zip Code
19122
Kelich, Joseph M; Yang, Weidong (2014) High-resolution imaging reveals new features of nuclear export of mRNA through the nuclear pore complexes. Int J Mol Sci 15:14492-504
Goryaynov, Alexander; Yang, Weidong (2014) Role of molecular charge in nucleocytoplasmic transport. PLoS One 9:e88792
Yang, Weidong (2013) Distinct, but not completely separate spatial transport routes in the nuclear pore complex. Nucleus 4:166-75
Ma, Jiong; Liu, Zhen; Michelotti, Nicole et al. (2013) High-resolution three-dimensional mapping of mRNA export through the nuclear pore. Nat Commun 4:2414
Goryaynov, Alexander; Ma, Jiong; Yang, Weidong (2012) Single-molecule studies of nucleocytoplasmic transport: from one dimension to three dimensions. Integr Biol (Camb) 4:10-21
Ma, Jiong; Goryaynov, Alexander; Sarma, Ashapurna et al. (2012) Self-regulated viscous channel in the nuclear pore complex. Proc Natl Acad Sci U S A 109:7326-31