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.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Study Section
Nuclear and Cytoplasmic Structure/Function and Dynamics Study Section (NCSD)
Program Officer
Lewis, Catherine D
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Temple University
Schools of Arts and Sciences
United States
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Ruba, Andrew; Luo, Wangxi; Yang, Weidong (2018) Application of High-speed Super-resolution SPEED Microscopy in Live Primary Cilium. J Vis Exp :
Li, Yichen; Luo, Wangxi; Yang, Weidong (2018) Nuclear Transport and Accumulation of Smad Proteins Studied by Single-Molecule Microscopy. Biophys J 114:2243-2251
Mudumbi, Krishna C; Yang, Weidong (2017) Determination of Membrane Protein Distribution on the Nuclear Envelope by Single-Point Single-Molecule FRAP. Curr Protoc Cell Biol 76:21.11.1-21.11.13
Ma, Jiong; Kelich, Joseph M; Junod, Samuel L et al. (2017) Super-resolution mapping of scaffold nucleoporins in the nuclear pore complex. J Cell Sci 130:1299-1306
Luo, Wangxi; Ruba, Andrew; Takao, Daisuke et al. (2017) Axonemal Lumen Dominates Cytosolic Protein Diffusion inside the Primary Cilium. Sci Rep 7:15793
Mudumbi, Krishna C; Schirmer, Eric C; Yang, Weidong (2016) Single-point single-molecule FRAP distinguishes inner and outer nuclear membrane protein distribution. Nat Commun 7:12562
Verhey, Kristen J; Yang, Weidong (2016) Permeability barriers for generating a unique ciliary protein and lipid composition. Curr Opin Cell Biol 41:109-16
Ma, Jiong; Kelich, Joseph M; Yang, Weidong (2016) SPEED Microscopy and Its Application in Nucleocytoplasmic Transport. Methods Mol Biol 1411:503-18
Ma, Jiong; Goryaynov, Alexander; Yang, Weidong (2016) Super-resolution 3D tomography of interactions and competition in the nuclear pore complex. Nat Struct Mol Biol 23:239-47
Ruba, Andrew; Yang, Weidong (2016) O-GlcNAc-ylation in the Nuclear Pore Complex. Cell Mol Bioeng 9:227-233

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