Molecular Size and Charge Effects on Nucleocytoplasmic Transport Studied By Single-Molecule Microscopy

In eukaryotic cells, essential genetic information is sequestered in the prominent organelle nucleus. To facilitate the selective bidirectional trafficking of genetic material, proteins and ribonucleoprotein cargoes across the nuclear envelope (NE), nucleus has evolved the highly sophisticated machinery, nuclear pore complex (NPC). Thousands of NPCs on the surface of the NE serve as highly-selective machinery for bidirectional nucleocytoplasmic transport. The selective barrier, which is formed by the natively unfolded phenylalanine-glycine (FG)-nucleoporins (Nups), in the NPC allows for two transport modes: passive diffusion of small signal-independent molecules (< 20-40 kDa) and transport-receptor facilitated translocation of signal-dependant cargoes. The molecular size of a cargo molecule is one of the important parameters that determine the final transport mode for its transport through the NPCs. Due to the elusive native configuration of the FG-Nup barrier, however, the dependence of the structure of the channel, the transport kinetics and the exact selective mechanism on the molecular size remain in dispute. The second important criterion for nucleocytoplasmic transport selectivity through the NPCs is the charges of the transiting molecules. Previously it was suggested that the positively charged FG-Nups form a selective barrier which would inhibit the transport of positively charged cargoes unless they are chaperoned by negatively charged transport receptors. However, the lack of complete knowledge about the precise structure of the FG-barrier in the NPC leaves much space for speculations on the charge effect of substrates on passive and facilitated transport.Finally, the surface hydrophobicity of substrates has been shown to be another important selection criterion. The hydrophobic interactions between the FG-barrier and transport receptors are believed to be the essential process for transport receptor-facilitated translocation through the NPCs. However, it is still unknown whether only the hydrophobic surface of a cargo molecule can enhance its successful transport, especially for those large molecules above the cut-off size. Thorough study of these selection criteria for nucleocytoplasmic transport under physiological conditions will finally provide better understanding of the structure and function of the NPC and eventually resolve the nucleocytoplasmic transport mechanism. It is known that molecules interact with the flexible FG-Nups at the millisecond or the sub-millisecond level in a three dimensional space of the NPC. To fully appreciate the structural complexity of the NPC and its mechanism, thus, one should be able to capture these extremely fast interactions with an outstanding precision in all three dimensions. None of the existing techniques is capable of doing that. A novel method is needed. We have developed an innovative single-point edge-excitation sub-diffraction (SPEED) microscopy in our lab, which allows us to track single transiting molecules with super spatiotemporal resolutions and create 3D spatial density map of the transient interactions in the NPC[1]. By the method, we plan to investigate the transport kinetics and three-dimensional spatial pathways of various transport probes with various sizes, hydrophobicities and charges in permeabilized Hela cells. Our major findings include: (i) there is a viscous self-regulated channel that serves as a sole gateway for passive diffusion of small- and medium-sized molecules, occluded by the distinct, but not completely spatially separated pathways of facilitated translocation on the periphery of the NPC; (ii) the net positive charge of the NPC indeed affects the efficiency of both passive diffusion and facilitated translocation depending on the charge of the cargo molecules, and also affects the spatial distribution of the molecules depending on their charges; (iii) the highly surface hydrophobic property of cargo molecules enhances their efficient nucleocytoplasmic transport.

Name thesis:degree_name

Doctor of Philosophy (Ph.D.)

Level thesis:degree_level

doctoral

Discipline thesis:degree_discipline

Biological Sciences

Grantor dc:publisher

Bowling Green State University

Year dc:date

2013

Author dc:creator

• Goryaynov, Alexander G.

Contributors dc:contributor

• Yang, Weidong

• Biophysics
• 3D imaging
• nucleoporins
• nuclear pore complex
• single-molecule fluorescence
• super-resolution microscopy

Statement dc:rights

• unrestricted
• This thesis or dissertation is protected by copyright: all rights reserved. It may not be copied or redistributed beyond the terms of applicable copyright laws.

Language dc:language

English

Repository record dc:identifier

http://rave.ohiolink.edu/etdc/view?acc_num=bgsu1357278635

OAI identifier oai:identifier

oai:etd.ohiolink.edu:bgsu1357278635