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Capturing directed molecular motion in the nuclear pore complex of live cells.

ABSTRACT: Nuclear pore complexes (NPCs) are gateways for nucleocytoplasmic exchange. Intrinsically disordered nucleoporins (Nups) form a selective filter inside the NPC, taking a central role in the vital nucleocytoplasmic transport mechanism. How such intricate meshwork relates to function and gives rise to a transport mechanism is still unclear. Here we set out to tackle this issue in intact cells by an established combination of fluorescence correlation spectroscopy and real-time tracking of the center of mass of single NPCs. We find the dynamics of nucleoporin Nup153 to be regulated so as to produce rapid, discrete exchange between two separate positions within the NPC. A similar behavior is also observed for both karyopherin?1 transport-receptor and cargoes destined to nuclear import. Thus, we argue that directed Nup-mediated molecular motion may represent an intrinsic feature of the overall selective gating through intact NPCs.

SUBMITTER: Cardarelli F

PROVIDER: S-EPMC3382504 | biostudies-literature | 2012 Jun

REPOSITORIES: biostudies-literature

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Capturing directed molecular motion in the nuclear pore complex of live cells.

Cardarelli Francesco F Lanzano Luca L Gratton Enrico E

Proceedings of the National Academy of Sciences of the United States of America 20120604 25

Nuclear pore complexes (NPCs) are gateways for nucleocytoplasmic exchange. Intrinsically disordered nucleoporins (Nups) form a selective filter inside the NPC, taking a central role in the vital nucleocytoplasmic transport mechanism. How such intricate meshwork relates to function and gives rise to a transport mechanism is still unclear. Here we set out to tackle this issue in intact cells by an established combination of fluorescence correlation spectroscopy and real-time tracking of the center ...[more]

PMID: 22665783

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Project description:Human immunodeficiency virus type 1 (HIV-1) displays the unique ability to infect nondividing cells. The capsid of HIV-1 is the viral determinant for viral nuclear import. To understand the cellular factors involved in the ability of HIV-1 to infect nondividing cells, we sought to find capsid mutations that allow the virus to infect dividing but not nondividing cells. Because the interaction of capsid with the nucleoporin protein 153 (Nup153) is important for nuclear import of HIV-1, we solved new crystal structures of hexameric HIV-1 capsid in complex with a Nup153-derived peptide containing a phenylalanine-glycine repeat (FG repeat), which we used to guide structure-based mutagenesis of the capsid-binding interface. HIV-1 viruses with mutations in these capsid residues were tested for their ability to infect dividing and nondividing cells. HIV-1 viruses with capsid N57 substitutions infected dividing but not nondividing cells. Interestingly, HIV-1 viruses with N57 mutations underwent reverse transcription but not nuclear translocation. The mutant capsids also lost the ability to interact with Nup153 and CPSF6. The use of small molecules PF74 and BI-2 prevented the interaction of FG-containing nucleoporins (Nups), such as Nup153, with the HIV-1 core. Analysis of integration sites in HIV-1 viruses with N57 mutations revealed diminished integration into transcriptionally active genes in a manner resembling that of HIV-1 in CPSF6 knockout cells or that of HIV-1-N74D. The integration pattern of the N57 mutant HIV-1 can be explained by loss of capsid interaction with CPSF6, whereas capsid interaction with Nup153 is required for HIV-1 to infect nondividing cells. Additionally, the observed viral integration profiles suggested that integration site selection is a multiparameter process that depends upon nuclear factors and the state of the cellular chromatin.IMPORTANCE One of the key advantages that distinguish lentiviruses, such as HIV-1, from all other retroviruses is its ability to infect nondividing cells. Interaction of the HIV-1 capsid with Nup153 and CPSF6 is important for nuclear entry and integration; however, the contribution of each of these proteins to nuclear import and integration is not clear. Using genetics, we demonstrated that these proteins contribute to different processes: Nup153 is essential for the HIV-1 nuclear import in nondividing cells, and CPSF6 is important for HIV-1 integration. In addition, nuclear factors such as CPSF6 and the state of the chromatin are known to be important for integration site selection; nevertheless, the preferential determinant influencing integration site selection is not known. This work demonstrates that integration site selection is a multiparameter process that depends upon nuclear factors and the state of the cellular chromatin.

Dataset Information

Capturing directed molecular motion in the nuclear pore complex of live cells.

Publications

Capturing directed molecular motion in the nuclear pore complex of live cells.

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