RESUMO
The nuclear pore complex (NPC), the sole gateway for nucleocytoplasmic exchange in eukaryotic cells, allows for the passive diffusion of small molecules and transport-receptor-facilitated translocation of signal-dependent cargo molecules. Whether small molecules passively diffuse through a single central channel or through multiple holes of a hydrogel network is a subject of debate. Additionally, whether the passive and facilitated transport systems occupy distinct or overlapping physical regions of the NPC remains unclear. Here, we directly test these models using three-dimensional super-resolution fluorescence microscopy of human cells. This approach reveals that a single viscous central channel in the NPC acts as the sole pathway for passive diffusion of various small molecules; transport receptors and their cargo complexes take distinct transport routes in the periphery, which is occluded by phenylalanine-glycine filaments. Furthermore, the passive and facilitated passageways in the NPC are closely correlated, and their conformations can be simultaneously regulated by Importin ß1 (a major transport receptor) and RanGTP (a critical regulator of transport directionality). These results strongly favor a self-regulated viscous channel configuration in native NPCs over the porous hydrogel meshwork model.
Assuntos
Núcleo Celular/metabolismo , Glicoproteínas de Membrana/metabolismo , Microscopia de Fluorescência/métodos , Poro Nuclear/metabolismo , Transporte Ativo do Núcleo Celular , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Imageamento Tridimensional/métodos , Glicoproteínas de Membrana/genética , Modelos Biológicos , beta Carioferinas/genética , beta Carioferinas/metabolismo , Proteína ran de Ligação ao GTP/genética , Proteína ran de Ligação ao GTP/metabolismoRESUMO
We present a study on the nuclear export efficiency and time of pre-ribosomal subunits in live mammalian cells, using high-speed single-molecule tracking and single-molecule fluorescence resonance energy transfer techniques. Our findings reveal that pre-ribosomal particles exhibit significantly higher nuclear export efficiency compared to other large cargos like mRNAs, with around two-thirds of interactions between the pre-60S or pre-40S and the nuclear pore complexes (NPCs) resulting in successful export to the cytoplasm. We also demonstrate that nuclear transport receptor (NTR) chromosomal maintenance 1 (CRM1) plays a crucial role in nuclear export efficiency, with pre-60S and pre-40S particle export efficiency decreasing by 11-17-fold when CRM1 is inhibited. Our results suggest that multiple copies of CRM1 work cooperatively to chaperone pre-ribosomal subunits through the NPC, thus increasing export efficiency and decreasing export time. Significantly, this cooperative NTR mechanism extends beyond pre-ribosomal subunits, as evidenced by the enhanced nucleocytoplasmic transport of proteins.
RESUMO
A selective barrier formed by intrinsically disordered Phe-Gly (FG) nucleoporins (Nups) allows transport receptor (TR)-facilitated translocation of signal-dependent cargos through the nuclear pore complexes (NPCs) of eukaryotic cells. However, the configuration of the FG-Nup barrier and its interactions with multiple TRs in native NPCs remain obscure. Here, we mapped the interaction sites of various TRs or FG segments within the FG-Nup barrier by using high-speed super-resolution microscopy and used these sites to reconstruct the three-dimensional tomography of the native barrier in the NPC. We found that each TR possesses a unique interaction zone within the FG-Nup barrier and that two major TRs, importin ß1 and Crm1, outcompete other TRs in binding FG Nups. Moreover, TRs may alter the tomography of the FG-Nup barrier and affect one another's pathways under circumstances of heavy competition.
Assuntos
Poro Nuclear/química , Poro Nuclear/metabolismo , Tomografia/métodos , Células HeLa , Humanos , Imageamento Tridimensional , Complexo de Proteínas Formadoras de Poros Nucleares/química , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas de Transporte Nucleocitoplasmático/química , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Mapeamento de Interação de ProteínasRESUMO
The nuclear pore complex (NPC) mediates the shuttle transport of macromolecules between the nucleus and cytoplasm in eukaryotic cells. The permeability barrier formed by intrinsically disordered phenylalanine-glycine-rich nucleoporins (FG-Nups) in the NPC functions as the critical selective control for nucleocytoplasmic transport. Signal-independent small molecules (< 40 kDa) passively diffuse through the pore, but passage of large cargo molecules is inhibited unless they are chaperoned by nuclear transport receptors (NTRs). NTRs are capable of interacting with FG-Nups and guide the cargos to cross the barrier by facilitated diffusion. The native conformation of the FG-Nups permeability barrier and the competition among multiple NTRs interacting with this barrier in the native NPCs are the 2 core questions still being highly debated in the field. Recently, we applied high-speed super-resolution fluorescence microscopy to map out the natural structure of the FG-Nups barrier and determined the competition among multiple NTRs as they interact with the barrier in the native NPCs. In this extra-view article, we will review the current understanding in the configuration and function of FG-Nups barrier and highlight the new evidence obtained recently to answer the core questions in nucleocytoplasmic transport.
Assuntos
Poro Nuclear/metabolismo , Transporte Ativo do Núcleo Celular , Humanos , Complexo de Proteínas Formadoras de Poros Nucleares/química , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Permeabilidade , Conformação ProteicaRESUMO
The nuclear pore complex (NPC) is one of the largest supramolecular structures in eukaryotic cells. Its octagonal ring-scaffold perforates the nuclear envelope and features a unique molecular machinery that regulates nucleocytoplasmic transport. NPCs are composed of ~30 different nucleoporins (Nups), averaged at 8, 16 or 32 copies per NPC. This estimate has not been confirmed for individual NPCs in living cells due to the inherent difficulty of counting proteins inside single supramolecular complexes. Here we used single-molecule SPEED microscopy to directly count the copy-number of twenty-four different Nups within individual NPCs of live yeast, and found agreement as well as significant deviation from previous estimates. As expected, we counted 8 copies of four peripheral Nups and 16 copies of fourteen scaffold Nups. Unexpectedly, we counted a maximum of 16 copies of Nsp1 and Nic96, rather than 32 as previously estimated; and found only 10-15 copies of six other Nups, rather than 8 or 16 copies as expected. This in situ molecular-counting technology can test structure-function models of NPCs and other supramolecular structures in cells.
Assuntos
Complexo de Proteínas Formadoras de Poros Nucleares/química , Poro Nuclear/química , Proteínas Nucleares/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/química , Transporte Ativo do Núcleo Celular , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Poro Nuclear/metabolismo , Poro Nuclear/ultraestrutura , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Multimerização Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Transport of genetic materials and proteins between the nucleus and cytoplasm of eukaryotic cells is mediated by nuclear pore complexes (NPCs). A selective barrier formed by phenylalanine-glycine (FG) nucleoporins (Nups) with net positive charges in the NPC allows for passive diffusion of signal-independent small molecules and transport-receptor facilitated translocation of signal-dependent cargo molecules. Recently, negative surface charge was postulated to be another essential criterion for selective passage through the NPC. However, the charge-driven mechanism in determining the transport kinetics and spatial transport route for either passive diffusion or facilitated translocation remains obscure. Here we employed high-speed single-molecule fluorescence microscopy with an unprecedented spatiotemporal resolution of 9 nm and 400 µs to uncover these mechanistic fundamentals for nuclear transport of charged substrates through native NPCs. We found that electrostatic interaction between negative surface charges on transiting molecules and the positively charged FG Nups, although enhancing their probability of binding to the NPC, never plays a dominant role in determining their nuclear transport mode or spatial transport route. A 3D reconstruction of transport routes revealed that small signal-dependent endogenous cargo protein constructs with high positive surface charges that are destined to the nucleus, rather than repelled from the NPC as suggested in previous models, passively diffused through an axial central channel of the NPC in the absence of transport receptors. Finally, we postulated a comprehensive map of interactions between transiting molecules and FG Nups during nucleocytoplasmic transport by combining the effects of molecular size, signal and surface charge.
Assuntos
Transporte Ativo do Núcleo Celular , Poro Nuclear/metabolismo , Difusão , Células HeLa , Humanos , Cinética , Microscopia de Fluorescência , Modelos Moleculares , Peso Molecular , Probabilidade , Conformação Proteica , Proteínas Ribossômicas/química , Proteínas Ribossômicas/metabolismoRESUMO
In eukaryotic cells, the bidirectional trafficking of proteins and genetic materials across the double-membrane nuclear envelope is mediated by nuclear pore complexes (NPCs). A highly selective barrier formed by the phenylalanine-glycine (FG)-nucleoporin (Nup) in the NPC allows for two transport modes: passive diffusion and transport receptor-facilitated translocation. Strict regulation of nucleocytoplasmic transport is crucial for cell survival, differentiation, growth and other essential activities. However, due to the limited knowledge of the native configuration of the FG-Nup barrier and the interactions between the transiting molecules and the barrier in the NPC, the precise nucleocytoplasmic transport mechanism remains unresolved. To refine the transport mechanism, single-molecule fluorescence microscopy methods have been employed to obtain the transport kinetics of individual fluorescent molecules through the NPC and to map the interactions between transiting molecules and the FG-Nup barrier. Important characteristics of nucleocytoplasmic transport, such as transport time, transport efficiency and spatial distribution of single transiting molecules in the NPC, have been obtained that could not be measured by either ensemble average methods or conventional electron microscopy. In this critical review, we discuss the development of various single-molecule techniques and their application to nucleocytoplasmic transport in vitro and in vivo. In particular, we highlight a recent advance from one-dimensional to three-dimensional single-molecule characterization of transport through the NPC and present a comprehensive understanding of the nucleocytoplasmic transport mechanism obtained by this new technical development (105 references).
Assuntos
Transporte Ativo do Núcleo Celular , Membrana Nuclear/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Poro Nuclear/metabolismo , Cinética , Microscopia de FluorescênciaRESUMO
The utility of single molecule fluorescence microscopy approaches has been proven to be of a great avail in understanding biological reactions over the last decade. The investigation of molecular interactions with high temporal and spatial resolutions deep within cells has remained challenging due to the inherently weak signals arising from individual molecules. Recent works by Yang et al. demonstrated that narrow-field epifluorescence microscopy allows visualization of nucleocytoplasmic transport at the single molecule level. By the single molecule approach, important kinetics, such as nuclear transport time and efficiency, for signal-dependent and independent cargo molecules have been obtained. Here we described a protocol for the methodological approach with an improved spatiotemporal resolution of 0.4 ms and 12 nm. The improved resolution enabled us to capture transient active transport and passive diffusion events through the nuclear pore complexes (NPC) in semi-intact cells. We expect this method to be used in elucidating other binding and trafficking events within cells.