Nucleocytoplasmic transport: a thermodynamic mechanism

The nuclear pore supports molecular communication between cytoplasm and nucleus in eukaryotic cells. Selective transport of proteins is mediated by soluble receptors, whose regulation by the small GTPase Ran leads to cargo accumulation in, or depletion from the nucleus, i.e., nuclear import or nuclear export. We consider the operation of this transport system by a combined analytical and experimental approach. Provocative predictions of a simple model were tested using cell-free nuclei reconstituted in Xenopus egg extract, a system well suited to quantitative studies. We found that accumulation capacity is limited, so that introduction of one import cargo leads to egress of another. Clearly, the pore per se does not determine transport directionality. Moreover, different cargo reach a similar ratio of nuclear to cytoplasmic concentration in steady-state. The model shows that this ratio should in fact be independent of the receptor-cargo affinity, though kinetics may be strongly influenced. Numerical conservation of the system components highlights a conflict between the observations and the popular concept of transport cycles. We suggest that chemical partitioning provides a framework to understand the capacity to generate concentration gradients by equilibration of the receptor-cargo intermediary.Comment: in press at HFSP Journal, vol 3 16 text pages, 1 table, 4 figures, plus Supplementary Material include

Components and regulation of nuclear transport processes

The spatial separation of DNA replication and gene transcription in the nucleus and protein translation in the cytoplasm is a uniform principle of eukaryotic cells. This compartmentalization imposes a requirement for a transport network of macromolecules to shuttle these components in and out of the nucleus. This nucleo-cytoplasmic transport of macromolecules is critical for both cell physiology and pathology. Consequently, investigating its regulation and disease-associated alterations can reveal novel therapeutic approaches to fight human diseases, such as cancer or viral infection. The characterization of the nuclear pore complex, the identification of transport signals and transport receptors, as well as the characterization of the Ran system (providing the energy source for efficient cargo transport) has greatly facilitated our understanding of the components, mechanisms and regulation of the nucleo-cytoplasmic transport of proteins in our cells. Here we review this knowledge with a specific emphasis on the selection of disease-relevant molecular targets for potential therapeutic intervention.COST Action [CM1106]; Fundacao para a Ciencia e a Tecnologia (FCT) [SFRH/BPD/84634/2012]info:eu-repo/semantics/publishedVersio

Fluorescence Anisotropy Reveals Order and Disorder of Protein Domains in the Nuclear Pore Complex

We present a new approach for studying individual protein domains within the nuclear pore complex (NPC) using fluorescence polarization microscopy. The NPC is a large macromolecular complex, the size and complexity of which presents experimental challenges. Using fluorescence anisotropy and exploiting the symmetry of the NPC and its organization in the nuclear envelope, we have resolved order and disorder of individual protein domains. Fluorescently tagging specific domains of individual nucleoporins revealed both rigid and flexible domains: the tips of the FG domains are disordered, whereas the NPC-anchored domains are ordered. Our technique allows the collection of structural information in vivo, providing the ability to probe the organization of protein domains within the NPC. This has particular relevance for the FG domain nucleoporins, which are crucial for nucleocytoplasmic transport

On the Octagonal Structure of the Nuclear Pore Complex: Insights from Coarse-Grained Models

The basic structure of the nuclear pore complex (NPC), conserved across almost all organisms from yeast to humans, persists in featuring an octagonal symmetry involving the nucleoporins that constitute the NPC ring. In this article, we seek to understand and evaluate the potential biomechanical reasons for this eightfold symmetry. Our analytical investigation shows that the eightfold symmetry maximizes the bending stiffness of each of the eight NPC spokes while our computational analyses identify the most likely deformation modes, frequencies, and associated kinetic energies of the NPC. These modes have energies close to other published findings using membrane analysis of the nuclear membrane pore opening, and deformation states in agreement with experimental observations. A better understanding of NPC mechanics is essential for characterizing the nucleocytoplasmic transport, which has a central importance in cell biology

A protocol for studying the kinetics of RNA within cultured cells: application to ribosomal RNA.

peer reviewedThis protocol describes a nonisotopic method for high-resolution investigation of the kinetics of RNA within the cell. This involves the incorporation of bromouridine-5'-triphosphate into RNA of living cells by lipofection followed by immunocytological detection of BrRNAs. The use of the same antibody identified either with fluorescence or with gold particles revealed the three-dimensional organization of sites containing labeled RNAs or their precise localization by using confocal and ultrastructural microscopy, respectively. Comparison of three-dimensional reconstruction obtained from the series of optical sections and ultrathin sections was extremely fruitful to describe topological and spatial dynamics of RNAs from their synthesis site inside the nucleus to the cytoplasm. Combined with immunolocalization of proteins involved in different nuclear activities and with highly resolved three-dimensional visualizations of the labelings, this method should also provide a significant contribution to our understanding of the functional, volumic organization of the cell nucleus. The entire protocol can be completed in approximately 10 d

Mapping the orientation of nuclear pore proteins in living cells with polarized fluorescence microscopy

The nuclear pore complex (NPC) perforates the nuclear envelope to facilitate selective transport between nucleus and cytoplasm. The NPC is composed of multiple copies of ~30 different proteins, termed nucleoporins, whose arrangement within the NPC is a major unsolved puzzle in structural biology. Various alternative models for NPC architecture have been proposed but not tested experimentally in intact NPCs. We present a method using polarized fluorescence microscopy to investigate nucleoporin orientation in live yeast and mammalian cells. Our results support an arrangement of both yeast Nic96 and human Nup133–Nup107 with their long axes approximately parallel to the nuclear envelope plane. This method can complement X-ray crystallography and electron microscopy to generate a high-resolution map of the entire NPC, and could monitor nucleoporin rearrangements during nucleocytoplasmic transport and NPC assembly. This strategy can also be adapted for other macromolecular machines