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Volume 122 of Methods in Cell Biology describes modern tools and techniques used to study nuclear pore complexes and nucleocytoplasmic transport in diverse eukaryotic model systems (including mammalian cells, Xenopus, C. elegans, yeast). The volume enables investigators to analyze nuclear pore complex structure, assembly, and dynamics; to evaluate protein and RNA trafficking through the nuclear envelope; and to design in vivo or in vitro assays appropriate to their research needs. Beyond the study of nuclear pores and transport as such, these protocols will also be helpful to scientists characterizing gene regulation, signal transduction, cell cycle, viral infections, or aging. The NPC being one of the largest multiprotein complexes in the cell, some protocols will also be of interest for people currently characterizing other macromolecular assemblies. This book is thus designed for laboratory use by graduate students, technicians, and researchers in many molecular and cellular disciplines. Describes modern tools and techniques used to study nuclear pore complexes and nucleocytoplasmic transport in diverse eukaryotic model systems (mammalian cells, Xenopus, C. elegans, yeast) Chapters are written by experts in the field Cutting-edge material
Bidirectional traffic of macromolecules across the nuclear envelope is an active and essential transport process in all eukaryotic cells. Work on various model systems has led to a tremendous increase in our understanding of nuclear transport in recent years. This volume summarizes our current knowledge of protein and RNA transport into and out of the nucleus. It contains nine up-to-date reviews which cover various aspects of nucleocytoplasmic transport, including the structure and function of the nuclear pore complex, the role of soluble transport factors in protein and RNA transport, and the regulation of protein transport through the nuclear pore.
Dysfunction of nuclear-cytoplasmic transport systems has been associated with many human diseases. Thus, understanding of how functional this transport system maintains, or through dysfunction fails to maintain remains the core question in cell biology. In eukaryotic cells, the nuclear envelope (NE) separates the genetic transcription in the nucleus from the translational machinery in the cytoplasm. Thousands of nuclear pore complexes (NPCs) embedded on the NE selectively mediate the bidirectional trafficking of macromolecules such as RNAs and proteins between these two cellular compartments. In this book, the authors integrate recent progress on the structure of NPC and the mechanism of nuclear-cytoplasmic transport system in vitro and in vivo.
Delivery of therapeutic proteomics and genomics represent an important area of drug delivery research. Genomics and proteomics approaches could be used to direct drug development processes by unearthing pathways involved in disease pathogenesis where intervention may be most successful. This book describes the basics of genomics and proteomics and highlights the various chemical, physical and biological approaches to protein and gene delivery. Covers a diverse array of topics from basic sciences to therapeutic applications of proteomics and genomics delivery Of interest to researchers in both academia and industry Highlights what’s currently known and where further research is needed
In eukaryotic cells, the nuclear genome and its transcriptional apparatus is separated from the site of protein synthesis by the nuclear envelope. Thus, a constant flow of proteins and nucleic acids has to cross the nuclear envelope in both directions. This transport in and out of the nucleus is mediated by nuclear pore complexes (NPCs) and occurs in an energy and signal-dependent manner. Thus, nucleocytoplasmic translocation of macro molecules across the nuclear envelope appears to be a highly specific and regulated process. Viruses that replicate their genome in the cell nucleus are therefore forced to develop efficient ways to deal with the intracellulZlr host cell transport machinery. Historically, investigation of Polyomavirus replication allowed identification ofsequences that mediate nuclear import, which led subsequently to our detailed understanding of the cellular factors that are involved in nuclear import. Transport ofmacromolecules in the opposite direction, however, is less well understood. The investigation of retroviral gene expression in recent years pro vided the first insights into the cellular mechanisms that regulate nuclear export. In particular, the detailed dissection of the function of the human immunodeficiency virus type I (HIV-I) Rev trans-activator protein identified CRMI, as a hona fide nuclear export receptor. CRM I appears to be involved in the nucleocytoplasmic translocation of the vast majority of viral and cellular proteins that have subsequently been found to contain a Rev-type leucine-rich nuclear export signal (NES).
The nuclear pore complex (NPC) is one of the largest known protein structures in the cell. Evolutionarily conserved in eukaryotes ranging from fungi to plants and animals, the NPC is the main transporter of molecules between the cell cytoplasm and nucleus. Maintaining the proper compartment-specific localization of proteins and RNA is crucial for normal cell function, and the nuclear pore accomplishes this task both robustly and efficiently. Over the past several decades, insight into the composition, organization, structure, and mechanism of the NPC has been gradually teased out through careful experimentation. However, many questions about the pore's function remain unanswered. In this dissertation, I describe efforts aimed at elucidating several aspects of the NPC. First, I investigate the transport properties of the pore, specifically looking at how the nuclear transport receptor importin-[beta] and the Ran GTPase interact not only with each other but also how they may affect the pore itself. The nucleoporin Nup153 is identified as an important player in the nuclear transport process which binds strongly to importin-[beta] in a Ran-sensitive manner. Using multiple experimental techniques, the properties of importin-[beta], and Nup153's interactions are characterized and shown to be capable of modulating the selective permeability barrier of the NPC. Next, I examine how members of a major class of nuclear pore proteins, the scaffold nucleoporins, are both structurally and functionally similar to the karyopherin family of soluble nuclear transport receptors. Structures of the proteins Nup188 and Nup192 are analyzed and shown to resemble those of karyopherins. Furthermore, in vitro assays indicate that at least a subset of the scaffold nucleoporins behave functionally as transport receptors, hinting at an evolutionary relationship between these two important classes of proteins. Finally, a calcium-mediated phenomenon affecting the permeability of the NPC is explored. I show that certain cytosolic proteases are activated by millimolar concentrations of calcium ion which leads irreversibly to an increase in the nuclear pore's permeability to large molecules. A model for physiological pathways implicated in this effect is proposed.
Nuclear pore complexes (NPCs) are large macromolecular gateways embedded in the nuclear envelope of Eukaryotic cells that serve to regulate bi-directional trafficking of particles to and from the nucleus. NPCs have been described as creating a selectively permeable barrier mediating the nuclear export of key endogenous cargoes such as mRNA, and pre-ribosomal subunits as well as allow for the nuclear import of nuclear proteins and some viral particles. Remarkably, other particles that are not qualified for nucleocytoplasmic transport are repelled from the NPC, unable to translocate. The NPC is made up of over 30 unique proteins, each present in multiples of eight copies. The two primary protein components of the NPC can be simplified as scaffold nucleoporins which form the main structure of the NPC and the phenylalanine-glycine (FG) motif containing nucleoporins (FG-Nups) which anchor to the scaffold and together create the permeability barrier within the pore. Advances in fluorescence microscopy techniques including single-molecule and super-resolution microscopy have made it possible to label and visualize the dynamic components of the NPC as well as track the rapid nucleocytoplasmic transport process of importing and exporting cargoes. The focus of this dissertation will be on live cell fluorescence microscopy application in probing the dynamic components of the NPC as well as tracking the processes of nucleocytoplasmic transport.