Histochemical determination of the fiber composition of locomotory muscles in a lizard, Dipsosaurus dorsalis

A histochemical survey was done on the fiber composition of 12 different locomotory muscles in the lizard Dipsosaurus dorsalis. Three types of fibers were found in all muscles: (1) fast-twitch-glycolytic (FG); (2) fast-twitch-oxidative-glycolytic (FOG); and (3) tonic fibers. Virtually all locomotory muscles contain some tonic fibers. Most muscles have bulk white regions (containing mostly FG fibers) and distinct red, oxidative regions (with FOG and tonic fibers). These red regions are predominantly located around the joints in the hind limb muscles, and probably serve a postural and joint-stabilizing function. The predominance of FG fibers in the bulk white regions is well-correlated with the rapid, anaerobically supported predator escape behavior of D. dosalis.     

Efficiency, selectivity, and robustness of nucleocytoplasmic transport

All materials enter or exit the cell nucleus through nuclear pore complexes (NPCs), efficient transport devices that combine high selectivity and throughput. NPC-associated proteins containing phenylalanine–glycine repeats (FG nups) have large, flexible, unstructured proteinaceous regions, and line the NPC. A central feature of NPC-mediated transport is the binding of cargo-carrying soluble transport factors to the unstructured regions of FG nups. Here, we model the dynamics of nucleocytoplasmic transport as diffusion in an effective potential resulting from the interaction of the transport factors with the flexible FG nups, using a minimal number of assumptions consistent with the most well-established structural and functional properties of NPC transport. We discuss how specific binding of transport factors to the FG nups facilitates transport, and how this binding and competition between transport factors and other macromolecules for binding sites and space inside the NPC accounts for the high selectivity of transport. We also account for why transport is relatively insensitive to changes in the number and distribution of FG nups in the NPC, providing an explanation for recent experiments where up to half the total mass of the FG nups has been deleted without abolishing transport. Our results suggest strategies for the creation of artificial nanomolecular sorting devices.     

FG nucleoporins feature unique patterns that distinguish them from other IDPs

FG nucleoporins (FG Nups) are intrinsically disordered proteins and are the putative regulators of nucleocytoplasmic transport. They allow fast, yet selective, transport of molecules through the nuclear pore complex, but the underlying mechanism of nucleocytoplasmic transport is not yet fully discovered. As a result, FG Nups have been the subject of extensive research in the past two decades. Although most studies have been focused on analyzing the conformation and function of FG Nups from a biophysical standpoint, some recent studies have investigated the sequence-function relationship of FG Nups, with a few investigating amino acid sequences of a large number of FG Nups to understand common characteristics that might enable their function. Previously, we identified an evolutionarily conserved feature in FG Nup sequences, which are extended subsequences with low charge density, containing only positive charges, and located toward the N-terminus of FG Nups. We named these patterns longest positive like charge regions (lpLCRs). These patterns are specific to positively charged residues, and negatively charged residues do not demonstrate such a pattern. In this study, we compare FG Nups with other disordered proteins obtained from the DisProt and UniProt database in terms of presence of lpLCRs. Our results show that the lpLCRs are virtually exclusive to FG Nups and are not observed in other disordered proteins. Also, lpLCRs are what differentiate FG Nups from DisProt proteins in terms of charge distribution, meaning that excluding lpLCRs from the sequences of FG Nups make them similar to DisProt proteins in terms of charge distribution. We also previously showed the biophysical effect of lpLCRs in conformation of FG Nups. The results of this study are in line with our previous findings and imply that lpLCRs are virtually exclusive and functionally significant characteristics of FG Nups and nucleocytoplasmic transport.     

Distinct functions of the Drosophila Nup153 and Nup214 FG domains in nuclear protein transport

The phenylanine-glycine (FG)-rich regions of several nucleoporins both bind to nuclear transport receptors and collectively provide a diffusion barrier to the nuclear pores. However, the in vivo roles of FG nucleoporins in transport remain unclear. We have inactivated 30 putative nucleoporins in cultured Drosophila melanogaster S2 cells by RNA interference and analyzed the phenotypes on importin alpha/beta-mediated import and CRM1-dependent protein export. The fly homologues of FG nucleoporins Nup358, Nup153, and Nup54 are selectively required for import. The FG repeats of Nup153 are necessary for its function in transport, whereas the remainder of the protein maintains pore integrity. Inactivation of the CRM1 cofactor RanBP3 decreased the nuclear accumulation of CRM1 and protein export. We report a surprisingly antagonistic relationship between RanBP3 and the Nup214 FG region in determining CRM1 localization and its function in protein export. Our data suggest that peripheral metazoan FG nucleoporins have distinct functions in nuclear protein transport events.     

Selective Increase in Dopamine Utilization in the Shell Subdivision of the Nucleus Accumbens by the Benzodiazepine Inverse Agonist FG 7142

Abstract: We examined the effects of the benzodiazepine inverse agonist FG 7142 on dopamine metabolism in the core and shell subdivisions of the nucleus accumbens. FG 7142 (15 mg/kg i.p.) or vehicle was administered to adult male rats 30 min before they were killed. Selected brain regions, including samples from the whole nucleus accumbens as well as core and shell subdivisions, were collected and assayed for tissue concentrations of dopamine and its major metabolite, 3,4-dihydroxyphenylacetic acid. Consistent with previous reports, FG 7142 administration increased dopamine utilization in the medial prefrontal cortex but not the whole nucleus accumbens. Examination of subdivisions revealed that FG 7142 produced increased dopamine utilization in the shell subdivision of the nucleus accumbens. No effect of FG 7142 on dopamine utilization in the core region of the nucleus accumbens was observed. These data are discussed in terms of in vivo microdialysis studies reporting increased dopamine release in the nucleus accumbens after FG 7142 administration.     

The role of the frontal ganglion in foregut movements of the moth,Manduca sexta

Two types of rhythmic foregut movements are described in fifth instar larvae of the moth, Manduca sexta. These consist of posteriorly-directed waves of peristalsis which move food toward the midgut, and synchronous constrictions of the esophageal region, which appear to retain food within the crop. We describe these movements and the muscles of the foregut that generate them.The firing patterns of a subset of these muscles, including a constrictor and dilator pair from both the esophageal and buccal regions of the foregut, are described for both types of foregut movement.The motor patterns for the foregut muscles require innervation by the frontal ganglion (FG), which lies anterior to the brain and contains about 35 neurons. Eliminating the ventral nerve cord, leaving the brain and FG intact, did not affect the muscle firing patterns in most cases. Eliminating both the brain and the ventral nerve cord, leaving only the FG to innervate the foregut, generally resulted in an increased period for both gut movements and muscle bursts. This manipulation also produced increases in burst durations for most muscles, and had variable effects on the phasing of muscle activity. Despite these changes, the foregut muscles still maintained a rhythmic firing pattern when innervated by the FG alone.Two nerves exit the FG to innervate the foregut musculature: the anteriorly-projecting frontal nerve, and the posteriorly-directed recurrent nerve. Cutting the frontal nerve immediately and irreversibly stopped all muscle activity in the buccal region, while cutting the recurrent nerve immediately stopped all muscle activity in the pharyngeal and esophageal regions. Recordings from the cut nerves leaving the FG showed that the ganglion was spontaneously active, with rhythmic activity continuing within the nerves. These observations indicate that all of the foregut muscle motoneurons are located within the FG, and the FG in isolation produces a rhythmic firing pattern in the motoneurons. We have identified several motoneurons within the FG, by cobalt backfills and/or simultaneous intracellular recordings and fills from putative motoneurons and their muscles.Abbreviations BC Buccal Constrictor - BC1 buccal constrictor motoneuron 1 - BC2 buccal constrictor motoneuron 2 - BD Buccal Dilator - BD1 buccal dilator motoneuron 1 - EC Esophageal Dilator - EC1 esophageal dilator motoneuron 1 - EC2 esophageal dilator motoneuron 2 - EC3 esophageal dilator motoneuron 3 - ejp excitatory junction potential - FG frontal ganglion - psp postsynaptic potential     

Neuromuscular organization of avian flight muscle: Morphology and contractile properties of motor units in the pectoralis (pars thoracicus) of pigeon (Columba livia)

We used acid digestion and glycogen depletion to determine fascicle organization, fiber morphology, and physiological and anatomical features of individual motor units of an in-series muscle, the pectoralis (pars thoracicus) of the pigeon (Columba livia). Most fascicles are attached at one end to connective tissue. Average fiber length in the four regions examined range from 42% to 66% of average fascicle length. More than 65% of fibers are blunt at one end of a fascicle and taper intrafascicularly. Fibers with blunt–blunt endings range from 13% to 31% of the population in different regions; taper–taper fibers range from 2% to 17%. Pigeon pectoralis fibers are distinguished histochemically into fast-twitch glycolytic (FG) and fast-twitch oxidative-glycolytic (FOG) populations. Three units composed of FG fibers (FG units) contract more quickly than three units composed of FOG fibers (FOG units) (range 31–37 vs 47–62 msec), produce more tetanic force (0.11–0.32 vs 0.02–0.05 N) and are more fatigable (<18% initial force vs >50% after repeated stimulation). Most motor units are confined to one of the four muscle regions. Territory of two FOG units is <30% of parent fascicle length. Territories of other units spanned parent fascicles; most fibers in these units do not extend the full fascicle length. Compared to FG units, FOG units have lower maximum innervation ratios and density indices (ratio of depleted/total FOG fibers in territory 8–14% vs 58–76% for FG units). These differences support the hypothesis that FG units are organized to produce substantial force and power for takeoff, landing and other ballistic movements whereas FOG units are suited for sustained flight when power requirements are reduced. Implications of findings for understanding the control of in-series muscles and the use of connective tissue elastic elements during wing movements are discussed. J.Morphol. 236:179–208, 1998. © 1998 Wiley-Liss, Inc.     

Systematic analysis of barrier-forming FG hydrogels from Xenopus nuclear pore complexes

Nuclear pore complexes (NPCs) control the traffic between cell nucleus and cytoplasm. While facilitating translocation of nuclear transport receptors (NTRs) and NTR·cargo complexes, they suppress passive passage of macromolecules ?30 kDa. Previously, we reconstituted the NPC barrier as hydrogels comprising S. cerevisiae FG domains. We now studied FG domains from 10 Xenopus nucleoporins and found that all of them form hydrogels. Related domains with low FG motif density also substantially contribute to the NPC's hydrogel mass. We characterized all these hydrogels and observed the strictest sieving effect for the Nup98‐derived hydrogel. It fully blocks entry of GFP‐sized inert objects, permits facilitated entry of the small NTR NTF2, but arrests importin β‐type NTRs at its surface. O‐GlcNAc modification of the Nup98 FG domain prevented this arrest and allowed also large NTR·cargo complexes to enter. Solid‐state NMR spectroscopy revealed that the O‐GlcNAc‐modified Nup98 gel lacks amyloid‐like β‐structures that dominate the rigid regions in the S. cerevisiae Nsp1 FG hydrogel. This suggests that FG hydrogels can assemble through different structural principles and yet acquire the same NPC‐like permeability.     

Proteomic analysis of urinary fibrinogen degradation products in patients with urothelial carcinomas

Despite many years of research efforts and continued progress in the identification of urine markers for detection of bladder cancer, none of the markers described to date has been able to replace cystoscopy and urine cytology, the current gold standards for diagnosis. Here, we present a comprehensive gel-based proteomic study in which we have analyzed the presence and origin of fibrinogen (FG) and its degradation products (FDPs) in the urine of patients with and without urothelial carcinoma (UCs), with the aim of evaluating the potential of two-dimensional (2D) gel FDP profiling for detecting bladder cancer. A total of 151 urine samples collected from patients with Ucs of varying degrees of atypia and stages of invasion were compared with a control group consisting of 34 healthy volunteers with no clinical history of bladder cancer. The level and degree of degradation of FG in the urine were determined by 2D gel Western blotting in combination with enhanced chemilumenscence detection. Elevated levels of urine FG/FDPs were found in 99% of patients bearing superficial tumors, in 97% of the cases with early invasive disease, and in 96% of patients with highly invasive tumors. 2D gel profiling of urine FG/FDPs showed that the FG chains exhibited differential susceptibility to in situ proteolysis, with the α-chain being the most susceptible and the γ-chain the most resistant. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry identified peptide sequence regions in several of the most representative and common FDPs, which can be of value for producing novel specific antibodies to detect FG/FDPs in the urine. In addition, we present evidence indicating that FG is not produced by normal or malignant urothelium, although it is present both in the stroma of malignant tissue and tumor lesions. Taken together, the data indicate that increased levels of FG/FDPs amounts in the urine are a characteristic feature of bladder cancer, and emphasize the value of 2D gel profiling of urine FG/FDPs for detecting low-grade, noninvasive UCs.     

Deciphering networks of protein interactions at the nuclear pore complex

The nuclear pore complex (NPC) gates the only known conduit for molecular exchange between the nucleus and cytoplasm of eukaryotic cells. Macromolecular transport across the NPC is mediated by nucleocytoplasmic shuttling receptors termed karyopherins (Kaps). Kaps interact with NPC proteins (nucleoporins) that contain FG peptide repeats (FG Nups) and altogether carry hundreds of different cargoes across the NPC. Previously we described a biochemical strategy to identify proteins that interact with individual components of the nucleocytoplasmic transport machinery. We used bacterially expressed fusions of glutathione S-transferase with nucleoporins or karyopherins as bait to capture interacting proteins from yeast extracts. Forty-five distinct proteins were identified as binding to one or several FG Nups and Kaps. Most of the detected interactions were expected, such as Kap-Nup interactions, but others were unexpected, such as the interactions of the multisubunit Nup84p complex with several of the FG Nups. Also unexpected were the interactions of various FG Nups with the nucleoporins Nup2p and Nup133p, the Gsp1p-GTPase-activating protein Rna1p, and the mRNA-binding protein Pab1p. Here we resolve how these interactions occur. We show that Pab1p associates nonspecifically with immobilized baits via RNA. More interestingly, we demonstrate that the Nup84p complex contains Nup133p as a subunit and binds to the FG repeat regions of Nups directly via the Nup85p subunit. Binding of Nup85p to the GLFG region of Nup116p was quantified in vitro (K(D) = 1.5 micro M) and was confirmed in vivo using the yeast two-hybrid assay. We also demonstrate that Nup2p and Rna1p can be tethered directly to FG Nups via the importin Kap95p-Kap60p and the exportin Crm1p, respectively. We discuss possible roles of these novel interactions in the mechanisms of nucleocytoplasmic transport.     

Characterization of Group A Streptococcal M23 Protein and Comparison of the M3 and M23 Protein’s Ligand-Binding Domains

The present study concerns the properties for binding of human plasma and extracellular matrix proteins and the relationship between M3 and M23 molecules. Here, it is demonstrated that M23 protein shows a multiple binding to fibrinogen (FG), fibronectin (FN), human serum albumin (HSA), immunoglobulin G (IgG), kininogen, and collagen type I (CI) in Western blot analysis. Some sets of truncated-recombinant M3 or M23 protein fragments were assayed for their capacity to bind FN, FG, IgG, HSA, and CI. The HSA binding activity resided in the C-repeat region of M3 protein, whereas fibrinogen-binding activity resided in the A-repeat region. The FG, FN, and IgG binding sites were mapped to the N-terminal portion of M23 protein, whereas HSA binding was localized in the B-repeat domain, which has homology with C-repeat domain in M3 molecule. Therefore, it is concluded that the FN, FG, and IgG binding regions in the M3 and M23 proteins are quite dissimilar at the amino acid sequence level, whereas HSA binding is localized to the conserved C-repeat domain in the M3 and M23 proteins.     

Fluoro-Gold: An alternative viability stain for multicolor flow cytometric analysis.

BACKGROUND: The viability stains propidium iodide (PI) and 7-amino-actinomycin D (7-AAD) are excited at 488 nm, as are the commonly used antibody conjugates fluorescein isothiocyanate (FITC), phycoerythrin (PE), and cyanine 5 dye covalently coupled to R-phycoerythrin (RPE-Cy5). When excited by a single laser, spectral overlap in the emission of PI and 7-AAD with RPE-Cy5 precludes the use of these viability stains for three-color immunophenotyping, particularly when evaluating low levels of marker expression in viable target cells. The ultraviolet excitable dye hydroxystilbamidine methanesulfonate (Fluoro-Gold, or FG) binds to DNA at the A-T-rich regions of the minor groove in permeabilized or dead cells. We assessed the suitability of this dye as a viability stain. METHODS: The ability of FG to detect nonviable cells in fresh and cryopreserved human apheresed peripheral blood cells was compared with that of PI and 7-AAD. The stability of FG staining and the effects of dye and cell concentration on the discrimination of nonviable cells was determined by measuring changes in the median fluorescence of viable and nonviable cells. RESULTS: FG labeling at dye concentrations of 2-8 microM is stable for at least 3 h over a wide range of cell concentrations (4 x 10(5) to 4 x 10(7) cells/ml). Costaining studies and linear regression analysis show that cell viability as determined by FG is strongly correlated with estimates using PI (r = 0.9636) and 7-AAD (r = 0.9879). CONCLUSIONS: FG is a reliable, alternative viability stain that can be used in conjunction with fluorochromes including FITC, PE, and RPE-Cy5 for multicolor analysis using dual-laser instruments.     

Diverse rhizobia that nodulate two species of Kummerowia in China

A total of 63 bacterial strains were isolated from root nodules of Kummerowia striata and K. stipulacea grown in different geographic regions of China. These bacteria could be divided into fast-growing (FG) rhizobia and slow-growing (SG) rhizobia according to their growth rate. Genetic diversity and taxonomic relationships among these rhizobia were revealed by PCR-based 16 S rDNA RFLP and sequencing, 16 S-IGS RFLP, SDS-PAGE of whole cell soluble proteins, BOX-PCR and symbiotic gene (nifH/nodC) analyses. The symbiotic FG strains were mainly isolated from temperate regions and they were identified as four genomic species in Rhizobium and Sinorhizobium meliloti based on the consensus of grouping results. The SG strains were classified as five genomic species within Bradyrhizobium and they were mainly isolated fron the subtropic and tropical regions. The phylogenetic analyses of nifH and nodC genes showed relationships similar to that of 16 S rDNA but the symbiotic genes of Bradyrhizobium strains isolated from Kummerowia were distinct from those isolated from Arachis and soybean. These results offered evidence for rhizobial biogeography and demonstrated that the Kummerowia-nodulating ability might have evolved independently in different regions in association with distinctive genomic species of rhizobia.     

A region in domain 1 of CD4 distinct from the primary gp120 binding site is involved in HIV infection and virus-mediated fusion.

The high affinity binding site for human immunodeficiency virus (HIV) envelope glycoprotein gp120 resides within the amino-terminal domain (D1) of CD4. Mutational and antibody epitope analyses have implicated the region encompassing residues 40-60 in D1 as the primary binding site for gp120. Outside of this region, a single residue substitution at position 87 abrogates syncytium formation without affecting gp120 binding. We describe two groups of CD4 monoclonal antibodies (mAbs) which recognize distinct epitopes associated with these regions in D1. These mAbs distinguish between the gp120 binding event and virus infection and virus-induced cell fusion. One cluster of mAbs, which bind at or near the high affinity gp120 binding site, blocked gp120 binding to CD4 and, as expected, also blocked HIV infection of CD4+ cells and virus-induced syncytium formation. A second cluster of mAbs, which recognize the CDR-3 like loop, did not block gp120 binding as demonstrated by their ability to form ternary complexes with CD4 and gp120. Yet, these mAbs strongly inhibited HIV infection of CD4+ cells and HIV-envelope/CD4-mediated syncytium formation. The structure of D1 has recently been solved at atomic resolution and in its general features resembles IgVk regions as predicted from sequence homology and mAb epitopes. In the D1 structure, the regions recognized by these two groups of antibodies correspond to the C'C" (Ig CDR2) and FG (Ig CDR3) hairpin loops, respectively, which are solvent-exposed beta turns protruding in two different directions on a face of D1 distal to the D2 domain. This face is straddled by the longer BC (Ig CDR1) loop which bisects the plain formed by C'C' and FG. This structure is consistent with C'C' and FG forming two distinct epitope clusters within D1. We conclude that the initial interaction between gp120 and CD4 is not sufficient for HIV infection and syncytium formation and that CD4 plays a critical role in the subsequent virus-cell and cell-cell membrane fusion events. We propose that the initial binding of CD4 to gp120 induces conformational changes in gp120 leading to subsequent interactions of the FG loop with other regions in gp120 or with the fusogenic gp41 potion of the envelope gp160 glycoprotein.     

Changes in Thalamic Connectivity in the Early and Late Stages of Amnestic Mild Cognitive Impairment: A Resting-State Functional Magnetic Resonance Study from ADNI

We used resting-state functional magnetic resonance imaging (fMRI) to investigate changes in the thalamus functional connectivity in early and late stages of amnestic mild cognitive impairment. Data of 25 late stages of amnestic mild cognitive impairment (LMCI) patients, 30 early stages of amnestic mild cognitive impairment (EMCI) patients and 30 well-matched healthy controls (HC) were analyzed from the Alzheimer’s disease Neuroimaging Initiative (ADNI). We focused on the correlation between low frequency fMRI signal fluctuations in the thalamus and those in all other brain regions. Compared to healthy controls, we found functional connectivity between the left/right thalamus and a set of brain areas was decreased in LMCI and/or EMCI including right fusiform gyrus (FG), left and right superior temporal gyrus, left medial frontal gyrus extending into supplementary motor area, right insula, left middle temporal gyrus (MTG) extending into middle occipital gyrus (MOG). We also observed increased functional connectivity between the left/right thalamus and several regions in LMCI and/or EMCI including left FG, right MOG, left and right precuneus, right MTG and left inferior temporal gyrus. In the direct comparison between the LMCI and EMCI groups, we obtained several brain regions showed thalamus-seeded functional connectivity differences such as the precentral gyrus, hippocampus, FG and MTG. Briefly, these brain regions mentioned above were mainly located in the thalamo-related networks including thalamo-hippocampus, thalamo-temporal, thalamo-visual, and thalamo-default mode network. The decreased functional connectivity of the thalamus might suggest reduced functional integrity of thalamo-related networks and increased functional connectivity indicated that aMCI patients could use additional brain resources to compensate for the loss of cognitive function. Our study provided a new sight to understand the two important states of aMCI and revealed resting-state fMRI is an appropriate method for exploring pathophysiological changes in aMCI.     

A gradient of affinity for the karyopherin Kap95p along the yeast nuclear pore complex

Karyopherins (Kaps) transport cargo across the nuclear pore complex (NPC) by interacting with nucleoporins that contain phenylalanine-glycine (FG) peptide repeats (FG Nups). As a test of the "affinity gradient" model for Kap translocation, we measured the apparent affinity of Kap95p to FG Nups representing three distinct regions of the S. cerevisiae NPC. We find that the affinity of Kap95p-Kap60p-cargo complexes to Nup1p (a nuclear basket Nup) is 225-fold higher than to Nup100p (a central scaffold Nup) and 4000-fold higher than to Nup42p (a cytoplasmic filament Nup), revealing a steep gradient of affinity for Kap95p complexes along the yeast NPC. A high affinity binding site for a Kap95p import complex was mapped to the C terminus of Nup1p, and, surprisingly, deletion of all FG repeats in that region did not eliminate binding of the complex. Instead, a 36-amino acid truncation of the C terminus of Nup1p reduced its affinity for the Kap95p import complex by 450-fold. Mutant yeast that express Nup1pDelta36 instead of full-length Nup1p display specific defects in Kap95p localization and Kap95p-mediated nuclear import. We conclude that a high affinity binding site for Kap95p at the nuclear basket increases the translocation efficiency of Kap95p import complexes across the NPC.     

Molecular and functional characterization of the p62 complex, an assembly of nuclear pore complex glycoproteins

Macromolecular trafficking across the nuclear envelope involves interactions between cytosolic transport factors and nuclear pore complex proteins. The p62 complex, an assembly of 62, 58, 54, and 45-kD O-linked glycoproteins-localized near the central gated channel of the nuclear pore complex, has been directly implicated in nuclear protein import. The cDNA cloning of rat p62 was reported previously. We have now carried out cDNA cloning of rat p58, p54, and p45. We found that p58 contains regions with FG (Phe, Gly) and PA (Pro, Ala) repeats at both its NH2 and COOH termini separated by a predicted alpha-helical coiled-coil region, while p54 has an NH2-terminal FG and PA repeat region and a COOH-terminal predicted coiled-coil region. p45 and p58 appear to be generated by alternative splicing, with p45 containing the NH2-terminal FG repeat region and the coiled-coil region of p58. Using immunogold electron microscopy, we found that p58/p45 and p54 are localized on both sides of the nuclear pore complex, like p62. Previous studies have shown that immobilized recombinant p62 can bind the cytosolic nuclear import factor NTF2 and thereby deplete transport activity from cytosol. We have now found that immobilized recombinant p58 and p54 also can deplete nuclear transport activity from cytosol, and that p62, p58, and p54 bind directly to the cytosolic nuclear import factors p97 and NTF2. At least in the case of p58, this involves FG repeat regions. Moreover, p58 can bind to a complex containing transport ligand, the nuclear localization sequence receptor (Srp1 alpha) and p97. These data support a model in which the p62 complex binds to a multicomponent particle consisting of transport ligand and cytosolic factors to achieve accumulation of ligand near the central gated channel of the nuclear pore complex.     

C‐terminal β‐strand swapping in a consensus‐derived fibronectin Type III scaffold

The crystal structures of six different fibronectin Type III consensus‐derived Tencon domains, whose solution properties exhibit no, to various degrees of, aggregation according to SEC, have been determined. The structures of the five variants showing aggregation reveal 3D domain swapped dimers. In all five cases, the swapping involves the C‐terminal β‐strand resulting in the formation of Tencon dimers in which the target‐binding surface is blocked. All of the variants differ in sequence in the FG loop, which is the hinge loop in the β‐strand‐swapped dimers. The six tencon variants have between 0 and 5 residues inserted between positions 77 and 78 in the FG loop. Analysis of the structures suggests that a non‐glycine residue at position 77 and insertions of <4 residues may destabilize the β‐turn in the FG loop promoting β‐strand swapping. Swapped dimers with an odd number of inserted residues may be less stable, particularly if they contain proline residues, because they cannot form perfect β‐bridges in the FG regions that link the swapped dimers. The Tencon β‐swapped variants with the longest FG sequences are observed to form higher order hexameric or helical oligomeric structures in the crystal correlating well with the aggregation properties of these domains observed in solution. Understanding the structural basis for domain‐swapped dimerization and oligomerization will support engineering efforts of the Tencon domain to produce variants with desired biophysical properties. Proteins 2014; 82:1359–1369. © 2013 Wiley Periodicals, Inc.     

Nanocompartmentalization of the Nuclear Pore Lumen

The nuclear pore complex (NPC) employs the intrinsically disordered regions (IDRs) from a family of phenylalanine-glycine-rich nucleoporins (FG-Nups) to control nucleocytoplasmic transport. It has been a long-standing mystery how the IDR-mediated mass exchange can be rapid yet selective. Here, we use a computational microscope to show that nanocompartmentalization of IDR subdomains leads to a remarkably elaborate gating structure as programmed by the amino acid sequences. In particular, we reveal a heterogeneous permeability barrier that combines an inner ring barrier with two vestibular condensates. Throughout the NPC, we find a polarized electrostatic potential and a diffuse thermoreversible FG network featuring mosaic FG territories with low FG-FG pairing fraction. Our theoretical anatomy of the central transporter sheds light into the sequence-structure-function relationship of the FG-Nups and provides a picture of nucleocytoplasmic mass exchange that allows a reconciliation of transport efficiency and specificity.