Indirect Excitons and Trions in MoSe2/WSe2 van der Waals Heterostructures.

Indirect excitons (IX) in semiconductor heterostructures are bosons, which can cool below the temperature of quantum degeneracy and can be effectively controlled by voltage and light. IX quantum Bose gases and IX devices were explored in GaAs heterostructures where an IX range of existence is limited to low temperatures due to low IX binding energies. IXs in van der Waals transition-metal dichalcogenide (TMD) heterostructures are characterized by large binding energies giving the opportunity for exploring excitonic quantum gases and for creating excitonic devices at high temperatures. TMD heterostructures also offer a new platform for studying single-exciton phenomena and few-particle complexes. In this work, we present studies of IXs in MoSe2/WSe2 heterostructures and report on two IX luminescence lines whose energy splitting and temperature dependence identify them as neutral and charged IXs. The experimentally found binding energy of the indirect charged excitons, that is, indirect trions, is close to the calculated binding energy of 28 meV for negative indirect trions in TMD heterostructures [Deilmann, T.; Thygesen, K. S. Nano Lett. 2018, 18, 1460]. We also report on the realization of IXs with a luminescence line width reaching 4 meV at low temperatures. An enhancement of IX luminescence intensity and the narrow line width are observed in localized spots.

Agreement of activity monitors for assessment of patients with sub-acute stroke in an inpatient rehabilitation facility.

PURPOSE: Determine the level of agreement of three activity monitors compared with the gold standard (video review) on the activity level of patients with stroke. METHODS: A prospective, observational, agreement study was performed on 47 individuals with sub-acute stroke in an inpatient rehabilitation facility. Data was collected during one physical therapy session. Individuals wore three device types; Actigraph (AG), Activpal (AP), and stepwatch activity monitor (SAM). Variables assessed were step counts for each limb (hemiparetic and non-hemiparetic) and percent time standing and other. ANALYSIS: Results from the activity monitors were compared to the video review and assessed for agreement using the intraclass correlation coefficient (ICC) and accuracy of mean difference from video observation. RESULTS: The step counts with the SAM on the non-hemiparetic limb had the highest ICC for step counts (ICC = 0.98, p < 0.001) and were overestimated with 21% accuracy. The SAM on the hemiparetic limb had 9.7% accuracy (ICC = 0.92, p < 0.001). For percent standing time all devices overestimated with poor reliability. For percent other activity time, the AP had the best accuracy and underestimated for both the hemiparetic limb (9.9% accuracy; ICC = 0.90, p < 0.001) and non-hemiparetic limb (8.3% accuracy; ICC = 0.84, p < 0.001). CONCLUSIONS: The use of multiple devices may be warranted to capture an accurate understanding of activity levels in this population of individuals with sub-acute stroke. There are concerns with all monitors and clinicians and researchers should be aware of what measures they are wanting to understand about their population.

The stepwatch activity monitor worn on the hemiparetic limb provided the best accuracy and excellent reliability for step counts in this population of subacute stroke.For percent standing time all devices overestimated with poor reliability.For percent other time, the AP had the best accuracy and good reliability on the non-hemiparetic limb.The use of multiple devices may be warranted to capture a more accurate understanding of activity level in this population of individuals with sub-acute stroke.Clinicians and researchers need to be aware of the biases of these devices in this population.

Molecular trafficking across the nuclear pore complex.

The nuclear pore complex is the gateway for protein and RNA transport between the cytoplasm and nucleus. Recent work has characterized signals and components involved in nuclear import of macromolecules and has described mechanisms for transport regulation. Advances in understanding the structure of the pore complex are starting to provide a framework for interpreting the biochemistry of nuclear import. Information on the export of RNA from the nucleus is only beginning to emerge.

Mechanisms of nuclear protein import.

The past two years have seen a significant increase in our understanding of nuclear protein import. Five cytosolic import factors have been identified, two of which have been shown to directly interact with components of the nuclear pore complex. These findings enable refinement of previous models for steps in the nuclear import pathway, and provide a framework for future research.

Taking from the cytoplasm and giving to the pore: soluble transport factors in nuclear protein import.

The past year has seen the publication of a number of papers describing the identification of cytosolic factors involved in import of proteins to the nucleus. Although, at first glance, this gives the impression that the study of nuclear transport has become extremely complicated, these factors in fact form a relatively small group of proteins that have been given different names. Characterization of these proteins is improving understanding of the nuclear import process and provides a starting point for further investigation of the steps that occur at the nuclear pore complex.

Integral membrane proteins and dynamic organization of the nuclear envelope.

The nuclear envelope is a complex structure consisting of nuclear membranes, nuclear pore complexes and lamina. Several integral membrane proteins specific to the nuclear pore membrane and the inner nuclear membrane are known. Pore membrane proteins are probably important for organization and assembly of the nuclear pore complex, while proteins of the inner nuclear membrane are likely to play major roles in the structure and dynamics of the nuclear lamina and chromatin. Biochemical studies are now identifying potential binding partners for some of these integral membrane proteins, and analysis of nuclear envelope assembly at the end of mitosis is providing important insights into their functions.

Two-way trafficking with Ran.

The small Ras-related GTPase Ran is directly involved in nuclear protein import and export. However, the question of how Ran functions in transport is highly controversial. Here, we suggest that Ran is important for the formation, vectorial movement and disassembly of many different classes of transport complexes that traverse the nuclear pore complex during import and export processes. Comparison of Ran with the translation elongation factor Ef-Tu raises the possibility that Ran might also be involved in a proofreading function related to the assembly of import complexes. Although aspects of this model are hypothetical and challenge some current dogma in the field, we believe that it can integrate most of the current data into a coherent picture of the import process.

Identification of NTF2, a cytosolic factor for nuclear import that interacts with nuclear pore complex protein p62.

Protein import into the nucleus is a multistep process that requires the activities of several cytosolic factors. In this study we have purified a cytosolic factor that interacts with the nuclear pore complex glycoprotein p62. Isolation involved biochemical complementation of cytosol depleted of this activity by preadsorption with recombinant p62 and the use of a novel flow cytometry-based assay for quantitation of nuclear import. The purified activity (NTF2) is an apparent dimer of approximately 14-kD subunits and is present at approximately 10(6) copies per cell. We obtained a cDNA encoding NTF2 and showed that the recombinant protein restores transport activity to p62-pretreated cytosol. Our data suggest that NTF2 acts at a relatively late stage of nuclear protein import, subsequent to the initial docking of nuclear import ligand at the nuclear envelope. NTF2 interacts with at least one additional cytosolic transport activity, indicating that it could be part of a multicomponent system of cytosolic factors that assemble at the pore complex during nuclear import.

Depletion of calcium from the lumen of endoplasmic reticulum reversibly inhibits passive diffusion and signal-mediated transport into the nucleus.

Nuclear pore complexes provide channels for molecular transport across the nuclear envelope. Translocation of most proteins and RNAs through the pore complex is mediated by signal- and ATP-dependent mechanisms, while transport of small molecules is accomplished by passive diffusion. We report here that depletion of calcium from the lumen of the endoplasmic reticulum and nuclear envelope with ionophores or the calcium pump inhibitor thapsigargin rapidly and potently inhibits signal mediated transport of proteins into the nucleus. Lumenal calcium depletion also inhibits passive diffusion through the pore complex. Signal-mediated protein import and passive diffusion are rapidly restored when the drugs depleting lumenal calcium are removed and cells are incubated at 37 degrees C in calcium-containing medium. These results indicate that loss of calcium from the lumen of the endoplasmic reticulum and nuclear envelope reversibly affects properties of pore complex components located on the nuclear/cytoplasmic membrane surfaces, and they provide direct functional evidence for conformational flexibility of the pore complex. These methods will be useful for achieving reversible inhibition of nucleocytoplasmic trafficking for in vivo functional studies, and for studying the structure of the passive diffusion channel(s) of the pore complex.

A fractionated cell-free system for analysis of prophase nuclear disassembly.

We describe a cell-free system in which a postribosomal supernatant (s140) from metaphase Chinese hamster ovary (CHO) cells induces prophase-like changes in isolated CHO cell nuclei, including chromatin condensation, and nuclear envelope and lamina disassembly. These events are strongly promoted by gamma-S-ATP and an ATP-regenerating system, and do not take place with an s140 derived from G2-phase cells. The metaphase cell s140 also induces disassembly of an isolated nuclear lamina fraction that is depleted of membranes, chromatin, and nuclear pore complexes. Disassembly of the isolated lamina is accompanied by phosphorylation of the major lamina proteins (lamins A, B, and C) to levels characteristic of metaphase cells. Kinetic analysis of lamina depolymerization indicates that cooperativity may be involved in this process. The biochemical properties of in vitro lamina disassembly suggest that the activity that depolymerizes the lamina during mitosis is soluble in metaphase cells, and support the notion that this activity is a lamin protein kinase.

A GTPase distinct from Ran is involved in nuclear protein import.

Signal-dependent transport of proteins into the nucleus is a multi-step process mediated by nuclear pore complexes and cytosolic transport factors. One of the cytosolic factors, Ran, is the only GTPase that has a characterized role in the nuclear import pathway. We have used a mutant form of Ran with altered nucleotide binding specificity to investigate whether any other GTPases are involved in nuclear protein import. D125N Ran (XTP-Ran) binds specifically to xanthosine triphosphate (XTP) and has a greatly reduced affinity for GTP, so it is no longer sensitive to inhibition by nonhydrolyzable analogues of GTP such as guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S). using in vitro transport assays, we have found that nuclear import supported by XTP-Ran is nevertheless inhibited by the addition of non-hydrolyzable GTP analogues. This in conjunction with the properties of the inhibitory effect indicates that at least one additional GTPase is involved in the import process. Initial characterization suggests that the inhibited GTPase plays a direct role in protein import and could be a component of the nuclear pore complex.

Nuclear protein import is inhibited by an antibody to a lumenal epitope of a nuclear pore complex glycoprotein.

Gp210 is a major transmembrane glycoprotein associated with the nuclear pore complex that is suggested to be important for organizing pore complex architecture and assembly. A mouse monoclonal IgG directed against an epitope in the lumenal domain of rat gp210 was expressed in cultured rat cells by microinjection of mRNA prepared from a hybridoma cell line. The expressed IgG, which becomes assembled into a functional antibody in the lumen of the endoplasmic reticulum, bound to the nuclear envelope in vivo. Expression of anti-gp210 antibody in interphase cells specifically reduced approximately fourfold the mediated nuclear import of a microinjected nuclear protein (nucleoplasmin) coupled to gold particles. The antibody also significantly decreased nuclear influx of a 10-kD dextran by passive diffusion. This transport inhibition did not result from removal of pore complexes from nuclear membranes or from gross alterations in pore complex structure, as shown by EM and immunocytochemistry. A physiological consequence of this transport inhibition was inhibition of cell progression from G2 into M phase. Hence, binding of this antibody to the lumenal side of gp210 must have a transmembrane effect on the structure and functions of the pore complex. These data argue that gp210 is directly or indirectly connected to pore complex constituents involved in mediated import and passive diffusion.

Gradient of increasing affinity of importin beta for nucleoporins along the pathway of nuclear import.

Nuclear import and export signals on macromolecules mediate directional, receptor-driven transport through the nuclear pore complex (NPC) by a process that is suggested to involve the sequential binding of transport complexes to different nucleoporins. The directionality of transport appears to be partly determined by the nucleocytoplasmic compartmentalization of components of the Ran GTPase system. We have analyzed whether the asymmetric localization of discrete nucleoporins can also contribute to transport directionality. To this end, we have used quantitative solid phase binding analysis to determine the affinity of an importin beta cargo complex for Nup358, the Nup62 complex, and Nup153, which are in the cytoplasmic, central, and nucleoplasmic regions of the NPC, respectively. These nucleoporins are proposed to provide progressively more distal binding sites for importin beta during import. Our results indicate that the importin beta transport complex binds to nucleoporins with progressively increasing affinity as the complex moves from Nup358 to the Nup62 complex and to Nup153. Antibody inhibition studies support the possibility that importin beta moves from Nup358 to Nup153 via the Nup62 complex during import. These results indicate that nucleoporins themselves, as well as the nucleocytoplasmic compartmentalization of the Ran system, are likely to play an important role in conferring directionality to nuclear protein import.

Nuclear pore complexes: dynamics in unexpected places.

In vivo studies on the dynamics of the nuclear pore complex (NPC) in yeast suggested that NPCs are highly mobile in the nuclear envelope. However, new evidence indicates that in mammalian cells NPCs are stably attached to a flexible lamina framework, but a peripheral component can exchange rapidly with an intranuclear pool.

Integral membrane proteins specific to the inner nuclear membrane and associated with the nuclear lamina.

We obtained a monoclonal antibody (RL13) that identifies three integral membrane proteins specific to the nuclear envelope of rat liver, a major 75-kD polypeptide and two more minor components of 68 and 55 kD. Immunogold labeling of isolated nuclear envelopes demonstrates that these antigens are localized specifically to the inner nuclear membrane, and that the RL13 epitope occurs on the inner membrane's nucleoplasmic surface where the nuclear lamina is found. When nuclear envelopes are extracted with solutions containing nonionic detergent and high salt to solubilize nuclear membranes and pore complexes, most of these integral proteins remain associated with the insoluble lamina. Since the polypeptides recognized by RL13 are relatively abundant, they may function as lamina attachment sites in the inner nuclear membrane. Major cross-reacting antigens are found by immunoblotting and immunofluorescence microscopy in all rat cells examined. Therefore, these integral proteins are biochemical markers for the inner nuclear membrane and will be useful models for studying nuclear membrane biogenesis.

Lamins A and C bind and assemble at the surface of mitotic chromosomes.

To study a possible interaction of nuclear lamins with chromatin, we examined assembly of lamins A and C at mitotic chromosome surfaces in vitro. When a postmicrosomal supernatant of metaphase CHO cells containing disassembled lamins A and C is incubated with chromosomes isolated from mitotic Chinese hamster ovary cells, lamins A and C undergo dephosphorylation and uniformly coat the chromosome surfaces. Furthermore, when purified rat liver lamins A and C are dialyzed with mitotic chromosomes into a buffer of physiological ionic strength and pH, lamins A and C coat chromosomes in a similar fashion. In both cases a lamin-containing supramolecular structure is formed that remains intact when the chromatin is removed by digestion with micrococcal nuclease and extraction with 0.5 M KCl. Lamins associate with chromosomes at concentrations approximately eightfold lower than the critical concentration at which they self-assemble into insoluble structures in the absence of chromosomes, indicating that chromosome surfaces contain binding sites that promote lamin assembly. These binding sites are destroyed by brief treatment of chromosomes with trypsin or micrococcal nuclease. Together, these data suggest the existence of a specific lamin-chromatin interaction in cells that may be important for nuclear envelope reassembly and interphase chromosome structure.

Inhibition of nuclear protein import by nonhydrolyzable analogues of GTP and identification of the small GTPase Ran/TC4 as an essential transport factor.

We have investigated a possible involvement of GTPases in nuclear protein import using an in vitro transport system involving digitonin-permeabilized cells supplemented with exogenous cytosol. Transport in this system was measured with a novel ELISA-based assay that allows rapid quantitative analysis. GTP gamma S and other nonhydrolyzable analogues of GTP were found to rapidly inhibit the rate of in vitro nuclear import. Transport inhibition by GTP gamma S was dependent on the concentrations of permeabilized cells and cytosol, and was strongly enhanced by a cytosolic factor(s). The predominant cytosolic component responsible for this inhibition was found in a 20-30-kD fraction in molecular sieving chromatography. Furthermore, a component(s) of this 20-30-kD fraction was itself required for efficient nuclear import. Biochemical complementation with bacterially expressed protein demonstrated that this essential GTP gamma S-sensitive transport factor was Ran/TC4, a previously described GTPase of the Ras superfamily found in both nucleus and cytoplasm. Ran/TC4 and its guanine nucleotide release protein RCC1 have previously been implicated in DNA replication, cell cycle checkpoint control, and RNA synthesis, processing and export. Our results suggest that Ran/TC4 serves to integrate nuclear protein import with these other nuclear activities.

Immunocytochemical localization of the major polypeptides of the nuclear pore complex-lamina fraction. Interphase and mitotic distribution.

This laboratory has previously isolated a fraction from rat liver nuclei consisting of nuclear pore complexes associated with the proteinaceous lamina which underlies the inner nuclear membrane. Using protein eluted from sodium dodecyl sulfate (SDS) gels, we have prepared antibodies in chickens to each of the three predominant pore complex-lamina bands. Ouchterlony double diffusion analysis shows that each of these individual bands cross-reacts strongly with all three antisera. In immunofluorescence localization performed on tissue culture cells with these antibodies, we obtain a pattern of intense staining at the periphery of the interphase nucleus, with little or no cytoplasmic reaction. Electron microscope immunoperoxidase staining of rat liver nuclei with these antibodies labels exclusively the nuclear periphery. Furthermore, reaction occurs in areas which contain the lamina, but not at the pore complexes. While our isolation procedure extracts the internal contents of nuclei completely, semiquantitative Ouchterlony analysis shows that it releases negligible amounts of these lamina antigens. Considered together, our results indicate that these three bands represent major components of a peripheral nuclear lamina, and are not structural elements of an internal "nuclear protein matrix." Fluorescence microscopy shows that the perinuclear interphase localization of these lamina proteins undergoes dramatic changes during mitosis. Concomitant with nuclear envelope disassembly in prophase, these antigens assume a diffuse localization throughout the cell. This distribution persists until telophase, when the antigens become progressively and completely localized at the surface of the daughter chromosome masses. We propose that the lamina is a biological polymer which can undergo reversible disassembly during mitosis.

Identification of a major polypeptide of the nuclear pore complex.

The nuclear pore complex is a prominent structural component of the nuclear envelope that appears to regulate nucleoplasmic molecular movement. Up to now, none of its polypeptides have been defined. To identify possible pore complex proteins, we fractionated rat liver nuclear envelopes and microsomal membranes with strong protein perturbants into peripheral and intrinsic membrane proteins, and compared these fractions on SDS gels. From this analysis, we identified a prominent 190-kilodalton intrinsic membrane polypeptide that occurs specifically in nuclear envelopes. Lectin binding studies indicate that this polypeptide (gp 190) is the major nuclear envelope glycoprotein. Upon treatment of nuclear envelopes with Triton X-100, gp 190 remains associated with a protein substructure of the nuclear envelope consisting of pore complexes and nuclear lamina. We prepared monospecific antibodies to gp 190 for immunocytochemical localization. Immunofluorescence staining of tissue culture cells suggests that gp 190 occurs exclusively in the nucleus during interphase. This polypeptide becomes dispersed throughout the cell in mitotic prophase when the nuclear envelope is disassembled, and subsequently returns to the nuclear surfaces during telophase when the nuclear envelope is reconstructed. Immunoferritin labeling of Triton-treated rat liver nuclei demonstrates that gp 190 occurs exclusively in the nuclear pore complex, in the regions of the cytoplasmic (and possibly nucleoplasmic) pore complex annuli. A polypeptide that cross-reacts with gp 190 is present in diverse vertebrate species, as shown by antibody labeling of nitrocellulose SDS gel transfers. On the basis of its biochemical characteristics, we suggest that gp 190 may be involved in anchoring the pore complex to nuclear envelope membranes.

Integral membrane proteins of the nuclear envelope are dispersed throughout the endoplasmic reticulum during mitosis.

We have analyzed the fate of several integral membrane proteins of the nuclear envelope during mitosis in cultured mammalian cells to determine whether nuclear membrane proteins are present in a vesicle population distinct from bulk ER membranes after mitotic nuclear envelope disassembly or are dispersed throughout the ER. Using immunofluorescence staining and confocal microscopy, we compared the localization of two inner nuclear membrane proteins (laminaassociated polypeptides 1 and 2 [LAP1 and LAP2]) and a nuclear pore membrane protein (gp210) to the distribution of bulk ER membranes, which was determined with lipid dyes (DiOC6 and R6) and polyclonal antibodies. We found that at the resolution of this technique, the three nuclear envelope markers become completely dispersed throughout ER membranes during mitosis. In agreement with these results, we detected LAP1 in most membranes containing ER markers by immunogold electron microscopy of metaphase cells. Together, these findings indicate that nuclear membranes lose their identity as a subcompartment of the ER during mitosis. We found that nuclear lamins begin to reassemble around chromosomes at the end of mitosis at the same time as LAP1 and LAP2 and propose that reassembly of the nuclear envelope at the end of mitosis involves sorting of integral membrane proteins to chromosome surfaces by binding interactions with lamins and chromatin.