Human Adenovirus Type 5 Infection Leads to Nuclear Envelope Destabilization and Membrane Permeability Independently of Adenovirus Death Protein.
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ABSTRACT:
SUBMITTER: Pfitzner S
PROVIDER: S-EPMC8657697 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The nuclear pore complex (NPC) mediates macromolecular exchange between nucleus and cytoplasm. It is a regulated channel whose functional properties are modulated in response to the physiological status of the cell. Identifying the factors responsible for regulating NPC activity is crucial to understand how intracellular signaling cues are integrated at the level of this channel to control nucleocytoplasmic trafficking. For proteins lacking active translocation signals the NPC acts as a molecular sieve limiting passage across the nuclear envelope (NE) to proteins with a MW below ~40 kD. Here, we investigate how this permeability barrier is altered in paradigms of cell death and cell survival, i.e., apoptosis induction via staurosporine, and enhanced viability via overexpression of Bcl-2. We monitor dynamic changes of the NPC's size-exclusion limit for passive diffusion by confocal time-lapse microscopy of cells undergoing apoptosis, and use different diffusion markers to determine how Bcl-2 expression affects steady-state NE permeability. We show that staurosporine triggers an immediate and gradual leakiness of the NE preceding the appearance of apoptotic hallmarks. Bcl-2 expression leads to a constitutive increase in NE permeability, and its localization at the NE is sufficient for the effect, evincing a functional role for Bcl-2 at the nuclear membrane. In both settings, NPC leakiness correlates with reduced Ca²? in internal stores, as demonstrated by fluorometric measurements of ER/NE Ca²? levels. By comparing two cellular models with opposite outcome these data pinpoint ER/NE Ca²? as a general and physiologically relevant regulator of the permeability barrier function of the NPC.
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Project description:After receptor-mediated endocytosis and endosomal escape, adenoviral capsids can travel via microtubule organizing centers to the nuclear envelope. Upon capsid disassembly, viral genome import into nuclei of interphase cells then occurs through nuclear pore complexes, involving the nucleoporins Nup214 and Nup358. Import also requires the activity of the classic nuclear export receptor CRM1, as it is blocked by the selective inhibitor leptomycin B. We have now used artificially enucleated as well as mitotic cells to analyze the role of an intact nucleus in different steps of the viral life cycle. In enucleated U2OS cells, viral capsids traveled to the microtubule organizing center, whereas their removal from this complex was blocked, suggesting that this step required nuclear factors. In mitotic cells, on the other hand, CRM1 promoted capsid disassembly and genome release, suggesting a role of this protein that does not require intact nuclear envelopes or nuclear pore complexes and is distinct from its function as a nuclear export receptor. Similar to enucleation, inhibition of CRM1 by leptomycin B also leads to an arrest of adenoviral capsids at the microtubule organizing center. In a small-scale screen using leptomycin B-resistant versions of CRM1, we identified a mutant, CRM1 W142A P143A, that is compromised with respect to adenoviral capsid disassembly in both interphase and mitotic cells. Strikingly, this mutant is capable of exporting cargo proteins out of the nucleus of living cells or digitonin-permeabilized cells, pointing to a role of the mutated region that is not directly linked to nuclear export. IMPORTANCE A role of nucleoporins and of soluble transport factors in adenoviral genome import into the nucleus of infected cells in interphase has previously been established. The nuclear export receptor CRM1 promotes genome import, but its precise function is not known. Using enucleated and mitotic cells, we showed that CRM1 does not simply function by exporting a crucial factor out of the nucleus that would then trigger capsid disassembly and genome import. Instead, CRM1 has an export-independent role, a notion that is also supported by a mutant, CRM1 W142A P143A, which is export competent but deficient in viral capsid disassembly, in both interphase and mitotic cells.
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Project description:We report on a simple, quantitative relationship between structure and permeability of a novel ultrathin nanoporous membrane based on nanocrystalline silicon. Large permeability of the free-standing nanomembrane to Ru(NH3)63+, O2, or 1,1'-ferrocenedimethanol, which was able to be measured for the first time by employing scanning electrochemical microscopy, is proportional to the density (67 mum-2) and average radius (5.6 nm) of nanopores. As solution electrolyte concentration decreases down to 0.01 M, the nanopores are selectively "closed" against Fe(CN)64- because of electrostatic repulsion against negative charges at the pore wall. Permeability of the silicon nanomembrane was compared to permeability of the nuclear envelope to find that the channel diameter of the nuclear pore complex that perforates the nuclear envelope is much larger than the average diameter of the silicon nanopores and concomitantly a hypothetical diameter of 10 nm.
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Project description:Heart muscle cells, cardiomyocytes, are highly differentiated cells that usually do not proliferate . During the non-proliferative state, extracellular signals control cardiomyocyte contractile function. However, during development and regeneration, cardiomyocytes enter the cell cycle and divide. It is unknown how cardiomyocytes modify their intracellular signaling to direct the cell cycle program. Here, we show that the nuclear lamina protein Lamin B2 (Lmnb2) regulates cardiomyocyte cell cycle activity using a gatekeeper mechanism. We identified Lmnb2 as a candidate for regulating intracellular signaling with deep transcriptional profiling of single cardiomyocytes. Lmnb2 was sufficient and necessary for cardiomyocyte cycling in the presence of serum. Lmnb2 increased the nucleoporin NUP98 and permeability of the nuclear membrane for phosphorylated ERK1/2. In vivo, the Lmnb2 gene was required for cardiomyocyte cell cycle activity during development. Increasing the expression of Lmnb2 in neonatal mice promoted cardiomyocyte M-phase and cytokinesis. LmnB2 gene transfer in neonatal mice that received a myocardial injury increased cardiomyocyte division and myocardial function in the injury border zone, indicating that the regenerated cardiomyocytes were functionally integrated. We propose a gatekeeper function of Lmnb2 that can be targeted to increase cardiomyocyte regeneration without the administration of exogenous growth factors.
2020-02-28 | GSE108360 | GEO
Project description:The nuclear envelope is an undisputed component of the intracellular mechanotransduction cascades which collect, process, and respond to mechanical stimuli from the environment. At the same time, the nuclear envelope performs the function of a selective barrier between the nuclear and cytoplasmic compartments. Although the mechanosensing and the barrier functions of the nuclear envelope have both been subjects of intense research, a possible reciprocal relationship between them is only beginning to emerge. In this report, the role of the nucleocytoplasmic permeability barrier is evaluated in nuclear mechanics. Using a combination of atomic force and confocal microscopy, the functional state of the nucleocytoplasmic permeability barrier and the nuclear mechanics is monitored. By modulating the stringency of the barrier and simulating the active transport imbalance across the nuclear envelope, the decisive impact of these parameters on nuclear mechanics is demonstrated. It is concluded that the nucleocytoplasmic barrier is the second essential component of the intracellular mechanostat function performed by the nuclear envelope.
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Project description:The nuclear envelope (NE) is one of the least characterized structures of eukaryotic cells. The study of its functional roles is hampered by the small number of proteins known to be specifically located to it. Here, we present a comprehensive characterization of the NE proteome. We applied different fractionation procedures and isolated protein subsets derived from distinct NE compartments. We identified 148 different proteins by 16-benzyl dimethyl hexadecyl ammonium chloride (16-BAC) gel electrophoresis and matrix-assisted laser desorption ionization (MALDI) mass spectrometry; among them were 19 previously unknown or noncharacterized. The identification of known proteins in particular NE fractions enabled us to assign novel proteins to NE substructures. Thus, our subcellular proteomics approach retains the screening character of classical proteomic studies, but also allows a number of predictions about subcellular localization and interactions of previously noncharacterized proteins. We demonstrate this result by showing that two novel transmembrane proteins, a 100-kDa protein with similarity to Caenorhabditis elegans Unc-84A and an unrelated 45-kDa protein we named LUMA, reside in the inner nuclear membrane and likely interact with the nuclear lamina. The utility of our approach is not restricted to the investigation of the NE. Our approach should be applicable to the analysis of other complex membrane structures of the cell as well.
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Project description:Adenovirus serotypes have been shown to cause drastic changes in nuclear organization, including the transcription machinery, during infection. This ability of adenovirus to subvert transcription in the host cell facilitates viral replication. Because nuclear actin and nuclear myosin I, myosin V and myosin VI have been implicated as direct regulators of transcription and important factors in the replication of other viruses, we sought to determine how nuclear actin and myosins are involved in adenovirus infection. We first confirmed reorganization of the host's transcription machinery to viral replication centers. We found that nuclear actin also reorganizes to sites of transcription through the intermediate but not the advanced late phase of viral infection. Furthermore, nuclear myosin I localized with nuclear actin and sites of transcription in viral replication centers. Intriguingly, nuclear myosins V and VI, which also reorganized to viral replication centers, exhibited different localization patterns, suggesting specialized roles for these nuclear myosins. Finally, we assessed the role of actin in adenovirus infection and found both cytoplasmic and nuclear actin likely play roles in adenovirus infection and replication. Together our data suggest the involvement of actin and multiple myosins in the nuclear replication and late viral gene expression of adenovirus.
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Project description:The nuclear envelope (NE) consists of two concentric nuclear membranes separated by the lumen, an ∼40-nm-wide fluid layer. NE proteins are implicated in important cellular processes ranging from gene expression to nuclear positioning. Although recent progress has been achieved in quantifying the assembly states of NE proteins in their native environment with fluorescence fluctuation spectroscopy, these studies raised questions regarding the association of NE proteins with nuclear membranes during the assembly process. Monitoring the interaction of proteins with membranes is important because the binding event is often associated with conformational changes that are critical to cellular signaling pathways. Unfortunately, the close physical proximity of both membranes poses a severe experimental challenge in distinguishing luminal and membrane-associated NE proteins. This study seeks to address this problem by introducing new, to our knowledge, fluorescence-based assays that overcome the restrictions imposed by the NE environment. We found that luminal proteins violate the Stokes-Einstein relation, which eliminates a straightforward use of protein mobility as a marker of membrane association within the NE. However, a surprising anomaly in the temperature-dependent mobility of luminal proteins was observed, which was developed into an assay for distinguishing between soluble and membrane-bound NE proteins. We further introduced a second independent tool for distinguishing both protein populations by harnessing the previously reported undulations of the nuclear membranes. These membrane undulations introduce local volume changes that produce an additional fluorescence fluctuation signal for luminal, but not for membrane-bound, proteins. After testing both methods using simple model systems, we apply the two assays to investigate a previously proposed model of membrane association for the luminal domain of SUN2, a constituent protein of the linker of nucleoskeleton and cytoskeleton complex. Finally, we investigate the effect of C- and N-terminal tagging of the luminal ATPase torsinA on its ability to associate with nuclear membranes.
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Project description:Partitioning of lipid precursors between membranes and storage is crucial for cell growth, and its disruption underlies pathologies such as cancer, obesity, and type 2 diabetes. However, the mechanisms and signals that regulate this process are largely unknown. In yeast, lipid precursors are mainly used for phospholipid synthesis in nutrient-rich conditions in order to sustain rapid proliferation but are redirected to triacylglycerol (TAG) stored in lipid droplets during starvation. Here we investigate how cells reprogram lipid metabolism in the endoplasmic reticulum. We show that the conserved phosphatidate (PA) phosphatase Pah1, which generates diacylglycerol from PA, targets a nuclear membrane subdomain that is in contact with growing lipid droplets and mediates TAG synthesis. We find that cytosol acidification activates the master regulator of Pah1, the Nem1-Spo7 complex, thus linking Pah1 activity to cellular metabolic status. In the absence of TAG storage capacity, Pah1 still binds the nuclear membrane, but lipid precursors are redirected toward phospholipids, resulting in nuclear deformation and a proliferation of endoplasmic reticulum membrane. We propose that, in response to growth signals, activation of Pah1 at the nuclear envelope acts as a switch to control the balance between membrane biogenesis and lipid storage.
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