The interphase genome's organization and protection provided by the nuclear envelope is dismantled during mitosis. Throughout the unending journey of time, all things experience their temporary nature.
The temporal and spatial regulation of parental pronuclei nuclear envelope breakdown (NEBD) during mitosis within the zygote is crucial for the integration of parental genomes. Essential for NEBD, the dismantling of the Nuclear Pore Complex (NPC) is pivotal to disrupting the nuclear permeability barrier, detaching NPCs from membranes situated near the centrosomes and those found between the neighboring pronuclei. Using a comprehensive methodology involving live-cell imaging, biochemical assays, and phosphoproteomic profiling, we investigated the dismantling of NPCs and identified the precise role of the mitotic kinase PLK-1 in this process. Our study shows that the NPC's disassembly is influenced by PLK-1, which selectively targets various NPC sub-complexes, such as the cytoplasmic filaments, central channel, and the inner ring. Evidently, PLK-1 is mobilized to and phosphorylates the intrinsically disordered regions of multiple multivalent linker nucleoporins, a mechanism which appears to be an evolutionarily conserved mediator of nuclear pore complex dismantling during mitosis. Repackage this JSON schema: sentences in a list format.
Intrinsically disordered regions of multiple multivalent nucleoporins are targeted by PLK-1, leading to the dismantling of nuclear pore complexes.
zygote.
Multivalent nucleoporins' intrinsically disordered regions are a specific site for PLK-1's activity, leading to the breakdown of nuclear pore complexes in the C. elegans zygote.

The FRQ-FRH complex (FFC), resulting from the binding of FREQUENCY (FRQ) with FRH (FRQ-interacting RNA helicase) and Casein Kinase 1 (CK1) within the Neurospora circadian clock's negative feedback loop, downregulates its own expression. This occurs by interacting with, and inducing phosphorylation of, the transcriptional activators White Collar-1 (WC-1) and WC-2, constituting the White Collar Complex (WCC). For repressive phosphorylations to occur, a physical connection between FFC and WCC is necessary; although the interaction-specific motif on WCC is identified, the complementary recognition motif(s) on FRQ remain(s) less clear. A series of frq segmental-deletion mutants was employed to assess FFC-WCC interaction, highlighting that diverse, dispersed regions of FRQ are critical for this interaction. Prior identification of a fundamental sequence motif on WC-1 highlighted its crucial role in WCC-FFC assembly, prompting our mutagenic investigation focusing on the negatively charged residues within FRQ. This led to the discovery of three indispensable Asp/Glu clusters in FRQ, essential for the formation of FFC-WCC complexes. Remarkably, despite substantial impairment of FFC-WCC interaction in numerous frq Asp/Glu-to-Ala mutants, the core clock surprisingly maintains a robust oscillation with a period essentially matching that of the wild type, suggesting that the clock's operation depends on the binding strength between positive and negative components within the feedback loop but not on the precise magnitude of that strength determining its period.

The manner in which membrane proteins are oligomerically organized within native cell membranes significantly impacts their function. High-resolution quantitative assessments of oligomeric assemblies and their transformations in response to diverse conditions are essential for a comprehensive understanding of membrane protein biology. To determine the oligomeric distribution of membrane proteins from native membranes, we have developed the single-molecule imaging technique, Native-nanoBleach, with a spatial precision of 10 nanometers. Amphipathic copolymers allowed us to capture target membrane proteins in native nanodiscs, preserving their proximal native membrane environment. By using membrane proteins that differed both structurally and functionally, and whose stoichiometries were well-defined, this method was created. We then quantified the oligomerization status of receptor tyrosine kinase TrkA and small GTPase KRas under growth-factor binding or oncogenic mutation conditions, respectively, utilizing Native-nanoBleach. Native-nanoBleach's single-molecule platform, extraordinarily sensitive, allows for the quantification of membrane protein oligomeric distributions in native membranes with unmatched spatial precision.

In a high-throughput screening (HTS) environment using live cells, FRET-based biosensors have been employed to pinpoint small molecules influencing the structure and function of the cardiac sarco/endoplasmic reticulum calcium ATPase (SERCA2a). Small-molecule drug-like activators of SERCA, which improve its function, represent our primary objective in treating heart failure. Prior investigations have presented an intramolecular FRET biosensor, derived from the human SERCA2a protein. A limited collection was screened with cutting-edge microplate readers, offering high speed, precision, and resolution in quantifying fluorescence lifetime or emission spectra. We now present the outcomes of a 50,000-compound screen, utilizing a unified biosensor. Subsequent Ca²⁺-ATPase and Ca²⁺-transport assays further assessed these hit compounds. selleck chemicals llc From a set of 18 hit compounds, we isolated eight structurally distinct compounds categorized into four classes, all acting as SERCA modulators; roughly half function as activators, and the other half as inhibitors. Though both activators and inhibitors demonstrate therapeutic utility, activators are crucial for future research in heart disease models, steering development of pharmaceutical therapies for heart failure.

HIV-1's retroviral Gag protein is instrumental in choosing unspliced viral RNA to be packaged within emerging virions. selleck chemicals llc Earlier studies revealed that the complete HIV-1 Gag molecule participates in nuclear transport, associating with unspliced viral RNA (vRNA) within transcription-active regions. To scrutinize the kinetics of HIV-1 Gag nuclear localization, we used biochemical and imaging techniques to assess the temporal characteristics of HIV-1's entry into the nucleus. We were further motivated to determine, with greater precision, Gag's subnuclear distribution in order to scrutinize the hypothesis that Gag would be found within euchromatin, the nucleus's actively transcribing region. In our observations, HIV-1 Gag's nuclear translocation was observed shortly after its cytoplasmic production, suggesting that the process of nuclear trafficking is independent of strict concentration dependence. The latently-infected CD4+ T cell line (J-Lat 106), treated with latency-reversal agents, displayed a preferential localization of HIV-1 Gag protein to transcriptionally active euchromatin compared to the heterochromatin-dense regions. HIV-1 Gag displayed a notable and more pronounced association with histone markers engaged in transcription, specifically close to the nuclear periphery, the area identified for HIV-1 provirus integration in prior studies. Although the exact function of Gag's association with histones in transcriptionally active chromatin remains ambiguous, the present finding, in line with previous observations, is suggestive of a potential role for euchromatin-associated Gag in selecting nascent, unspliced viral RNA during the initial stage of virion assembly.
HIV-1 Gag's preferential selection of unspliced viral RNA, as per the conventional retroviral assembly theory, occurs in the cytoplasm. Our earlier investigations into HIV-1 Gag’s activity showed that it enters the nucleus and binds to unspliced HIV-1 RNA at transcription sites, leading us to infer a potential role for genomic RNA selection within the nucleus. Our present investigation documented the nuclear entry of HIV-1 Gag and its co-localization with unspliced viral RNA within a timeframe of eight hours post-expression. A study using CD4+ T cells (J-Lat 106) treated with latency reversal agents, as well as a HeLa cell line stably expressing an inducible Rev-dependent provirus, determined that HIV-1 Gag specifically localized with histone marks associated with enhancer and promoter regions of active euchromatin near the nuclear periphery, which may promote HIV-1 proviral integration. These observations support the proposition that HIV-1 Gag's interaction with euchromatin-associated histones facilitates its localization to actively transcribing regions, leading to the packaging of recently synthesized viral genomic RNA.
In the cytoplasm, the traditional model of retroviral assembly proposes the HIV-1 Gag's selection of unspliced vRNA. Our prior studies showcased that HIV-1 Gag penetrates the nucleus and associates with unspliced HIV-1 RNA at sites of transcription, thereby suggesting a potential nuclear role in the selection of viral genomic RNA. Eight hours post-expression, a concurrent nuclear entry of HIV-1 Gag and co-localization with unspliced viral RNA was observed in this study. J-Lat 106 CD4+ T cells, subjected to latency reversal agent treatment, and a HeLa cell line expressing an inducible Rev-dependent provirus, displayed a preferential localization of HIV-1 Gag proteins near the nuclear periphery in association with histone marks characteristic of active enhancer and promoter regions within euchromatin. This distribution potentially reflects a predilection for proviral integration sites. HIV-1 Gag's strategy of leveraging euchromatin-associated histones to target sites of active transcription, as observed, corroborates the hypothesis that this mechanism facilitates the collection and packaging of newly synthesized viral genomic RNA.

Mycobacterium tuberculosis (Mtb), a prime example of a successful human pathogen, possesses a multitude of factors that enable it to subvert host immunity and reprogram host metabolism. In contrast, the strategies pathogens employ to manipulate the metabolic processes of their hosts remain poorly characterized. We demonstrate that the novel glutamine metabolism inhibitor, JHU083, suppresses Mycobacterium tuberculosis growth in both laboratory and live animal models. selleck chemicals llc JHU083-treated mice demonstrated weight gain, prolonged survival, a 25-log reduction in lung bacterial load 35 days post-infection, and a decrease in lung tissue abnormalities.