During the mitotic phase, the nuclear envelope, responsible for protecting and organizing the interphase genome, is disassembled. Throughout the course of history, everything experiences its fleeting moments.
Within the zygote, the unification of parental genomes relies on the mitosis-linked, spatially and temporally regulated breakdown of the nuclear envelopes (NEBD) of parental pronuclei. Critical to NEBD is the disassembly of Nuclear Pore Complexes (NPCs), a necessary step for rupturing the nuclear permeability barrier, freeing NPCs from membranes near the centrosomes and those located between the juxtaposed pronuclei. We utilized a combined strategy involving live cell imaging, biochemical studies, and phosphoproteomics to characterize NPC disassembly and uncover the specific function of mitotic kinase PLK-1 in this process. We demonstrate that PLK-1's mechanism of NPC disassembly targets crucial NPC sub-complexes, such as the cytoplasmic filaments, the central channel, and the inner ring. Significantly, PLK-1 is drawn to and phosphorylates intrinsically disordered regions within multiple multivalent linker nucleoporins, a mechanism apparently serving as an evolutionarily conserved driving force behind NPC disassembly during the mitotic stage. Repackage this JSON schema: sentences in a list format.
Nuclear pore complexes are dismantled by PLK-1, which acts upon the intrinsically disordered regions of multiple multivalent nucleoporins.
zygote.
The intrinsically disordered regions of multivalent nucleoporins are the targets of PLK-1, a protein that disrupts nuclear pore complexes in the C. elegans zygote.

In the Neurospora circadian clock's regulatory loop, FREQUENCY (FRQ), a central component, unites with FRH (FRQ-interacting RNA helicase) and Casein Kinase 1 (CK1) to form the FRQ-FRH complex (FFC). This complex dampens its own production by interacting with and initiating phosphorylation of the transcriptional activators White Collar-1 (WC-1) and WC-2, elements of the White Collar Complex (WCC). The physical coupling between FFC and WCC is a prerequisite for the repressive phosphorylations, and despite the known motif on WCC essential for this interaction, the reciprocal recognition motif(s) on FRQ remain(s) vaguely understood. A systematic assessment of FFC-WCC was undertaken employing frq segmental-deletion mutants, validating the requirement of multiple, dispersed FRQ regions for proper interaction with WCC. 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. In a surprising finding, even with substantial reductions in FFC-WCC interaction due to Asp/Glu-to-Ala mutations in the frq gene, the core clock maintained robust oscillation at a period nearly identical to wild type, suggesting that while the binding force between positive and negative components in the feedback loop is essential for the clock's operation, it does not solely define the oscillation period.

A critical role in regulating the function of membrane proteins is played by their oligomeric organization within native cell membranes. High-resolution quantitative measurements of oligomeric assemblies and their alterations under various conditions are crucial for comprehending the intricacies of membrane protein biology. The single-molecule imaging technique, Native-nanoBleach, is introduced for determining the oligomeric distribution of membrane proteins from native membranes with a spatial resolution of 10 nanometers. Target membrane proteins were encapsulated within native nanodiscs, maintaining their proximal native membrane environment, thanks to amphipathic copolymers. Membrane proteins, diverse in their structural and functional roles and exhibiting known stoichiometries, formed the basis for this method. Employing Native-nanoBleach, we evaluated the degree of oligomerization of the receptor tyrosine kinase TrkA and small GTPase KRas, in the presence of growth factor binding or oncogenic mutations, respectively. With unprecedented spatial resolution, Native-nanoBleach's sensitive single-molecule platform quantifies the oligomeric distribution of membrane proteins within native membranes.

To identify small molecules affecting the structure and function of the cardiac sarco/endoplasmic reticulum calcium ATPase (SERCA2a), we have used FRET-based biosensors in a sturdy high-throughput screening (HTS) platform involving live cells. To tackle heart failure, our principal aim is to find small-molecule activators that are drug-like and can improve the function of SERCA. Our prior work highlighted the utility of an intramolecular FRET biosensor constructed using human SERCA2a. A small validation set was evaluated using novel microplate readers, which precisely measure fluorescence lifetime or emission spectra at high speed and resolution. We report the results of a 50,000-compound screen, which utilized the same biosensor, followed by functional assessment of the hit compounds via Ca²⁺-ATPase and Ca²⁺-transport assays. Procyanidin C1 Focusing on 18 hit compounds, our analysis yielded eight structurally unique compounds and four categories of SERCA modulators. About half of these compounds acted as activators, and the other half as inhibitors. Activators and inhibitors, while both possessing therapeutic potential, serve as a foundation for future testing in heart disease models, leading to the development of pharmaceutical treatments for heart failure.

HIV-1's retroviral Gag protein is centrally involved in the process of selecting unspliced viral genomic RNA for packaging in new virions. Procyanidin C1 Earlier experiments revealed that the full HIV-1 Gag protein undergoes nuclear trafficking, where it interacts with unprocessed viral RNA (vRNA) at transcription sites. To delve further into the kinetics of HIV-1 Gag nuclear localization, we employed biochemical and imaging methods to analyze the temporal aspect of HIV-1's nuclear entry. Our investigation also included the goal of achieving a more accurate assessment of Gag's subnuclear distribution, to explore the proposition that Gag would be associated with the euchromatin, the nucleus's transcriptionally active component. We documented the nuclear localization of HIV-1 Gag soon after its synthesis in the cytoplasm, implying that nuclear trafficking mechanisms are not strictly concentration-based. Within the latently infected CD4+ T cell line (J-Lat 106), following exposure to latency-reversal agents, HIV-1 Gag protein showed a significant preference for the euchromatin fraction, which is active in transcription, compared to the dense heterochromatin region. An interesting observation is the more robust association of HIV-1 Gag with transcriptionally active histone markers situated near the nuclear periphery, where the HIV-1 proviral DNA has been previously shown to integrate. While the exact purpose of Gag's relationship with histones within actively transcribing chromatin is unclear, this discovery, in agreement with previous reports, proposes a potential role for euchromatin-associated Gag molecules in the selection of newly synthesized unspliced viral RNA during the initial steps of virion assembly.
The established model of retroviral assembly suggests that HIV-1 Gag protein selection of unedited viral RNA commences within the cellular cytoplasm. Nonetheless, our prior investigations revealed that HIV-1 Gag translocates to the nucleus and interacts with unspliced HIV-1 RNA at transcriptional loci, implying a potential role for nuclear genomic RNA selection. This study revealed the nuclear translocation of HIV-1 Gag protein, concurrently with unspliced viral RNA, occurring within eight hours of expression. Latency reversal agents, acting on CD4+ T cells (J-Lat 106), along with a HeLa cell line containing a stably expressed inducible Rev-dependent provirus, caused HIV-1 Gag to preferentially localize with histone marks correlated to active enhancer and promoter regions within euchromatin near the nuclear periphery, potentially favoring 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.
HIV-1 Gag's selection of unspliced vRNA, in the traditional retroviral assembly model, starts in the cytoplasm. Our preceding studies highlighted that HIV-1 Gag enters the nucleus and binds to unprocessed HIV-1 RNA at the transcription initiation sites, thus suggesting a nuclear stage for genomic RNA selection. Following expression, we observed the nuclear entry of HIV-1 Gag and its concurrent localization with unspliced viral RNA, completing this process within eight hours. In our study using J-Lat 106 CD4+ T cells treated with latency reversal agents, and a HeLa cell line expressing a stably induced Rev-dependent provirus, we found HIV-1 Gag to be preferentially localized near the nuclear periphery, situated with histone marks indicative of enhancer and promoter regions in active euchromatin. This co-localization could reflect favored HIV-1 proviral integration sites. Evidence suggests that HIV-1 Gag's ability to seize euchromatin-associated histones to focus on active transcription sites supports the idea that this facilitates the collection and packaging of newly synthesized 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. However, the pathways by which pathogens affect the host's metabolic machinery are not completely understood. JHU083, a groundbreaking glutamine metabolism antagonist, proves effective in reducing Mtb proliferation in both laboratory and animal studies. Procyanidin C1 Mice receiving JHU083 treatment experienced weight gain, enhanced survival, a significant 25 log decrease in lung bacterial burden at 35 days post-infection, and reduced lung tissue abnormalities.