The interphase genome's structured environment, the nuclear envelope, is broken down during the process of mitosis. In the intricate tapestry of life, each element eventually fades away.
During mitosis, the breakdown of the parental pronuclei's nuclear envelopes (NEBD) is precisely controlled in space and time to facilitate the union of the parental genomes within a zygote. The dismantling of the Nuclear Pore Complex (NPC) during NEBD is essential for rupturing the nuclear permeability barrier and separating NPCs from the membranes near the centrosomes and those intervening the joined 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. Through our analysis, we reveal that PLK-1 disassembles the NPC by focusing on its multiple sub-complexes, specifically the cytoplasmic filaments, the 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.
PLK-1's strategy to dismantle nuclear pore complexes involves targeting intrinsically disordered regions in multiple multivalent nucleoporins.
zygote.
Multiple multivalent nucleoporins' intrinsically disordered regions are precisely targeted by PLK-1, which consequently leads to the breakdown of nuclear pore complexes in C. elegans zygotes.

FREQUENCY (FRQ), the key player in the Neurospora circadian negative feedback loop, joins forces with FRH (FRQ-interacting RNA helicase) and Casein Kinase 1 (CK1) to create the FRQ-FRH complex (FFC). This complex curtails its own expression by engaging with and triggering the phosphorylation of White Collar-1 (WC-1) and WC-2 (constituents of the White Collar Complex, WCC), its transcriptional activators. The physical interaction of FFC and WCC is fundamental to the repressive phosphorylations; while the required motif on WCC for this interaction is well-defined, the corresponding recognition motif(s) on FRQ are still largely unknown. Through the use of frq segmental-deletion mutants, the FFC-WCC interaction was examined, confirming the role of multiple, scattered regions on FRQ in mediating the association. Following the recognition of a critical sequence motif in WC-1 regarding WCC-FFC assembly, a mutagenic approach was undertaken to analyze the negatively charged residues of FRQ. This research process led to the discovery of three indispensable Asp/Glu clusters in FRQ, which are necessary for the creation of FFC-WCC structures. Surprisingly, the core clock's robust oscillation, with a period essentially matching wild type, persisted in several frq Asp/Glu-to-Ala mutants characterized by a pronounced decrease in FFC-WCC interaction, implying that the binding strength between positive and negative feedback loop components is essential to the clock's function, but not as a determinant of the oscillation period.

Native cell membranes' protein function is determined by the oligomeric arrangements of membrane proteins they contain. Precise high-resolution quantitative analyses of oligomeric assemblies and their modifications in different conditions are fundamental to advancing our knowledge 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. Employing amphipathic copolymers, we encapsulated target membrane proteins in native nanodiscs, retaining their proximal native membrane environment. This method's development relied on the utilization of membrane proteins exhibiting both functional and structural diversity, as well as predetermined stoichiometric amounts. For evaluating the oligomerization status of TrkA, a receptor tyrosine kinase, and KRas, a small GTPase, under growth factor binding or oncogenic mutations, we used Native-nanoBleach. The sensitive single-molecule platform of Native-nanoBleach allows for an unprecedented spatial resolution in quantifying the oligomeric distribution of membrane proteins within native membranes.

Our investigation, employing FRET-based biosensors within a robust high-throughput screening (HTS) setup on live cells, has revealed small molecules that modify the structure and activity of the cardiac sarco/endoplasmic reticulum calcium ATPase (SERCA2a). To effectively treat heart failure, our primary objective is the identification of small-molecule drug-like activators that enhance SERCA function. In our previous research, an intramolecular FRET biosensor based on the human SERCA2a protein was employed. High-speed and high-resolution microplate readers were used to validate this approach through screening a small subset, determining fluorescence lifetime or emission spectra. The 50,000-compound screen, using the same biosensor platform, is reported here, with hit compounds subsequently evaluated through Ca²⁺-ATPase and Ca²⁺-transport assays. ACY-775 Our investigation centered on 18 hit compounds; from these, eight structurally unique compounds were identified, belonging to four classes of SERCA modulators. Approximately half act as activators, and 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.

Human immunodeficiency virus type 1 (HIV-1)'s retroviral Gag protein plays a critical role in the selection of unspliced viral genomic RNA for incorporation into nascent virions. ACY-775 In previous work, we ascertained that the entire HIV-1 Gag protein exhibits nuclear trafficking, where it engages with unspliced viral RNA (vRNA) at transcription sites. 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. To examine the hypothesis of Gag's association with euchromatin, the transcriptionally active region of the nucleus, a more precise determination of Gag's subnuclear distribution was also undertaken. Our research demonstrated that HIV-1 Gag relocated to the nucleus soon after its creation in the cytoplasm, suggesting that nuclear trafficking does not adhere to a strict concentration dependency. Analysis of latently infected CD4+ T cells (J-Lat 106), treated with latency-reversal agents, demonstrated that HIV-1 Gag protein was predominantly found in the transcriptionally active euchromatin portion of the cell, compared to the heterochromatin-rich regions. A compelling discovery is that HIV-1 Gag had a stronger connection to transcriptionally active histone markers situated near the nuclear periphery, a location previously implicated in the insertion of the HIV-1 provirus. 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.
In the prevailing model of retroviral assembly, the initial stage of HIV-1 Gag selecting unspliced viral RNA takes place in the 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's findings illustrated the nuclear import of HIV-1 Gag protein and its co-localization with unspliced viral RNA, happening within eight hours post-expression. Our research on CD4+ T cells (J-Lat 106) treated with latency reversal agents, alongside a HeLa cell line that stably expresses an inducible Rev-dependent provirus, revealed that HIV-1 Gag preferentially clustered near the nuclear periphery with histone marks related to active enhancer and promoter regions within euchromatin, a location positively correlated with HIV-1 proviral integration sites. These observations provide support for the hypothesis that HIV-1 Gag, through its association with euchromatin-associated histones, facilitates localization at active transcriptional sites to promote the capture of newly synthesized viral genomic RNA for packaging.
Retroviral assembly, according to the traditional view, sees HIV-1 Gag's selection of unspliced vRNA commencing in the cellular cytoplasm. Our previous research exemplified the nuclear import of HIV-1 Gag and its binding to the unspliced HIV-1 RNA at transcription areas, implying the potential for genomic RNA selection to take place within the nucleus. Our observations revealed the presence of HIV-1 Gag within the nucleus, co-localized with unspliced viral RNA, evidenced within eight hours post-expression. Latency-reversal agents administered to J-Lat 106 CD4+ T cells, in combination with a HeLa cell line engineered to stably express an inducible Rev-dependent provirus, revealed a preferential localization of HIV-1 Gag proteins near the nuclear periphery, specifically with histone marks associated with enhancer and promoter regions of active euchromatin. This proximity is suggestive of favored HIV-1 proviral integration locations. The data suggest that HIV-1 Gag's exploitation of euchromatin-associated histones to concentrate at active transcription sites supports the hypothesis that this enhances the acquisition and packaging of newly synthesized genomic RNA for viral use.

With its status as one of the most successful human pathogens, Mycobacterium tuberculosis (Mtb) has evolved numerous factors to counteract host immunity and modify metabolic pathways in the host. Nevertheless, the intricacies of how pathogens disrupt a host's metabolic processes are still unclear. We present evidence that JHU083, a novel glutamine metabolism antagonist, inhibits the multiplication of Mtb in laboratory and animal-based settings. ACY-775 JHU083-treated mice displayed weight gain, enhanced survival, a 25-log reduction in lung bacillary burden 35 days post-infection, and mitigation of lung pathology.