Phosphatidylinositol phosphates are powerful signaling molecules that orchestrate signaling and direct membrane trafficking in the cytosol. Interestingly, phosphatidylinositol phosphates also localize within the membrane-less compartments of the cell nucleus, where they participate in the regulation of gene expression. Nevertheless, current models of gene expression, which include condensates of proteins and nucleic acids, do not include nuclear phosphatidylinositol phosphates. This gap is partly a result of the missing detailed analysis of the subnuclear distribution of phosphatidylinositol phosphates and their relationships with gene expression. Here, we used quantitative dual-color direct stochastic optical reconstruction microscopy to analyze the nanoscale co-patterning between RNA polymerase II transcription initiation and elongation markers with respect to phosphatidylinositol 4,5- or 3,4-bisphosphate in the nucleoplasm and nuclear speckles and compared it with randomized data and cells with inhibited transcription. We found specific co-patterning of the transcription initiation marker P-S5 with phosphatidylinositol 4,5-bisphosphate in the nucleoplasm and with phosphatidylinositol 3,4-bisphosphate at the periphery of nuclear speckles. We showed the specific accumulation of the transcription elongation marker PS-2 and of nascent RNA in the proximity of phosphatidylinositol 3,4-bisphosphate associated with nuclear speckles. Taken together, this shows that the distinct spatial associations between the consecutive stages of RNA polymerase II transcription and nuclear phosphatidylinositol phosphates exhibit specificity within the gene expression compartments. Thus, in analogy to the cellular membranes, where phospholipid composition orchestrates signaling pathways and directs membrane trafficking, we propose a model in which the phospholipid identity of gene expression compartments orchestrates RNA polymerase II transcription.

Lamins, the nuclear intermediate filaments, are important regulators of nuclear structural integrity as well as nuclear functional processes such as DNA transcription, replication and repair, and epigenetic regulations. A portion of phosphorylated lamin A/C localizes to the nuclear interior in interphase, forming a lamin A/C pool with specific properties and distinct functions. Nucleoplasmic lamin A/C molecular functions are mainly dependent on its binding partners; therefore, revealing new interactions could give us new clues on the lamin A/C mechanism of action. In the present study, we show that lamin A/C interacts with nuclear phosphoinositides (PIPs), and with nuclear myosin I (NM1). Both NM1 and nuclear PIPs have been previously reported as important regulators of gene expression and DNA damage/repair. Furthermore, phosphorylated lamin A/C forms a complex with NM1 in a phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2)-dependent manner in the nuclear interior. Taken together, our study reveals a previously unidentified interaction between phosphorylated lamin A/C, NM1, and PI(4,5)P2 and suggests new possible ways of nucleoplasmic lamin A/C regulation, function, and importance for the formation of functional nuclear microdomains.

DNA-intercalated motifs (iMs) are facile scaffolds for the design of various pH-responsive nanomachines, including biocompatible pH sensors. First, DNA pH sensors relied on complex intermolecular scaffolds. Here, we used a simple unimolecular dual-labeled iM scaffold and minimized it by replacing the redundant loop nucleosides with abasic or alkyl linkers. These modifications improved the thermal stability of the iM and increased the rates of its pH-induced conformational transitions. The best effects were obtained upon the replacement of all three native loops with short and flexible linkers, such as the propyl one. The resulting sensor showed a pH transition value equal to 6.9 ± 0.1 and responded rapidly to minor acidification (tau1/2 <1 s for 7.2 → 6.6 pH jump). We demonstrated the applicability of this sensor for pH measurements in the nuclei of human lung adenocarcinoma cells (pH = 7.4 ± 0.2) and immortalized embryonic kidney cells (pH = 7.0 ± 0.2). The sensor stained diffusely the nucleoplasm and piled up in interchromatin granules. These findings highlight the prospects of iMs in the studies of normal and pathological pH-dependent processes in the nucleus, including the formation of biomolecular condensates.

The existence of the nucleus distinguishes prokaryotes and eukaryotes. Apart from containing most of the genetic material, the nucleus possesses several nuclear bodies composed of protein and RNA molecules. The nucleus is separated from the cytoplasm by a double membrane, regulating the trafficking of molecules in- and outwards. Here, we investigate the composition and function of the different plant nuclear bodies and molecular clues involved in nuclear trafficking. The behavior of the nucleolus, Cajal bodies, dicing bodies, nuclear speckles, cyclophilin-containing bodies, photobodies and DNA damage foci is analyzed in response to different abiotic stresses. Furthermore, we research the literature to collect the different protein localization signals that rule nucleocytoplasmic trafficking. These signals include the different types of nuclear localization signals (NLSs) for nuclear import, and the nuclear export signals (NESs) for nuclear export. In contrast to these unidirectional-movement signals, the existence of nucleocytoplasmic shuttling signals (NSSs) allows bidirectional movement through the nuclear envelope. Likewise, nucleolar signals are also described, which mainly include the nucleolar localization signals (NoLSs) controlling nucleolar import. In contrast, few examples of nucleolar export signals, called nucleoplasmic localization signals (NpLSs) or nucleolar export signals (NoESs), have been reported. The existence of consensus sequences for these localization signals led to the generation of prediction tools, allowing the detection of these signals from an amino acid sequence. Additionally, the effect of high temperatures as well as different post-translational modifications in nuclear and nucleolar import and export is discussed.

Lamina-associated polypeptide 1 (LAP1) is a nuclear envelope (NE) protein whose function remains poorly characterized. In a recent LAP1 protein interactome study, a putative regulatory role in the DNA damage response (DDR) has emerged and telomeric repeat-binding factor 2 (TRF2), a protein intimately associated with this signaling pathway, was among the list of LAP1 interactors. To gain insights into LAP1\'s physiological properties, the interaction with TRF2 in human cells exposed to DNA-damaging agents was investigated. The direct LAP1:TRF2 binding was validated in vitro by blot overlay and in vivo by co-immunoprecipitation after hydrogen peroxide and bleomycin treatments. The regulation of this protein interaction by LAP1 phosphorylation was demonstrated by co-immunoprecipitation and mass spectrometry following okadaic acid exposure. The involvement of LAP1 and TRF2 in the DDR was confirmed by their increased nuclear protein levels after bleomycin treatment, evaluated by immunoblotting, as well as by their co-localization with DDR factors at the NE and within the nucleoplasm, assessed by immunocytochemistry. Effectively, we showed that the LAP1:TRF2 complex is established during a cellular response against DNA damage. This work proposes a novel functional role for LAP1 in the DDR, revealing a potential biological mechanism that may be disrupted in LAP1-associated pathologies.

Liquid-liquid phase separation (LLPS) has been recognized as one of the key cellular organizing principles and was shown to be responsible for formation of membrane-less organelles such as nucleoli. Although nucleoli were found to behave like liquid droplets, many ramifications of LLPS including nucleolar dynamics and interactions with the surrounding liquid remain to be revealed. Here, we study the motion of human nucleoli in vivo, while monitoring the shape of the nucleolus-nucleoplasm interface. We reveal two types of nucleolar pair dynamics: an unexpected correlated motion prior to coalescence and an independent motion otherwise. This surprising kinetics leads to a nucleolar volume distribution, [Formula: see text], unaccounted for by any current theory. Moreover, we find that nucleolus-nucleoplasm interface is maintained by ATP-dependent processes and susceptible to changes in chromatin transcription and packing. Our results extend and enrich the LLPS framework by showing the impact of the surrounding nucleoplasm on nucleoli in living cells.

Parvoviruses are an important platform for gene and cancer therapy. Their cell entry and the following steps, including nuclear import, are inefficient, limiting their use in therapeutic applications. Two models exist on parvoviral nuclear entry: the classical import of the viral capsid using nuclear transport receptors of the importin (karyopherin) family or the direct attachment of the capsid to the nuclear pore complex leading to the local disintegration of the nuclear envelope. Here, by laser scanning confocal microscopy and in situ proximity ligation analyses combined with coimmunoprecipitation, we show that infection requires importin β-mediated access to the nuclear pore complex and nucleoporin 153-mediated interactions on the nuclear side. The importin β-capsid interaction continued within the nucleoplasm, which suggests a mixed model of nuclear entry in which the classical nuclear import across the nuclear pore complex is accompanied by transient ruptures of the nuclear envelope, also allowing the passive entry of importin β-capsid complexes into the nucleus.IMPORTANCE Parvoviruses are small DNA viruses that deliver their DNA into the postmitotic nuclei, which is an important step for parvoviral gene and cancer therapies. Limitations in virus-receptor interactions or endocytic entry do not fully explain the low transduction/infection efficiency, indicating a bottleneck after virus entry into the cytoplasm. We thus investigated the transfer of parvovirus capsids from the cytoplasm to the nucleus, showing that the nuclear import of the parvovirus capsid follows a unique strategy, which differs from classical nuclear import and those of other viruses.

Proper partitioning of homologous chromosomes during meiosis relies on the coordinated execution of multiple interconnected events: Homologs must locate, recognize, and align with their correct pairing partners. Further, homolog pairing must be coupled to assembly of the synaptonemal complex (SC), a meiosis-specific tripartite structure that maintains stable associations between the axes of aligned homologs and regulates formation of crossovers between their DNA molecules to create linkages that enable their segregation. Here, we identify HAL-3 (Homolog Alignment 3) as an important player in coordinating these key events during Caenorhabditis elegans meiosis. HAL-3, and the previously identified HAL-2, are interacting and interdependent components of a protein complex that localizes to the nucleoplasm of germ cells. hal-3 (or hal-2) mutants exhibit multiple meiotic prophase defects including failure to establish homolog pairing, inappropriate loading of SC subunits onto unpaired chromosome axes, and premature loss of synapsis checkpoint protein PCH-2. Further, loss of hal function results in misregulation of the subcellular localization and activity of Polo-like kinases (PLK-1 and PLK-2), which dynamically localize to different defined subnuclear sites during wild-type prophase progression to regulate distinct cellular events. Moreover, loss of PLK-2 activity partially restores tripartite SC structure in a hal mutant background, suggesting that the defect in pairwise SC assembly in hal mutants reflects inappropriate PLK activity. Together, our data support a model in which the nucleoplasmic HAL-2/HAL-3 protein complex constrains both localization and activity of meiotic Polo-like kinases, thereby preventing premature interaction with stage-inappropriate targets.

The intercellular communication mediated by extracellular vesicles (EVs) has gained international interest during the last decade. Interfering with the mechanisms regulating this cellular process might find application particularly in oncology where cancer cell-derived EVs play a role in tumour microenvironment transformation. Although several mechanisms were ascribed to explain the internalization of EVs, little is our knowledge about the fate of their cargos, which are crucial to mediate their function. We recently demonstrated a new intracellular pathway in which a fraction of endocytosed EV-associated proteins is transported into the nucleoplasm of the host cell via a subpopulation of late endosomes penetrating into the nucleoplasmic reticulum. Silencing tetraspanin CD9 both in EVs and recipient cells strongly decreased the endocytosis of EVs and abolished the nuclear transfer of their cargos. Here, we investigated whether monovalent Fab fragments derived from 5H9 anti-CD9 monoclonal antibody (referred hereafter as CD9 Fab) interfered with these cellular processes. To monitor the intracellular transport of proteins, we used fluorescent EVs containing CD9-green fluorescent protein fusion protein and various melanoma cell lines and bone marrow-derived mesenchymal stromal cells as recipient cells. Interestingly, CD9 Fab considerably reduced EV uptake and the nuclear transfer of their proteins in all examined cells. In contrast, the divalent CD9 antibody stimulated both events. By impeding intercellular communication in the tumour microenvironment, CD9 Fab-mediated inhibition of EV uptake, combined with direct targeting of cancerous cells could lead to the development of novel anti-melanoma therapeutic strategies.

The 3D spatiotemporal organization of the human genome inside the cell nucleus remains a major open question in cellular biology. In the time between two cell divisions, chromatin-the functional form of DNA in cells-fills the nucleus in its uncondensed polymeric form. Recent in vivo imaging experiments reveal that the chromatin moves coherently, having displacements with long-ranged correlations on the scale of micrometers and lasting for seconds. To elucidate the mechanism(s) behind these motions, we develop a coarse-grained active polymer model where chromatin is represented as a confined flexible chain acted upon by molecular motors that drive fluid flows by exerting dipolar forces on the system. Numerical simulations of this model account for steric and hydrodynamic interactions as well as internal chain mechanics. These demonstrate that coherent motions emerge in systems involving extensile dipoles and are accompanied by large-scale chain reconfigurations and nematic ordering. Comparisons with experiments show good qualitative agreement and support the hypothesis that self-organizing long-ranged hydrodynamic couplings between chromatin-associated active motor proteins are responsible for the observed coherent dynamics.