The luminal AAA+ ATPase torsinA mediates distinct mechanisms of nuclear-cytoplasmic communication by adopting different functional assembly states.

Chemical and mechanical nuclear-cytoplasmic communication across the nuclear envelope (NE) is largely mediated by the nuclear pore complex (NPC) and the linker of nucleoskeleton and cytoskeleton (LINC) complex, respectively. While NPC and LINC complex assembly are functionally related, the mechanisms responsible for this relationship remain poorly understood. Here, we investigated how the luminal ATPases associated with various cellular activities (AAA+) protein torsinA promotes NPC and LINC complex assembly using fluorescence fluctuation spectroscopy (FFS), quantitative photobleaching analyses, and functional cellular assays. We report that torsinA controls LINC complex-dependent nuclear-cytoskeletal coupling as a soluble hexameric AAA+ protein and interphase NPC biogenesis as a membrane-associated helical polymer. These findings help resolve the conflicting models of torsinA function that were recently proposed based on in vitro structural studies. Our results will enable future studies of the role of defective nuclear-cytoplasmic communication in DYT1 dystonia and other diseases caused by mutations in torsinA.

Pre-meiotic pairing of homologous chromosomes during Drosophila male meiosis

In the early stages of meiosis, maternal and paternal chromosomes pair with their homologous partner and recombine to ensure exchange of genetic information and proper segregation. These events can vary drastically between species and between males and females of the same species. In Drosophila, in contrast to females, males do not form synaptonemal complexes (SCs), do not recombine and have no crossing-over; yet, males are able to segregate their chromosomes properly. Here, we investigated the early steps of homologues pairing in Drosophila males. We found that homologues are not paired in germline stem cells (GSCs) and become paired in the mitotic region before meiotic entry, similarly to females. Surprisingly, male germline cells express SC proteins, which localize to centromeres and promote pairing. We further found that the SUN/KASH (LINC) complex and microtubules are required for homologues pairing as in females. Chromosome movements are however much slower than in females and we demonstrate that this slow dynamic is compensated in males by having longer cell cycles. In agreement, slowing down cell cycles was sufficient to rescue pairing-defective mutants in female meiosis. Our results demonstrate that although meiosis differs significantly between males and females, sex-specific cell cycle kinetics are integrated with similar molecular mechanisms to achieve proper homologues pairing.

Nuclear pore protein NUP210 depletion suppresses metastasis through heterochromatin-mediated disruption of tumor cell mechanical response

Mechanical signals from the extracellular microenvironment have been implicated in tumor and metastatic progression. Here, we identify nucleoporin NUP210 as a metastasis susceptibility gene for human estrogen receptor positive (ER+) breast cancer and a cellular mechanosensor. Nup210 depletion suppresses lung metastasis in mouse models of breast cancer. Mechanistically, NUP210 interacts with LINC complex protein SUN2 which connects the nucleus to the cytoskeleton. In addition, the NUP210/SUN2 complex interacts with chromatin via the short isoform of BRD4 and histone H3.1/H3.2 at the nuclear periphery. In Nup210 knockout cells, mechanosensitive genes accumulate H3K27me3 heterochromatin modification, mediated by the polycomb repressive complex 2 and differentially reposition within the nucleus. Transcriptional repression in Nup210 knockout cells results in defective mechanotransduction and focal adhesion necessary for their metastatic capacity. Our study provides an important role of nuclear pore protein in cellular mechanosensation and metastasis.

Stage-resolved Hi-C analyses reveal meiotic chromosome organizational features influencing homolog alignment

During meiosis, chromosomes exhibit dramatic changes in morphology and intranuclear positioning. How these changes influence homolog pairing, alignment, and recombination remain elusive. Using Hi-C, we systematically mapped 3D genome architecture throughout all meiotic prophase substages during mouse spermatogenesis. Our data uncover two major chromosome organizational features varying along the chromosome axis during early meiotic prophase, when homolog alignment occurs. First, transcriptionally active and inactive genomic regions form alternating domains consisting of shorter and longer chromatin loops, respectively. Second, the force-transmitting LINC complex promotes the alignment of ends of different chromosomes over a range of up to 20% of chromosome length. Both features correlate with the pattern of homolog interactions and the distribution of recombination events. Collectively, our data reveal the influences of transcription and force on meiotic chromosome structure and suggest chromosome organization may provide an infrastructure for the modulation of meiotic recombination in higher eukaryotes.

Two-step regulation of centromere distribution by condensin II and the nuclear envelope proteins

The arrangement of centromeres within the nucleus differs among species and cell types. However, neither the mechanisms determining centromere distribution nor its biological significance are currently well understood. In this study, we demonstrate the importance of centromere distribution for the maintenance of genome integrity through the cytogenic and molecular analysis of mutants defective in centromere distribution. We propose a two-step regulatory mechanism that shapes the non-Rabl-like centromere distribution in Arabidopsis thaliana through condensin II and the linker of the nucleoskeleton and cytoskeleton (LINC) complex. Condensin II is enriched at centromeres and, in cooperation with the LINC complex, induces the scattering of centromeres around the nuclear periphery during late anaphase/telophase. Then, after entering interphase, the positions of the scattered centromeres are stabilized by nuclear lamina proteins of the CROWDED NUCLEI (CRWN) family. We also found that, despite their strong impact on centromere distribution, condensin II and CRWNs have little effect on chromatin organization involved in the control of gene expression, indicating the robustness of chromatin organization regardless of the type of centromere distribution.

Phosphorylation of luminal region of the SUN-domain protein Mps3 promotes nuclear envelope localization during meiosis

During meiosis, protein ensembles in the nuclear envelope (NE) containing SUN- and KASH-domain proteins, called linker nucleocytoskeleton and cytoskeleton (LINC) complex, promote the chromosome motion. Yeast SUN-domain protein, Mps3, forms multiple meiosis-specific ensembles on NE, which show dynamic localisation for chromosome motion; however, the mechanism by which these Mps3 ensembles are formed during meiosis remains largely unknown. Here, we showed that the cyclin-dependent protein kinase (CDK) and Dbf4-dependent Cdc7 protein kinase (DDK) regulate meiosis-specific dynamics of Mps3 on NE, particularly by mediating the resolution of Mps3 clusters and telomere clustering. We also found that the luminal region of Mps3 juxtaposed to the inner nuclear membrane is required for meiosis-specific localisation of Mps3 on NE. Negative charges introduced by meiosis-specific phosphorylation in the luminal region of Mps3 alter its interaction with negatively charged lipids by electric repulsion in reconstituted liposomes. Phospho-mimetic substitution in the luminal region suppresses the localisation of Mps3 via the inactivation of CDK or DDK. Our study revealed multi-layered phosphorylation-dependent regulation of the localisation of Mps3 on NE for meiotic chromosome motion and NE remodelling.

Ensconsin-dependent changes in microtubule organization and LINC complex-dependent changes in nucleus-nucleus interactions result in quantitatively distinct myonuclear positioning defects

Nuclear movement is a fundamental process of eukaryotic cell biology. Skeletal muscle presents an intriguing model to study nuclear movement because its development requires the precise positioning of multiple nuclei within a single cytoplasm. Furthermore, there is a high correlation between aberrant nuclear positioning and poor muscle function. Although many genes that regulate nuclear movement have been identified, the mechanisms by which these genes act is not known. Using Drosophila melanogaster muscle development as a model system, and a combination of live-embryo microscopy and laser ablation of nuclei, we have found that clustered nuclei encompass at least two phenotypes that are caused by distinct mechanisms. Specifically, Ensconsin is necessary for productive force production to drive any movement of nuclei whereas Bocksbeutel and Klarsicht are necessary to form distinct populations of nuclei that move to different cellular locations. Mechanisitcally, Ensconsin regulates the number of growing microtubules that are used to move nuclei whereas Bocksbeutel and Klarsicht regulate interactions between nuclei. [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text]

Mena regulates the LINC complex to control actin–nuclear lamina associations, trans-nuclear membrane signalling and cancer gene expression

Interactions between cells and the extracellular matrix, mediated by integrin adhesion complexes (IACs), play key roles in cancer progression and metastasis. We investigated systems-level changes in the integrin adhesome during metastatic progression of a patient-derived cutaneous squamous cell carcinoma (cSCC), and found that the actin regulatory protein Mena is enriched in IACs in metastatic cSCC cells. Mena is connected within a subnetwork of actin-binding proteins to the LINC complex component nesprin-2, with which it interacts and co-localises at the nuclear envelope of metastatic cells. Moreover, Mena potentiates the interactions of nesprin-2 with the actin cytoskeleton and the nuclear lamina. CRISPR-mediated Mena depletion causes altered nuclear morphology, reduces tyrosine phosphorylation of the nuclear membrane protein emerin and downregulates expression of the immunomodulatory gene PTX3 via the recruitment of its enhancer to the nuclear periphery. We have uncovered an unexpected novel role for Mena at the nuclear membrane, where it controls the LINC complex, nuclear architecture, chromatin repositioning and cancer gene expression. This is the first description of an adhesion protein regulating gene transcription via direct signalling across the nuclear envelope.