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The focal adhesion protein Vinculin (VCL) is ascribed to various cytoplasmic functions; however, its nuclear role has so far been ambiguous. We observed that VCL localizes to the nuclei of mouse primary spermatocytes undergoing first meiotic division. Specifically, VCL localizes along the meiosis-specific structure synaptonemal complex (SC) during prophase I and the centromeric regions, where it remains until metaphase I. To study the role of VCL in meiotic division, we prepared a conditional knock-out mouse (VCLcKO). We found that the VCLcKO male mice were semi-fertile, with a decreased number of offspring compared to wild-type animals. This study of events in late prophase I indicated premature splitting of homologous chromosomes, accompanied by an untimely loss of SCP1. This caused erroneous kinetochore formation, followed by failure of the meiotic spindle assembly and metaphase I arrest. To assess the mechanism of VCL involvement in meiosis, we searched for its possible interacting partners. A mass spectrometry approach identified several putative interactors which belong to the ubiquitin–proteasome pathway (UPS). The depletion of VLC leads to the dysregulation of a key subunit of the proteasome complex in the meiotic nuclei and an altered nuclear SUMOylation level. Taken together, we show for the first time the presence of VCL in the nucleus of spermatocytes and its involvement in proper meiotic progress. It also suggests the direction for future studies regarding the role of VCL in spermatogenesis through regulation of UPS.

BACKGROUND: Nuclear myosin I (NM1) is a nuclear isoform of the well-known "cytoplasmic" Myosin 1c protein (Myo1c). Located on the 11(th) chromosome in mice, NM1 results from an alternative start of transcription of the Myo1c gene adding an extra 16 amino acids at the N-terminus. Previous studies revealed its roles in RNA Polymerase I and RNA Polymerase II transcription, chromatin remodeling, and chromosomal movements. Its nuclear localization signal is localized in the middle of the molecule and therefore directs both Myosin 1c isoforms to the nucleus. METHODOLOGY/PRINCIPAL FINDINGS: In order to trace specific functions of the NM1 isoform, we generated mice lacking the NM1 start codon without affecting the cytoplasmic Myo1c protein. Mutant mice were analyzed in a comprehensive phenotypic screen in cooperation with the German Mouse Clinic. Strikingly, no obvious phenotype related to previously described functions has been observed. However, we found minor changes in bone mineral density and the number and size of red blood cells in knock-out mice, which are most probably not related to previously described functions of NM1 in the nucleus. In Myo1c/NM1 depleted U2OS cells, the level of Pol I transcription was restored by overexpression of shRNA-resistant mouse Myo1c. Moreover, we found Myo1c interacting with Pol II. The ratio between Myo1c and NM1 proteins were similar in the nucleus and deletion of NM1 did not cause any compensatory overexpression of Myo1c protein. CONCLUSION/SIGNIFICANCE: We observed that Myo1c can replace NM1 in its nuclear functions. Amount of both proteins is nearly equal and NM1 knock-out does not cause any compensatory overexpression of Myo1c. We therefore suggest that both isoforms can substitute each other in nuclear processes.

BACKGROUND: Nuclear myosin I (NM1) was the first molecular motor identified in the cell nucleus. Together with nuclear actin, they participate in crucial nuclear events such as transcription, chromatin movements, and chromatin remodeling. NM1 is an isoform of myosin 1c (Myo1c) that was identified earlier and is known to act in the cytoplasm. NM1 differs from the "cytoplasmic" myosin 1c only by additional 16 amino acids at the N-terminus of the molecule. This amino acid stretch was therefore suggested to direct NM1 into the nucleus. METHODOLOGY/PRINCIPAL FINDINGS: We investigated the mechanism of nuclear import of NM1 in detail. Using over-expressed GFP chimeras encoding for truncated NM1 mutants, we identified a specific sequence that is necessary for its import to the nucleus. This novel nuclear localization sequence is placed within calmodulin-binding motif of NM1, thus it is present also in the Myo1c. We confirmed the presence of both isoforms in the nucleus by transfection of tagged NM1 and Myo1c constructs into cultured cells, and also by showing the presence of the endogenous Myo1c in purified nuclei of cells derived from knock-out mice lacking NM1. Using pull-down and co-immunoprecipitation assays we identified importin beta, importin 5 and importin 7 as nuclear transport receptors that bind NM1. Since the NLS sequence of NM1 lies within the region that also binds calmodulin we tested the influence of calmodulin on the localization of NM1. The presence of elevated levels of calmodulin interfered with nuclear localization of tagged NM1. CONCLUSIONS/SIGNIFICANCE: We have shown that the novel specific NLS brings to the cell nucleus not only the "nuclear" isoform of myosin I (NM1 protein) but also its "cytoplasmic" isoform (Myo1c protein). This opens a new field for exploring functions of this molecular motor in nuclear processes, and for exploring the signals between cytoplasm and the nucleus.

Abstract Metabolic reprogramming is one of the hallmarks of tumorigenesis. Here, we show that nuclear myosin 1 (NM1) serves as a key regulator of cellular metabolism. NM1 directly affects mitochondrial oxidative phosphorylation (OXPHOS) by regulating mitochondrial transcription factors TFAM and PGC1α, and its deletion leads to underdeveloped mitochondria inner cristae and mitochondrial redistribution within the cell. These changes are associated with reduced OXPHOS gene expression, decreased mitochondrial DNA copy number, and deregulated mitochondrial dynamics, which lead to metabolic reprogramming of NM1 KO cells from OXPHOS to aerobic glycolysis.This, in turn, is associated with a metabolomic profile typical for cancer cells, namely increased amino acid-, fatty acid-, and sugar metabolism, and increased glucose uptake, lactate production, and intracellular acidity. NM1 KO cells form solid tumors in a mouse model, suggesting that the metabolic switch towards aerobic glycolysis provides a sufficient carcinogenic signal. We suggest that NM1 plays a role as a tumor suppressor and that NM1 depletion may contribute to the Warburg effect at the onset of tumorigenesis.

The interaction between SARS-CoV-2 non-structural protein Nsp9 and the nanobody 2NSP90 was investigated by NMR spectroscopy using the paramagnetic perturbation methodology PENELOP (Paramagnetic Equilibrium vs Nonequilibrium magnetization Enhancement or LOss Perturbation). The Nsp9 monomer is an essential component of the replication and transcription complex (RTC) that reproduces the viral gRNA for subsequent propagation. Therefore preventing Nsp9 recruitment in RTC would represent an efficient antiviral strategy that could be applied to different coronaviruses, given the Nsp9 relative invariance. The NMR results were consistent with a previous characterization suggesting a 4:4 Nsp9-to-nanobody stoichiometry with the occurrence of two epitope pairs on each of the Nsp9 units that establish the inter-dimer contacts of Nsp9 tetramer. The oligomerization state of Nsp9 was also analyzed by molecular dynamics simulations and both dimers and tetramers resulted plausible. A different distribution of the mapped epitopes on the tetramer surface with respect to the former 4:4 complex could also be possible, as well as different stoichiometries of the Nsp9-nanobody assemblies such as the 2:2 stoichiometry suggested by the recent crystal structure of the Nsp9 complex with 2NSP23 (PDB ID: 8dqu), a nanobody exhibiting essentially the same affinity as 2NSP90. The experimental NMR evidence, however, ruled out the occurrence in liquid state of the relevant Nsp9 conformational change observed in the same crystal structure.

The actin-based molecular motors, myosins, have also been linked to transcription, but their precise role has remained elusive. Here the authors show RNA polymerase II (RNAPII) is lost from chromatin upon myosin perturbation and that myosin acts as a molecular anchor to maintain RNAPII spatial organisation.

β-actin loss can affect gene expression and heterochromatin organization. Here the authors conduct a comprehensive genomic analysis of β-actin knockout mouse embryonic fibroblasts (MEFs) to investigate the impact of changes in β-actin levels on 3d genome architecture and chromatin remodeling activities of BAF and polycomb proteins.

Actin cytoskeletal dynamics drive cellular shape changes, linking numerous cell functions to physiological and pathological cues. Mutations in actin regulators that are differentially expressed or enriched in immune cells cause severe human diseases known as primary immunodeficiencies underscoring the importance of efficienct actin remodeling in immune cell homeostasis. Here we discuss recent findings on how immune cells sense the mechanical properties of their environement. Moreover, while the organization and biochemical regulation of cytoplasmic actin have been extensively studied, nuclear actin reorganization is a rapidly emerging field that has only begun to be explored in immune cells. Based on the critical and multifaceted contributions of cytoplasmic actin in immune cell functionality, nuclear actin regulation is anticipated to have a large impact on our understanding of immune cell development and functionality.

Summary: In eukaryotic cells, actin regulates both cytoplasmic and nuclear functions. However, whether actin-based structures are present in the mitochondria and are involved in mitochondrial functions has not been investigated. Here, using wild-type β-actin +/+ and knockout (KO) β-actin −/− mouse embryonic fibroblasts we show evidence for the defect in maintaining mitochondrial membrane potential (MMP) in β-actin-null cells. MMP defects were associated with impaired mitochondrial DNA (mtDNA) transcription and nuclear oxidative phosphorylation (OXPHOS) gene expression. Using super-resolution microscopy we provided direct evidence on the presence of β-actin-containing structures inside mitochondria. Large aggregates of TFAM-stained nucleoids were observed in bulb-shaped mitochondria in KO cells, suggesting defects in mitochondrial nucleoid segregation without β-actin. The observation that mitochondria-targeted β-actin rescued mtDNA transcription and MMP suggests an indispensable functional role of a mitochondrial β-actin pool necessary for mitochondrial quality control. : Molecular Biology; Cell Biology; Functional Aspects of Cell Biology Subject Areas: Molecular Biology, Cell Biology, Functional Aspects of Cell Biology

Cell-penetrating peptides (CPPs) are short peptides that are able to efficiently penetrate cellular lipid bilayers. Although CPPs have been used as carriers in conjugation with certain cargos to target specific genes and pathways, how rationally designed CPPs per se affect global gene expression has not been investigated. Therefore, following time course treatments with 4 CPPs-penetratin, PepFect14, mtCPP1 and TP10, HeLa cells were transcriptionally profiled by RNA sequencing. Results from these analyses showed a time-dependent response to different CPPs, with specific sets of genes related to ribosome biogenesis, microtubule dynamics and long-noncoding RNAs being differentially expressed compared to untreated controls. By using an image-based high content phenotypic profiling platform we confirmed that differential gene expression in CPP-treated HeLa cells strongly correlates with changes in cellular phenotypes such as increased nucleolar size and dispersed microtubules, compatible with altered ribosome biogenesis and cell growth. Altogether these results suggest that cells respond to different cell penetrating peptides by alteration of specific sets of genes, which are possibly part of the common response to such stimulus.