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Human papillomaviruses (HPVs) utilize an atypical mode of nuclear import during cell entry. Residing in the Golgi apparatus until mitosis onset, a subviral complex composed of the minor capsid protein L2 and viral DNA (L2/vDNA) is imported into the nucleus after nuclear envelope breakdown by associating with mitotic chromatin. In this complex, L2 plays a crucial role in the interactions with cellular factors that enable delivery and ultimately tethering of the viral genome to mitotic chromatin. To date, the cellular proteins facilitating these steps remain unknown. Here, we addressed which cellular proteins may be required for this process. Using label-free mass spectrometry, biochemical assays, microscopy, and functional virological assays, we discovered that L2 engages a hitherto unknown protein complex of Ran-binding protein 10 (RanBP10), karyopherin alpha2 (KPNA2), and dynein light chain DYNLT3 to facilitate transport towards mitotic chromatin. Thus, our study not only identifies novel cellular interactors and mechanism that facilitate a poorly understood step in HPV entry, but also a novel cellular transport complex.

Integrin-mediated cell adhesion and mechanotransduction are considered key innovations in animal evolution. Here, we show that these processes represent a specialization of an evolutionarily conserved force coupling mechanism that originated in unicellular organisms and is mediated by the actin-binding protein talin. In contrast to heterodimeric integrin receptors, talin is widely distributed in unicellular organisms, including amoebae. By comparing the molecular mechanics of talin-A from amoeboid cells with that of mammalian talin-1, we uncover a conserved role for talin in transmitting pN-scale forces, even in unicellular organisms lacking canonical integrin receptors but expressing the functional homologue SibA. Our data indicate that the critical evolutionary steps towards integrin-mediated cell adhesion in metazoan organisms were the specialization of talin as an adaptor protein allowing the activation of integrin receptors, the regulation of biochemical signaling by paxillin, FAK and YAP, and the control of cell adhesion turnover by KANK recruitment. Collectively, these experiments suggest a central but thus far underappreciated role for talin in the evolution of eukaryotic cell-substrate adhesion and force transmission.

Amyotrophic lateral sclerosis (ALS) is a lethal disease characterized by motor neuron degeneration and associated with aggregation of nuclear RNA-binding proteins (RBPs), including FUS. How FUS aggregation and neurodegeneration are prevented in healthy motor neurons remain critically unanswered questions. Here, we use a combination of ALS patient autopsy tissue and induced pluripotent stem cell-derived neurons to study the effects of FUS mutations on RBP homeostasis. We show that FUS’ tendency to aggregate is normally buffered by interacting RBPs, but this buffering is lost when FUS mislocalizes to the cytoplasm due to ALS mutations. The presence of aggregation-prone FUS in the cytoplasm causes imbalances in RBP homeostasis that exacerbate neurodegeneration. However, enhancing autophagy using small molecules reduces cytoplasmic FUS, restores RBP homeostasis and rescues motor function in vivo. We conclude that disruption of RBP homeostasis plays a critical role in FUS-ALS and can be treated by stimulating autophagy.

During influenza A virus (IAV) infections, viral proteins are targeted by cellular E3 ligases for modification with ubiquitin. Here, we decipher and functionally explore the ubiquitination landscape of the IAV polymerase proteins during infection of human alveolar epithelial cells by applying mass spectrometry analysis of immuno-purified K-ε-GG (di-glycyl)-remnant-bearing peptides. We have identified 59 modified lysines across the three subunits, PB2, PB1 and PA of the viral polymerase of which 17 distinctively affect mRNA transcription, vRNA replication and the generation of recombinant viruses via non-proteolytic mechanisms. Moreover, further functional and in silico analysis indicate that ubiquitination at K578 in the PB1 thumb domain is mechanistically linked to dynamic structural transitions of the viral polymerase that are required for vRNA replication. Mutations K578A and K578R differentially affect the generation of recombinant viruses by impeding cRNA and vRNA synthesis, NP binding as well as polymerase dimerization. Collectively, our results demonstrate that the ubiquitin-mediated charge neutralization at PB1-K578 disrupts the interaction to an unstructured loop in the PB2 N-terminus that is required to coordinate polymerase dimerization and facilitate vRNA replication. This provides evidence that IAV exploits the cellular ubiquitin system to modulate the activity of the viral polymerase for viral replication. Influenza A virus replication relies on host cell-derived ubiquitination of the viral polymerase. Here, Günl et al. show that site-specific ubiquitination of PB1-K578 is acquired during infection and facilitates spatiotemporal control of polymerase dimerization and NP binding.

Multiple sclerosis (MS) is the most frequent demyelinating disease in young adults and despite significant advances in immunotherapy, disease progression still cannot be prevented. Promotion of remyelination, an endogenous repair mechanism resulting in the formation of new myelin sheaths around demyelinated axons, represents a promising new treatment approach. However, remyelination frequently fails in MS lesions, which can in part be attributed to impaired differentiation of oligodendroglial progenitor cells into mature, myelinating oligodendrocytes. The reasons for impaired oligodendroglial differentiation and defective remyelination in MS are currently unknown. To determine whether intrinsic oligodendroglial factors contribute to impaired remyelination in relapsing–remitting MS (RRMS), we compared induced pluripotent stem cell-derived oligodendrocytes (hiOL) from RRMS patients and controls, among them two monozygous twin pairs discordant for MS. We found that hiOL from RRMS patients and controls were virtually indistinguishable with respect to remyelination-associated functions and proteomic composition. However, while analyzing the effect of extrinsic factors we discovered that supernatants of activated peripheral blood mononuclear cells (PBMCs) significantly inhibit oligodendroglial differentiation. In particular, we identified CD4 + T cells as mediators of impaired oligodendroglial differentiation; at least partly due to interferon-gamma secretion. Additionally, we observed that blocked oligodendroglial differentiation induced by PBMC supernatants could not be restored by application of oligodendroglial differentiation promoting drugs, whereas treatment of PBMCs with the immunomodulatory drug teriflunomide prior to supernatant collection partly rescued oligodendroglial differentiation. In summary, these data indicate that the oligodendroglial differentiation block is not due to intrinsic oligodendroglial factors but rather caused by the inflammatory environment in RRMS lesions which underlines the need for drug screening approaches taking the inflammatory environment into account. Combined, these findings may contribute to the development of new remyelination promoting strategies.

Activities as diverse as migration, proliferation and patterning occur simultaneously and in a coordinated fashion during tissue morphogenesis. In the growing vasculature, the formation of motile, invasive and filopodia-carrying endothelial sprouts is balanced with the stabilization of blood-transporting vessels. Here, we show that sprouting endothelial cells in the retina have high rates of VEGF uptake, VEGF receptor endocytosis and turnover. These internalization processes are opposed by atypical protein kinase C activity in more stable and mature vessels. aPKC phosphorylates Dab2, a clathrin-associated sorting protein that, together with the transmembrane protein ephrin-B2 and the cell polarity regulator PAR-3, enables VEGF receptor endocytosis and downstream signal transduction. Accordingly, VEGF receptor internalization and the angiogenic growth of vascular beds are defective in loss-of-function mice lacking key components of this regulatory pathway. Our work uncovers how vessel growth is dynamically controlled by local VEGF receptor endocytosis and the activity of cell polarity proteins. The sprouting activity of filopodia emerging from endothelial sprouting cells needs to be compensated for in mature stable vessels. Adams and colleagues find that sprouting cells in mouse retinal vasculature show high VEGF uptake and VEGF receptor turnover, both essential for sprouting. These are inhibited by an aPKC-mediated decrease in VEGF receptor endocytosis in mature vessels, through a mechanism implicating clathrin-associated proteins, the transmembrane protein ephrin-B2 and the polarity factor PAR-3.

The current trends in the research and development of self-driving technology aim for Level 3+ autonomy, where the vehicle controls both lateral and longitudinal motions of the dynamic driving task, while the driver is permitted to divert their attention, as long as they are able to react properly to a handover request initiated by the vehicle. At this level of autonomy, situation awareness of the human driver has become one of the most important metrics of safety. This paper presents the results of a user study to evaluate handover performance at Level 3 autonomy. The study investigates whether the level of situation awareness during critical handover situations has a direct impact on task performance, with higher situation awareness expected to lead to better outcomes during emergency interventions. The study is performed in a simulated environment, using the CARLA driving simulator and the master console of the da Vinci Surgical System. The test subjects were asked to answer the questions of a questionnaire during the experiment; the answers for those questions and the measured control signals were analyzed to gain further knowledge on the safety of the handover process.

Paludiculture, the productive use of wet or rewetted peatlands, offers an option for continued land use by farmers after rewetting formerly drained peatlands, while reducing the greenhouse gas emissions from peat soils. Biodiversity conservation may benefit, but research on how biodiversity responds to paludiculture is scarce. We conducted a multi-taxon study investigating vegetation, breeding bird and arthropod diversity at six rewetted fen sites dominated by Carex or Typha species. Sites were either unharvested, low- or high-intensity managed, and were located in Mecklenburg-Vorpommern in northeastern Germany. Biodiversity was estimated across the range of Hill numbers using the iNEXT package, and species were checked for Red List status. Here we show that paludiculture sites can provide biodiversity value even while not reflecting historic fen conditions; managed sites had high plant diversity, as well as Red Listed arthropods and breeding birds. Our study demonstrates that paludiculture has the potential to provide valuable habitat for species even while productive management of the land continues.

Mitosis induces cellular rearrangements like spindle formation, Golgi fragmentation, and nuclear envelope breakdown. Similar to certain retroviruses, nuclear delivery during entry of human papillomavirus (HPV) genomes is facilitated by mitosis, during which minor capsid protein L2 tethers viral DNA to mitotic chromosomes. However, the mechanism of viral genome delivery and tethering to condensed chromosomes is barely understood. It is unclear, which cellular proteins facilitate this process or how this process is regulated. This work identifies crucial phosphorylations on HPV minor capsid protein L2 occurring at mitosis onset. L2’s chromosome binding region (CBR) is sequentially phosphorylated by the master mitotic kinases CDK1 and PLK1. L2 phosphorylation, thus, regulates timely delivery of HPV vDNA to mitotic chromatin during mitosis. In summary, our work demonstrates a crucial role of mitotic kinases for nuclear delivery of viral DNA and provides important insights into the molecular mechanism of pathogen import into the nucleus during mitosis. Human papillomavirus (HPV) coopts mitosis for nuclear entry by tethering the viral DNA to mitotic chromosomes, a process facilitated by the viral minor capsid protein L2. Here, Rizzato et al. show that L2 contains conserved phosphorylation motifs within the chromosome-binding region and provide evidence that host master mitotic kinases CDK1 and PLK1 sequentially mediate phosphorylation of L2 at mitosis onset to allow timely tethering of viral DNA to mitotic chromosomes.

The DND microRNA-mediated repression inhibitor 1 ( DND1 ) is a conserved RNA binding protein (RBP) that plays important roles in survival and fate maintenance of primordial germ cells (PGCs) and in the development of the male germline in zebrafish and mice. Dead end was shown to be expressed in human pluripotent stem cells (PSCs), PGCs and spermatogonia, but little is known about its specific role concerning pluripotency and human germline development. Here we use CRISPR/Cas mediated knockout and PGC-like cell (PGCLC) differentiation in human iPSCs to determine if DND1 (1) plays a role in maintaining pluripotency and (2) in specification of PGCLCs. We generated several clonal lines carrying biallelic loss of function mutations and analysed their differentiation potential towards PGCLCs and their gene expression on RNA and protein levels via RNA sequencing and mass spectrometry. The generated knockout iPSCs showed no differences in pluripotency gene expression, proliferation, or trilineage differentiation potential, but yielded reduced numbers of PGCLCs as compared with their parental iPSCs. RNAseq analysis of mutated PGCLCs revealed that the overall gene expression remains like non-mutated PGCLCs. However, reduced expression of genes associated with PGC differentiation and maintenance (e.g., NANOS3 , PRDM1 ) was observed. Together, we show that DND1 iPSCs maintain their pluripotency but exhibit a reduced differentiation to PGCLCs. This versatile model will allow further analysis of the specific mechanisms by which DND1 influences PGC differentiation and maintenance.