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Membrane contact sites (MCSs) link organelles to coordinate cellular functions across space and time. Although viruses remodel organelles for their replication cycles, MCSs remain largely unexplored during infections. Here, we design a targeted proteomics platform for measuring MCS proteins at all organelles simultaneously and define functional virus-driven MCS alterations by the ancient beta-herpesvirus human cytomegalovirus (HCMV). Integration with super-resolution microscopy and comparisons to herpes simplex virus (HSV-1), Influenza A, and beta-coronavirus HCoV-OC43 infections reveals time-sensitive contact regulation that allows switching anti- to pro-viral organelle functions. We uncover a stabilized mitochondria-ER encapsulation structure (MENC). As HCMV infection progresses, MENCs become the predominant mitochondria-ER contact phenotype and sequentially recruit the tethering partners VAP-B and PTPIP51, supporting virus production. However, premature ER-mitochondria tethering activates STING and interferon response, priming cells against infection. At peroxisomes, ACBD5-mediated ER contacts balance peroxisome proliferation versus membrane expansion, with ACBD5 impacting the titers of each virus tested. Membrane contact sites link organelles to coordinate cell functions. Comparing herpesvirus, influenza, and coronavirus infections, the authors define the virus-driven rewiring of organelle contact sites, uncovering ER-mitochondria encapsulation structures as well as a role for ER contacts in pro-viral peroxisome remodeling.

The dynamic regulation of mitochondria shape via fission and fusion is critical for cellular responses to stimuli. In homeostatic cells, two modes of mitochondrial fission, midzone and peripheral, provide a decision fork between either proliferation or clearance of mitochondria. However, the relationship between specific mitochondria shapes and functions remains unclear in many biological contexts. While commonly associated with decreased bioenergetics, fragmented mitochondria paradoxically exhibit elevated respiration in several disease states, including infection with the prevalent pathogen human cytomegalovirus (HCMV) and metastatic melanoma. Here, incorporating super-resolution microscopy with mass spectrometry and metabolic assays, we use HCMV infection to establish a molecular mechanism for maintaining respiration within a fragmented mitochondria population. We establish that HCMV induces fragmentation through peripheral mitochondrial fission coupled with suppression of mitochondria fusion. Unlike uninfected cells, the progeny of peripheral fission enter mitochondria-ER encapsulations (MENCs) where they are protected from degradation and bioenergetically stabilized during infection. MENCs also stabilize pro-viral inter-mitochondria contacts (IMCs), which electrochemically link mitochondria and promote respiration. Demonstrating a broader relevance, we show that the fragmented mitochondria within metastatic melanoma cells also form MENCs. Our findings establish a mechanism where mitochondria fragmentation can promote increased respiration, a feature relevant in the context of human diseases. During HCMV infection, mitochondrial progenies derived from peripheral fragmentation can be protected from mitophagy and result in elevated respiration through a mechanism leveraging ER-mitochondria and inter-mitochondria membrane contacts.

The complexity of intracellular signalling requires both a diversity of molecular players and the sequestration of activity to unique compartments within the cell. Recent findings on the role of liquid–liquid phase separation provide a distinct mechanism for the spatial segregation of proteins to regulate signalling pathway crosstalk. Here, we discover that DACT1 is induced by TGFβ and forms protein condensates in the cytoplasm to repress Wnt signalling. These condensates do not localize to any known organelles but, rather, exist as phase-separated proteinaceous cytoplasmic bodies. The deletion of intrinsically disordered domains within the DACT1 protein eliminates its ability to both form protein condensates and suppress Wnt signalling. Isolation and mass spectrometry analysis of these particles revealed a complex of protein machinery that sequesters casein kinase 2—a Wnt pathway activator. We further demonstrate that DACT1 condensates are maintained in vivo and that DACT1 is critical to breast and prostate cancer bone metastasis. Esposito et al. show that TGF-β-induced DACT1 forms biomolecular condensates that sequester CK2 to repress Wnt signalling and modulate bone metastasis in cancer.

The complexity of intracellular signalling requires both a diversity of molecular players and the sequestration of activity to unique compartments within the cell. Recent findings on the role of liquid-liquid phase separation provide a distinct mechanism for the spatial segregation of proteins to regulate signalling pathway crosstalk. Here, we discover that DACT1 is induced by TGF[beta] and forms protein condensates in the cytoplasm to repress Wnt signalling. These condensates do not localize to any known organelles but, rather, exist as phase-separated proteinaceous cytoplasmic bodies. The deletion of intrinsically disordered domains within the DACT1 protein eliminates its ability to both form protein condensates and suppress Wnt signalling. Isolation and mass spectrometry analysis of these particles revealed a complex of protein machinery that sequesters casein kinase 2–a Wnt pathway activator. We further demonstrate that DACT1 condensates are maintained in vivo and that DACT1 is critical to breast and prostate cancer bone metastasis.

Bifunctional diazirine lipids are valuable tools for mapping protein-lipid interactions and cellular localization by photocrosslinking. Yet, the crosslinking efficiency of these probes has not been systematically evaluated. Here, we use the lipid transfer protein STARD10, which binds phospholipids in a 1:1 stoichiometry within a hydrophobic pocket, to measure the upper limit of the photo-crosslinking efficiency of bifunctional lipid probes. We characterize reaction products using native and denaturing mass spectrometry. Our results show that approximately 5% of photoactivated lipids form covalent protein-lipid crosslinks, while the majority follow intramolecular reaction trajectories, resulting in the formation of products featuring alkene, ketone and hydroxyl moieties. These findings provide essential context for the use of bifunctional probes to uncover the cell biology of lipids and highlight the need for continuous improvement to experimental workflows.Competing Interest StatementThe authors have declared no competing interest.Funder Information DeclaredPaul G. Allen Family Foundation, https://ror.org/01degd278, Allen Distiguished Investigator AwardWellcome Trust, 221795/Z/20/ZGerman National Academy of Sciences Leopoldina, LPDS 2023-0

Biological membranes often feature an unequal and actively maintained concentration gradients of lipids between bilayer leaflets, termed lipid asymmetry.1–3 Lipid asymmetry has been linked to many cellular processes4,5, but the mechanisms that connect lipid trans-bilayer concentration gradients to cellular functions are poorly understood. Here we systematically map the cellular processes affected by dysregulation of lipid asymmetry by knocking out flippases, floppases, and scramblases in mammalian cells. We identify broad alterations to core metabolic pathways including nutrient uptake, neutral lipid turnover, pentose phosphate pathway and glycolysis. Lipidomics, respirometry, lipid and metabolic imaging, and live-cell calorimetry revealed elevated neutral lipid and ATP consumption rates which are coupled with increased heat loss per unit of biomass synthesized despite slower growth. This suggests that compensatory maintenance of lipid asymmetry strains the cellular energy budget, inducing a shift from a growth-promoting anabolic to a more energy-using catabolic state. Our data indicate that lipid asymmetry and its active maintenance is key for proper cell energetics, highlighting its putative role as a cellular store of potential energy akin to proton and ion transmembrane gradients.

Objective Neuromyelitis optica spectrum disorders (NMOSD) represent rare autoimmune diseases of the central nervous system largely targeting optic nerve(s) and spinal cord. The present analysis used real-world data to identify clinical and epidemiological correlates of treatment change in patients with NMOSD. Methods CIRCLES is a longitudinal, observational study of NMOSD conducted at 15 centers across North America. Patients with ≥ 60 days of follow-up and receiving on-study maintenance treatment were evaluated. The mean annual relapse rate (ARR) was estimated using negative binomial models; the likelihood of treatment change was estimated using Cox proportional hazards models. Relapses were included as time-varying covariates to estimate the relationship to treatment change. Results Of 542 patients included, 171 (31.5%) experienced ≥ 1 relapse on the study and 133 patients (24.5%) had ≥ 1 change in the treatment regimen. Two categories of variables significantly correlated with the likelihood of treatment change: (1) relapse: any on-study relapse (hazard ratio [HR] = 2.91; p  < 0.001), relapse phenotypes (HR range = 2.15–5.49; p  < 0.001), and pre-study ARR > 0.75 (HR 2.28; p  < 0.001); 2) disease phenotype: brain syndrome only vs transverse myelitis involvement at onset (HR 2.44; p  = 0.008), disease duration < 1 vs > 5 years (HR 1.66; p  = 0.028), or autoimmune comorbidity (HR 1.55; p  = 0.015). A subset of these factors significantly correlated with shorter time to first rituximab discontinuation. Conclusions In CIRCLES, relapse patterns and disease phenotype significantly correlated with changes in the maintenance treatment regimen. Such findings may facilitate the identification of patients with NMOSD who are likely to benefit from treatment change to reduce relapse risk or disease burden and enhance the quality of life.

B.1.351 is the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant most resistant to antibody neutralization. We demonstrate how the dose and number of immunizations influence protection. Nonhuman primates received two doses of 30 or 100 µg of Moderna’s mRNA-1273 vaccine, a single immunization of 30 µg, or no vaccine. Two doses of 100 µg of mRNA-1273 induced 50% inhibitory reciprocal serum dilution neutralizing antibody titers against live SARS-CoV-2 p.Asp614Gly and B.1.351 of 3,300 and 240, respectively. Higher neutralizing responses against B.1.617.2 were also observed after two doses compared to a single dose. After challenge with B.1.351, there was ~4- to 5-log 10 reduction of viral subgenomic RNA and low to undetectable replication in bronchoalveolar lavages in the two-dose vaccine groups, with a 1-log 10 reduction in nasal swabs in the 100-µg group. These data establish that a two-dose regimen of mRNA-1273 will be critical for providing upper and lower airway protection against major variants of concern. The beta variant (B.1.351) is to date the most resistant to neutralization of the SARS-CoV-2 variants. Using nonhuman primates, Seder and colleagues demonstrate that double vaccination with a high dose of the lipid nanoparticle vaccine mRNA-1273 protects against infection with the beta variant.

Racially and ethnically minoritized populations have been historically excluded and underrepresented in research. This paper will describe best practices in multicultural and multilingual awareness-raising strategies used by the Recruitment Innovation Center to increase minoritized enrollment into clinical trials. The Passive Immunity Trial for Our Nation will be used as a primary example to highlight real-world application of these methods to raise awareness, engage community partners, and recruit diverse study participants.

Immune correlates of protection can be used as surrogate endpoints for vaccine efficacy. The nonhuman primate (NHP) model of SARS-CoV-2 infection replicates key features of human infection and may be used to define immune correlates of protection following vaccination. Here, NHP received either no vaccine or doses ranging from 0.3 - 100 μg of mRNA-1273, a mRNA vaccine encoding the prefusion-stabilized SARS-CoV-2 spike (S-2P) protein encapsulated in a lipid nanoparticle. mRNA-1273 vaccination elicited robust circulating and mucosal antibody responses in a dose-dependent manner. Viral replication was significantly reduced in bronchoalveolar lavages and nasal swabs following SARS-CoV-2 challenge in vaccinated animals and was most strongly correlated with levels of anti-S antibody binding and neutralizing activity. Consistent with antibodies being a correlate of protection, passive transfer of vaccine-induced IgG to naïve hamsters was sufficient to mediate protection. Taken together, these data show that mRNA-1273 vaccine-induced humoral immune responses are a mechanistic correlate of protection against SARS-CoV-2 infection in NHP.Immune correlates of protection can be used as surrogate endpoints for vaccine efficacy. The nonhuman primate (NHP) model of SARS-CoV-2 infection replicates key features of human infection and may be used to define immune correlates of protection following vaccination. Here, NHP received either no vaccine or doses ranging from 0.3 - 100 μg of mRNA-1273, a mRNA vaccine encoding the prefusion-stabilized SARS-CoV-2 spike (S-2P) protein encapsulated in a lipid nanoparticle. mRNA-1273 vaccination elicited robust circulating and mucosal antibody responses in a dose-dependent manner. Viral replication was significantly reduced in bronchoalveolar lavages and nasal swabs following SARS-CoV-2 challenge in vaccinated animals and was most strongly correlated with levels of anti-S antibody binding and neutralizing activity. Consistent with antibodies being a correlate of protection, passive transfer of vaccine-induced IgG to naïve hamsters was sufficient to mediate protection. Taken together, these data show that mRNA-1273 vaccine-induced humoral immune responses are a mechanistic correlate of protection against SARS-CoV-2 infection in NHP.mRNA-1273 vaccine-induced antibody responses are a mechanistic correlate of protection against SARS-CoV-2 infection in NHP.ONE-SENTENCE SUMMARYmRNA-1273 vaccine-induced antibody responses are a mechanistic correlate of protection against SARS-CoV-2 infection in NHP.