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SignificanceOur data challenge the generally accepted view that Kv2.1 proteins regulate arterial smooth muscle function by regulating their membrane potential. Rather, we discovered that Kv2.1 plays both conductive and structural roles with opposing functional consequences on arterial myocytes, with the former predominating in males, the latter in females. Opening of Kv2.1 channels opposes vasoconstriction by inducing membrane hyperpolarization. In addition to this conductive function, Kv2.1 promotes the structural clustering of CaV1.2 channels, thereby enhancing Ca2+ influx and inducing vasoconstriction. These two functions are highlighted by differences in the regulation of membrane potential, intracellular Ca2+, and myogenic tone between males and females. Our data suggest that these disparities derive from sex-specific variations in Kv2.1 expression levels in male versus female myocytes. The accepted role of the protein Kv2.1 in arterial smooth muscle cells is to form K+ channels in the sarcolemma. Opening of Kv2.1 channels causes membrane hyperpolarization, which decreases the activity of L-type CaV1.2 channels, lowering intracellular Ca2+ ([Ca2+]i) and causing smooth muscle relaxation. A limitation of this model is that it is based exclusively on data from male arterial myocytes. Here, we used a combination of electrophysiology as well as imaging approaches to investigate the role of Kv2.1 channels in male and female arterial myocytes. We confirmed that Kv2.1 plays a canonical conductive role but found it also has a structural role in arterial myocytes to enhance clustering of CaV1.2 channels. Less than 1% of Kv2.1 channels are conductive and induce membrane hyperpolarization. Paradoxically, by enhancing the structural clustering and probability of CaV1.2–CaV1.2 interactions within these clusters, Kv2.1 increases Ca2+ influx. These functional impacts of Kv2.1 depend on its level of expression, which varies with sex. In female myocytes, where expression of Kv2.1 protein is higher than in male myocytes, Kv2.1 has conductive and structural roles. Female myocytes have larger CaV1.2 clusters, larger [Ca2+]i, and larger myogenic tone than male myocytes. In contrast, in male myocytes, Kv2.1 channels regulate membrane potential but not CaV1.2 channel clustering. We propose a model in which Kv2.1 function varies with sex: in males, Kv2.1 channels control membrane potential but, in female myocytes, Kv2.1 plays dual electrical and CaV1.2 clustering roles. This contributes to sex-specific regulation of excitability, [Ca2+]i, and myogenic tone in arterial myocytes.

Nanobodies (nAbs) are small, minimal antibodies that have distinct attributes that make them uniquely suited for certain biomedical research, diagnostic and therapeutic applications. Prominent uses include as intracellular antibodies or intrabodies to bind and deliver cargo to specific proteins and/or subcellular sites within cells, and as nanoscale immunolabels for enhanced tissue penetration and improved spatial imaging resolution. Here, we report the generation and validation of nAbs against a set of proteins prominently expressed at specific subcellular sites in mammalian brain neurons. We describe a novel hierarchical validation pipeline to systematically evaluate nAbs isolated by phage display for effective and specific use as intrabodies and immunolabels in mammalian cells including brain neurons. These nAbs form part of a robust toolbox for targeting proteins with distinct and highly spatially-restricted subcellular localization in mammalian brain neurons, allowing for visualization and/or modulation of structure and function at those sites.

The accepted role of the protein Kv2.1 in arterial smooth muscle cells is to form K⁺ channels in the sarcolemma. Opening of Kv2.1 channels causes membrane hyperpolarization, which decreases the activity of L-type CaV1.2 channels, lowering intracellular Ca2+ ([Ca2+]i) and causing smooth muscle relaxation. A limitation of this model is that it is based exclusively on data from male arterial myocytes. Here, we used a combination of electrophysiology as well as imaging approaches to investigate the role of Kv2.1 channels in male and female arterial myocytes. We confirmed that Kv2.1 plays a canonical conductive role but found it also has a structural role in arterial myocytes to enhance clustering of CaV1.2 channels. Less than 1% of Kv2.1 channels are conductive and induce membrane hyperpolarization. Paradoxically, by enhancing the structural clustering and probability of CaV1.2–CaV1.2 interactionswithin these clusters, Kv2.1 increases Ca2+ influx. These functional impacts of Kv2.1 depend on its level of expression, which varies with sex. In female myocytes, where expression of Kv2.1 protein is higher than in male myocytes, Kv2.1 has conductive and structural roles. Female myocytes have larger CaV1.2 clusters, larger [Ca2+]i, and larger myogenic tone than male myocytes. In contrast, in male myocytes, Kv2.1 channels regulate membrane potential but not CaV1.2 channel clustering. We propose a model in which Kv2.1 function varies with sex: in males, Kv2.1 channels control membrane potential but, in female myocytes, Kv2.1 plays dual electrical and CaV1.2 clustering roles. This contributes to sex-specific regulation of excitability, [Ca2+]i, and myogenic tone in arterial myocytes.

The accepted role of the protein Kv2.1 in arterial smooth muscle cells is to form K+ channels in the sarcolemma. Opening of Kv2.1 channels causes membrane hyperpolarization, which decreases the activity of L-type CaV1.2 channels, lowering intracellular Ca2+ ([Ca2+]i) and causing smooth muscle relaxation. A limitation of this model is that it is based exclusively on data from male arterial myocytes. Here, we used a combination of electrophysiology as well as imaging approaches to investigate the role of Kv2.1 channels in male and female arterial myocytes. We confirmed that Kv2.1 plays a canonical conductive role but found it also has a structural role in arterial myocytes to enhance clustering of CaV1.2 channels. Less than 1% of Kv2.1 channels are conductive and induce membrane hyperpolarization. Paradoxically, by enhancing the structural clustering and probability of CaV1.2-CaV1.2 interactions within these clusters, Kv2.1 increases Ca2+ influx. These functional impacts of Kv2.1 depend on its level of expression, which varies with sex. In female myocytes, where expression of Kv2.1 protein is higher than in male myocytes, Kv2.1 has conductive and structural roles. Female myocytes have larger CaV1.2 clusters, larger [Ca2+]i, and larger myogenic tone than male myocytes. In contrast, in male myocytes, Kv2.1 channels regulate membrane potential but not CaV1.2 channel clustering. We propose a model in which Kv2.1 function varies with sex: in males, Kv2.1 channels control membrane potential but, in female myocytes, Kv2.1 plays dual electrical and CaV1.2 clustering roles. This contributes to sex-specific regulation of excitability, [Ca2+]i, and myogenic tone in arterial myocytes.

Endoplasmic reticulum (ER) and plasma membrane (PM) form junctions crucial to ion and lipid signaling and homeostasis. The Kv2.1 ion channel is unique among PM proteins in organizing ER-PM junctions. Here, we show that this organizing function is conserved between Kv2 family members that differ in their biophysical properties, modulation and cellular expression. Manipulation of actin cytoskeleton surrounding Kv2 ER-PM junctions affects their spatial organization. Kv2-containing ER-PM junctions overlap with those formed by canonical ER-PM tethers. ER-PM junction organization by Kv2 channels is unchanged by point mutations that eliminate ion conduction, but abolished by those that eliminate PM clustering without impacting ion channel function. Kv2.2 is distinct in lacking the reversible modulation of junction organization present in Kv2.1. Brain neurons in Kv2 double knockout mice have altered ER-PM junctions, demonstrating a conserved in vivo function for Kv2 family members distinct from their canonical role as ion-conducting channels shaping neuronal excitability.

Nanobodies (nAbs) are small, minimal antibodies that have distinct attributes that make them uniquely suited for certain biomedical research, diagnostic and therapeutic applications. Prominent uses include as intracellular antibodies or intrabodies to deliver cargo to specific proteins and/or subcellular sites within cells, and as nanoscale immunolabels for enhanced tissue penetration and improved spatial imaging resolution. Here, we report the generation and validation of nAbs against a set of proteins prominently expressed at specific subcellular sites in brain neurons. We describe a novel hierarchical validation pipeline to systematically evaluate nAbs isolated by phage display for effective and specific use as intrabodies and immunolabels in mammalian cells including brain neurons. These nAbs form part of a robust toolbox for targeting proteins with distinct and highly spatially-restricted subcellular localization in mammalian brain neurons, allowing for visualization and/or modulation of structure and function at those sites.

Coral restoration programs are expanding to revive coral populations and ecosystem services, but local and global stressors still threaten coral survival. In the Caribbean, the ESA-listed staghorn coral Acropora cervicornis has experienced profound declines due to multiple stressors, including disease and nutrient pollution. We studied the impact of these two stressors on ten A. cervicornis genotypes for which disease susceptibility was previously ranked in a disease transmission experiment. Results showed that elevated ammonium, disease, and their combination negatively affected A. cervicornis survivorship, with variable susceptibility among genotypes. Three genotypes were susceptible to elevated ammonium alone and experienced mortality in up to 80% of their fragments. Exposure to a disease homogenate under ambient ammonium caused mortality in 100% of the fragments in four coral genotypes, intermediate mortality in five (33-66% of their fragments), and no mortality in one genotype. However, all genotypes experienced mortality (30-100% of their fragments) when exposed to both elevated ammonium and disease. Despite the detrimental effects of ammonium on coral survivorship, corals under elevated ammonium presented higher photochemical efficiency ( F v /F m ) of the algal symbionts. Disease susceptibility did not align with the genotypic ranking established in a previous study, suggesting that, while genotypes may vary in their disease resistance, rankings may change due to environmental factors or disease type. Regardless of individual susceptibility, our results suggest that water quality improvement is necessary for increasing A. cervicornis survivorship.

The effects of turbidity and sedimentation stress on early life stages of corals are poorly understood, particularly in Atlantic species. Dredging operations, beach nourishment, and other coastal construction activities can increase sedimentation and turbidity in nearby coral reef habitats and have the potential to negatively affect coral larval development and metamorphosis, reducing sexual reproduction success. In this study, we investigated the performance of larvae of the threatened Caribbean coral species Orbicella faveolata exposed to suspended sediments collected from a reef site in southeast Florida recently impacted by dredging (Port of Miami), and compared it to the performance of larvae exposed to sediments collected from the offshore, natal reef of the parent colonies. In a laboratory experiment, we tested whether low and high doses of each of these sediment types affected the survival, settlement, and respiration of coral larvae compared to a no-sediment control treatment. In addition, we analyzed the sediments used in the experiments with 16S rRNA gene amplicon sequencing to assess differences in the microbial communities present in the Port versus Reef sediments, and their potential impact on coral performance. Overall, only O . faveolata larvae exposed to the high-dose Port sediment treatment had significantly lower survival rates compared to the control treatment, suggesting an initial tolerance to elevated suspended sediments. However, significantly lower settlement rates were observed in both Port treatments (low- and high-dose) compared to the control treatment one week after exposure, suggesting strong latent effects. Sediments collected near the Port also contained different microbial communities than Reef sediments, and higher relative abundances of the bacteria Desulfobacterales, which has been associated with coral disease. We hypothesize that differences in microbial communities between the two sediments may be a contributing factor in explaining the observed differences in larval performance. Together, these results suggest that the settlement success and survival of O . faveolata larvae are more readily compromised by encountering port inlet sediments compared to reef sediments, with potentially important consequences for the recruitment success of this species in affected areas.

The staghorn coral, Acropora cervicornis, was once abundant in the Caribbean, but now is listed as critically endangered. To recover A. cervicornis populations, restoration efforts have focused on preserving genetic diversity and increasing coral cover. However, identifying stress-resistant corals can help to increase restoration success, by allocating genotypes to reefs where they are more likely to survive. We assessed the performance (growth, survivorship, and photochemical efficiency) and characterized the microbiome (prokaryotes) of six A. cervicornis genotypes that were maintained at control temperatures (~ 26 °C) and either ambient nutrients or elevated nutrients (elevated NH4, and elevated NH4 + PO4) for > 2 months. We then compared how these parameters changed when the corals were exposed to heat stress (3 weeks at ~ 31.5 °C). We found that exposure to elevated nutrients reduced A. cervicornis performance under control temperatures and heat stress. However, there was a wide range of variation among genotypes, with three genotypes maintaining relatively higher survivorship and growth rates when exposed to nutrients alone, and nutrients followed by heat stress. Heat stress alone changed the microbial composition among genotypes more than elevated nutrients alone, but heat stress also interacted with nutrient pre-exposure to affect microbial communities. The relative abundance of Midichloriaceae and Spirochaetaceae varied by coral genotype and a high abundance of these bacterial taxa was a positive predictor of coral survivorship rate, suggesting a microbial signature that could aid in identifying resistant A. cervicornis genotypes. Our findings suggest there is significant variation among genotypes in the response of A. cervicornis to elevated nutrients and temperatures. Resistant genotypes may be identifiable via their microbiomes and prioritized for outplanting at sites that experience nutrient pollution. Large-scale microbiome screening may help expedite targeted outplanting and could be tested and extended to facilitate the identification of genotypes with other resistance characteristics.

Coral cover is declining worldwide due to multiple interacting threats. We compared the effects of elevated nutrients and temperature on three Caribbean corals: Acropora cervicornis, Orbicella faveolata, and Siderastrea siderea. Colonies hosting different algal symbionts were exposed to either ambient nutrients (A), elevated NH4 (N), or elevated NH4 + PO4 (N + P) at control temperatures (26 °C) for > 2 months, followed by a 3-week thermal challenge (31.5 °C). A. cervicornis hosted Symbiodinium (S. fitti) and was highly susceptible to the combination of elevated nutrients and temperature. During heat stress, A. cervicornis pre-exposed to elevated nutrients experienced 84%–100% mortality and photochemical efficiency (Fv/Fm) declines of 41–50%. In comparison, no mortality and lower Fv/Fm declines (11–20%) occurred in A. cervicornis that were heat-stressed but not pre-exposed to nutrients. O. faveolata and S. siderea response to heat stress was determined by their algal symbiont community and was not affected by nutrients. O. faveolata predominantly hosted Durusdinium trenchii or Breviolum, but only corals hosting Breviolum were susceptible to heat, experiencing 100% mortality, regardless of nutrient treatment. S. siderea colonies predominantly hosted Cladocopium C1 (C. goreaui), Cladocopium C3, D. trenchii, or variable proportions of Cladocopium C1 and D. trenchii. This species was resilient to elevated nutrients and temperature, with no significant mortality in any of the treatments. However, during heat stress, S. siderea hosting Cladocopium C3 suffered higher reductions in Fv/Fm (41–56%) compared to S. siderea hosting Cladocopium C1 and D. trenchii (17–26% and 10–16%, respectively). These differences in holobiont susceptibility to elevated nutrients and heat may help explain historical declines in A. cervicornis starting decades earlier than other Caribbean corals. Our results suggest that tackling only warming temperatures may be insufficient to ensure the continued persistence of Caribbean corals, especially A. cervicornis. Reducing nutrient inputs to reefs may also be necessary for these iconic coral species to survive.