Kv2.1 mediates spatial and functional coupling of L-type calcium channels and ryanodine receptors in mammalian neurons.

The voltage-gated K+ channel Kv2.1 serves a major structural role in the soma and proximal dendrites of mammalian brain neurons, tethering the plasma membrane (PM) to endoplasmic reticulum (ER). Although Kv2.1 clustering at neuronal ER-PM junctions (EPJs) is tightly regulated and highly conserved, its function remains unclear. By identifying and evaluating proteins in close spatial proximity to Kv2.1-containing EPJs, we discovered that a significant role of Kv2.1 at EPJs is to promote the clustering and functional coupling of PM L-type Ca2+ channels (LTCCs) to ryanodine receptor (RyR) ER Ca2+ release channels. Kv2.1 clustering also unexpectedly enhanced LTCC opening at polarized membrane potentials. This enabled Kv2.1-LTCC-RyR triads to generate localized Ca2+ release events (i.e., Ca2+ sparks) independently of action potentials. Together, these findings uncover a novel mode of LTCC regulation and establish a unique mechanism whereby Kv2.1-associated EPJs provide a molecular platform for localized somatodendritic Ca2+ signals in mammalian brain neurons

A toolbox of nanobodies developed and validated for use as intrabodies and nanoscale immunolabels in mammalian brain neurons.

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

A novel gene cluster allows preferential utilization of fucosylated milk oligosaccharides in Bifidobacterium longum subsp. longum SC596

La microbiota intestinal infantil es a menudo colonizada por dos subespecies de Bifidobacterium longum: subsp. infantis (B. infantis) y subsp. longum (B. longum). El crecimiento competitivo de B. infantis en el intestino del neonato ha sido vinculado a la utilización de oligosacáridos (HMO) en la leche humana. Sin embargo, poco se sabe sobre cómo B. longum en el consumo de HMO. En este estudio, las cepas B. longum transmitidas mostraron diferentes fenotipos de crecimiento de HMO. Mientras que todas las cepas utilizadas eficientemente con lacto-N-tetraosa, focalizaron ciertas cepas, que, además, metabolizaron el HMO. El B. longum SC596 creció vigorosamente en el HMO, y la glycocaracterización reveló una preferencia por el consumo de HMO fucosilados. El transcriptomes SC596 durante la etapa temprana de crecimiento sobre HMO se asemeja más al crecimiento de la fucosilactosa, pasando más tarde a un patrón similar al crecimiento en el HMO neutro. El B. longum SC596 contiene un gen novedoso cerrado dedicado a la utilización de HMO fucosilado, incluyendo los genes para la importación de moléculas fucosiladas, metabolismo fucoso y dos α-fucosidasas. Este grupo mostró una inducción modular durante el crecimiento temprano de HMO y fucosilactosa. Este trabajo aclara el genoma y la variabilidad fisiológica del bebé portador de B. longum en el consumo de HMO, que se asemeja a B. infantis. La capacidad de consumir preferentemente HMO fucosilado sugiere una ventaja competitiva para estas singulares de las cepas de B. longum en el lactante alimentado con tripa.The infant intestinal microbiota is often colonized by two subspecies of Bifidobacterium longum: subsp. infantis (B. infantis) and subsp. longum (B. longum). Competitive growth of B. infantis in the neonate intestine has been linked to the utilization of human milk oligosaccharides (HMO). However, little is known how B. longum consumes HMO. In this study, infant-borne B. longum strains exhibited varying HMO growth phenotypes. While all strains efficiently utilized lacto-N-tetraose, certain strains additionally metabolized fucosylated HMO. B. longum SC596 grew vigorously on HMO, and glycoprofiling revealed a preference for consumption of fucosylated HMO. Transcriptomes of SC596 during early-stage growth on HMO were more similar to growth on fucosyllactose, transiting later to a pattern similar to growth on neutral HMO. B. longum SC596 contains a novel gene cluster devoted to the utilization of fucosylated HMO, including genes for import of fucosylated molecules, fucose metabolism and two α-fucosidases. This cluster showed a modular induction during early growth on HMO and fucosyllactose. This work clarifies the genomic and physiological variation of infant-borne B. longum to HMO consumption, which resembles B. infantis. The capability to preferentially consume fucosylated HMO suggests a competitive advantage for these unique B. longum strains in the breast-fed infant gut.• National Institutes of Health Awards AT007079, HD065122 y AT008759 • Peter J. Shields Endowed Chair in Dairy Food Science • Conicyt Fondecyt: Beca de iniciación 11130518 • Ministerio de Educación y Ciencia y Universidad de Extremadura.peerReviewe

Chemical Approaches To Perturb, Profile, and Perceive Glycans

Glycosylation is an essential form of post-translational modification that regulates intracellular and extracellular processes. Regrettably, conventional biochemical and genetic methods often fall short for the study of glycans, because their structures are often not precisely defined at the genetic level. To address this deficiency, chemists have developed technologies to perturb glycan biosynthesis, profile their presentation at the systems level, and perceive their spatial distribution. These tools have identified potential disease biomarkers and ways to monitor dynamic changes to the glycome in living organisms. Still, glycosylation remains the underexplored frontier of many biological systems. In this Account, we focus on research in our laboratory that seeks to transform the study of glycan function from a challenge to routine practice

Identification of VAPA and VAPB as Kv2 Channel-Interacting Proteins Defining Endoplasmic Reticulum–Plasma Membrane Junctions in Mammalian Brain Neurons

Membrane contacts between endoplasmic reticulum (ER) and plasma membrane (PM), or ER-PM junctions, are ubiquitous in eukaryotic cells and are platforms for lipid and calcium signaling and homeostasis. Recent studies have revealed proteins crucial to the formation and function of ER-PM junctions in non-neuronal cells, but little is known of the ER-PM junctions prominent in aspiny regions of mammalian brain neurons. The Kv2.1 voltage-gated potassium channel is abundantly clustered at ER-PM junctions in brain neurons and is the first PM protein that functions to organize ER-PM junctions. However, the molecular mechanism whereby Kv2.1 localizes to and remodels these junctions is unknown. We used affinity immunopurification and mass spectrometry-based proteomics on brain samples from male and female WT and Kv2.1 KO mice and identified the resident ER vesicle-associated membrane protein-associated proteins isoforms A and B (VAPA and VAPB) as prominent Kv2.1-associated proteins. Coexpression with Kv2.1 or its paralog Kv2.2 was sufficient to recruit VAPs to ER-PM junctions. Multiplex immunolabeling revealed colocalization of Kv2.1 and Kv2.2 with endogenous VAPs at ER-PM junctions in brain neurons from male and female mice in situ and in cultured rat hippocampal neurons, and KO of VAPA in mammalian cells reduces Kv2.1 clustering. The association of VAPA with Kv2.1 relies on a "two phenylalanines in an acidic tract" (FFAT) binding domain on VAPA and a noncanonical phosphorylation-dependent FFAT motif comprising the Kv2-specific clustering or PRC motif. These results suggest that Kv2.1 localizes to and organizes neuronal ER-PM junctions through an interaction with VAPs.SIGNIFICANCE STATEMENT Our study identified the endoplasmic reticulum (ER) proteins vesicle-associated membrane protein-associated proteins isoforms A and B (VAPA and VAPB) as proteins copurifying with the plasma membrane (PM) Kv2.1 ion channel. We found that expression of Kv2.1 recruits VAPs to ER-PM junctions, specialized membrane contact sites crucial to distinct aspects of cell function. We found endogenous VAPs at Kv2.1-mediated ER-PM junctions in brain neurons and other mammalian cells and that knocking out VAPA expression disrupts Kv2.1 clustering. We identified domains of VAPs and Kv2.1 necessary and sufficient for their association at ER-PM junctions. Our study suggests that Kv2.1 expression in the PM can affect ER-PM junctions via its phosphorylation-dependent association to ER-localized VAPA and VAPB

Milk Glycans and Their Interaction with the Infant-Gut Microbiota

Human milk is a unique and complex fluid that provides infant nutrition and delivers an array of bioactive molecules that serve various functions. Glycans, abundant in milk, can be found as free oligosaccharides or as glycoconjugates. Milk glycans are increasingly linked to beneficial outcomes in neonates through protection from pathogens and modulation of the immune system. Indeed, these glycans influence the development of the infant and the infant-gut microbiota. Bifidobacterium species commonly are enriched in breastfed infants and are among a limited group of bacteria that readily consume human milk oligosaccharides (HMOs) and milk glycoconjugates. Given the importance of bifidobacteria in infant health, numerous studies have examined the molecular mechanisms they employ to consume HMOs and milk glycans, thus providing insight into this unique enrichment and shedding light on a range of translational opportunities to benefit at-risk infants

Milk Glycans and Their Interaction with the Infant-Gut Microbiota

Human milk is a unique and complex fluid that provides infant nutrition and delivers an array of bioactive molecules that serve various functions. Glycans, abundant in milk, can be found as free oligosaccharides or as glycoconjugates. Milk glycans are increasingly linked to beneficial outcomes in neonates through protection from pathogens and modulation of the immune system. Indeed, these glycans influence the development of the infant and the infant-gut microbiota. Bifidobacterium species commonly are enriched in breastfed infants and are among a limited group of bacteria that readily consume human milk oligosaccharides (HMOs) and milk glycoconjugates. Given the importance of bifidobacteria in infant health, numerous studies have examined the molecular mechanisms they employ to consume HMOs and milk glycans, thus providing insight into this unique enrichment and shedding light on a range of translational opportunities to benefit at-risk infants

Kv2.1 mediates spatial and functional coupling of L-type calcium channels and ryanodine receptors in mammalian neurons.

The voltage-gated K+ channel Kv2.1 serves a major structural role in the soma and proximal dendrites of mammalian brain neurons, tethering the plasma membrane (PM) to endoplasmic reticulum (ER). Although Kv2.1 clustering at neuronal ER-PM junctions (EPJs) is tightly regulated and highly conserved, its function remains unclear. By identifying and evaluating proteins in close spatial proximity to Kv2.1-containing EPJs, we discovered that a significant role of Kv2.1 at EPJs is to promote the clustering and functional coupling of PM L-type Ca2+ channels (LTCCs) to ryanodine receptor (RyR) ER Ca2+ release channels. Kv2.1 clustering also unexpectedly enhanced LTCC opening at polarized membrane potentials. This enabled Kv2.1-LTCC-RyR triads to generate localized Ca2+ release events (i.e., Ca2+ sparks) independently of action potentials. Together, these findings uncover a novel mode of LTCC regulation and establish a unique mechanism whereby Kv2.1-associated EPJs provide a molecular platform for localized somatodendritic Ca2+ signals in mammalian brain neurons