Meeting report - An online gathering about the latest on Molecular Membrane Biology

Gordon Research Conferences (GRC) are among the meetings that are particularly important as they are rather small and foster scientific exchange. Many established researchers participate and meet for a week, often in some remote location. The 2021 molecular membrane biology Gordon conference, usually held at Proctor Academy (New Hampshire, USA) was one of the many meetings affected when the SARS-CoV2 pandemic resulted in an immediate shut down of all public life and world travel. Scientists and scientific organizations reacted immediately and discovered the advantages of online meetings. Several members of the membrane biology community felt that a complete halt of scientific conferences would particularly harm young investigators in their career path as they would not get the chance to present their data in front of the scientific community. As a result, we decided to take the initiative and try to find a format for an online meeting in 2021. Before starting, we sent around a survey to ask for interest, and received overwhelming enthusiasm and support – a testament to the vitality of the membrane trafficking community. We agreed on a format that would mimic somehow the in-person meeting: poster sessions, followed by a dense set of talks with sufficient time for discussion, a meet-the-speaker slot, then another set of talks and eventually another poster session or meeting time. Talks were presented mostly by early career researchers, postdocs or assistant professors. To foster discussion and presentation of unpublished data, talks were live and not recorded. Gather.town was used as a platform for poster sessions and social interactions. Very importantly, the meeting was completely free as funding was secured via four journals (Journal of Cell Biology, Journal of Cell Science, FEBS Journal and Traffic Journal) and three German DFG-funded research consortia (SFB 944, 958 and TR 186), which covered the user fees for all participants

Nutritional and immunological factors in breast milk: A role in the intergenerational transmission from maternal psychopathology to child development

Perinatal psychopathologies affect more than 25% of women during and after their gestational period. These psychiatric disorders can potentially determine important biological variations in their organisms, affecting many different physiological and metabolic pathways. Of relevance, any of these changes occurring in the mother can alter the normal composition of breast milk, particularly the concentration of nutritional and inflammatory components, which play a role in child brain functioning and development. Indeed, there is evidence showing that changes in milk composition can contribute to cognitive impairments and alterations in mental abilities in children. This review aims to shed light on the unique intergenerational role played by breast milk composition, from maternal psychopathologies to child development

A novel physiological role for ARF1 in the formation of bidirectional tubules from the Golgi.

Capitalizing on CRISPR/Cas9 gene-editing techniques and super-resolution nanoscopy, we explore the role of the small GTPase ARF1 in mediating transport steps at the Golgi. Besides its well-established role in generating COPI vesicles, we find that ARF1 is also involved in the formation of long (∼3 µm), thin (∼110 nm diameter) tubular carriers. The anterograde and retrograde tubular carriers are both largely free of the classical Golgi coat proteins coatomer (COPI) and clathrin. Instead, they contain ARF1 along their entire length at a density estimated to be in the range of close packing. Experiments using a mutant form of ARF1 affecting GTP hydrolysis suggest that ARF1[GTP] is functionally required for the tubules to form. Dynamic confocal and stimulated emission depletion imaging shows that ARF1-rich tubular compartments fall into two distinct classes containing 1) anterograde cargoes and clathrin clusters or 2) retrograde cargoes and coatomer clusters

Recombinant biosensors for multiplex and super-resolution imaging of phosphoinositides

Phosphoinositides are a small family of phospholipids that act as signaling hubs and key regulators of cellular function. Detecting their subcellular distribution is crucial to gain insights into membrane organization and is commonly done by the overexpression of biosensors. However, this leads to cellular perturbations and is challenging in systems that cannot be transfected. Here, we present a toolkit for the reliable, fast, multiplex, and super-resolution detection of phosphoinositides in fixed cells and tissue, based on recombinant biosensors with self-labeling SNAP tags. These are highly specific and reliably visualize the subcellular distributions of phosphoinositides across scales, from 2D or 3D cell culture to Drosophila tissue. Further, these probes enable super-resolution approaches, and using STED microscopy, we reveal the nanoscale organization of PI(3)P on endosomes and PI(4)P on the Golgi. Finally, multiplex staining reveals an unexpected presence of PI(3,5)P2-positive membranes in swollen lysosomes following PIKfyve inhibition. This approach enables the versatile, high-resolution visualization of multiple phosphoinositide species in an unprecedented manner.</p

Recombinant biosensors for multiplex and super-resolution imaging of phosphoinositides

Phosphoinositides are a small family of phospholipids that act as signaling hubs and key regulators of cellular function. Detecting their subcellular distribution is crucial to gain insights into membrane organization and is commonly done by the overexpression of biosensors. However, this leads to cellular perturbations and is challenging in systems that cannot be transfected. Here, we present a toolkit for the reliable, fast, multiplex, and super-resolution detection of phosphoinositides in fixed cells and tissue, based on recombinant biosensors with self-labeling SNAP tags. These are highly specific and reliably visualize the subcellular distributions of phosphoinositides across scales, from 2D or 3D cell culture to Drosophila tissue. Further, these probes enable super-resolution approaches, and using STED microscopy, we reveal the nanoscale organization of PI(3)P on endosomes and PI(4)P on the Golgi. Finally, multiplex staining reveals an unexpected presence of PI(3,5)P2-positive membranes in swollen lysosomes following PIKfyve inhibition. This approach enables the versatile, high-resolution visualization of multiple phosphoinositide species in an unprecedented manner.</p

A versatile Halo- and SNAP-tagged BMP/TGFβ receptor library for quantification of cell surface ligand binding

TGFβs, BMPs and Activins regulate numerous developmental and homeostatic processes and signal through hetero-tetrameric receptor complexes composed of two types of serine/threonine kinase receptors. Each of the 33 different ligands possesses unique affinities towards specific receptor types. However, the lack of specific tools hampered simultaneous testing of ligand binding towards all BMP/TGFβ receptors. Here we present a N-terminally Halo- and SNAP-tagged TGFβ/BMP receptor library to visualize receptor complexes in dual color. In combination with fluorescently labeled ligands, we established a Ligand Surface Binding Assay (LSBA) for optical quantification of receptor-dependent ligand binding in a cellular context. We highlight that LSBA is generally applicable to test (i) binding of different ligands such as Activin A, TGFβ1 and BMP9, (ii) for mutant screens and (iii) evolutionary comparisons. This experimental set-up opens opportunities for visualizing ligand-receptor binding dynamics, essential to determine signaling specificity and is easily adaptable for other receptor signaling pathways

Two-colour live-cell nanoscale imaging of intracellular targets.

Stimulated emission depletion (STED) nanoscopy allows observations of subcellular dynamics at the nanoscale. Applications have, however, been severely limited by the lack of a versatile STED-compatible two-colour labelling strategy for intracellular targets in living cells. Here we demonstrate a universal labelling method based on the organic, membrane-permeable dyes SiR and ATTO590 as Halo and SNAP substrates. SiR and ATTO590 constitute the first suitable dye pair for two-colour STED imaging in living cells below 50 nm resolution. We show applications with mitochondria, endoplasmic reticulum, plasma membrane and Golgi-localized proteins, and demonstrate continuous acquisition for up to 3 min at 2-s time resolution