Biology subjects

Haucke, V.

Publications and source records attributed to Haucke, V..

24 records · Page 2Linked to original sources

bioRxiv2022

The molecular organization of flat and curved caveolae indicates bendable structural units at the plasma membrane.

Caveolae are small coated inner plasma membrane invaginations found in many cell types. Their diverse functions span from endocytosis to signaling, regulating key cellular processes including lipid uptake, pathogen entry, and membrane tension. Caveolae undergo shape changes from flat to curved. It is unclear which proteins regulate this process. To address this gap, we studied the shapes of caveolae with platinum replica electron microscopy in six common cell types. Next, we developed a correlative multi-color stimulated emission depletion (STED) fluorescence and platinum replica EM imaging (CLEM) method to image caveolae-associated proteins at caveolae of different shapes at the nanoscale. Caveolins and cavins were found at all caveolae, independent of their curvature. EHD2, a classic caveolar neck protein, was strongly detected at both curved and flat caveolae. Both pacsin2 and the regulator EHBP1 were found only at a subset of caveolae. Pacsin2 was localized primarily to areas surrounding flat caveolae, whereas EHBP1 was mostly detected at spheres. Contrary to classic models, dynamin was absent from caveolae and localized only to clathrin-coated structures. Cells lacking dynamin showed no substantial changes to caveolae, suggesting that dynamin is not directly involved in caveolae curvature. Together, we provide a mechanistic map for the molecular control of caveolae shape by eight of the major caveolae-associated coat and regulatory proteins. We propose a model where caveolins, cavins, and EHD2 assemble as a cohesive structural unit regulated by more intermittent associations with pacsin2 and EHBP1. These complexes can flatten and curve, capturing membrane to enable lipid traffic and changes to the surface area of the cell.

cell biology↗

bioRxiv2021

Clathrin-independent endocytic retrieval of SV proteins mediated by the clathrin adaptor AP-2 at mammalian central synapses

Neurotransmission is based on the exocytic fusion of synaptic vesicles (SVs) followed by endocytic membrane retrieval and the reformation of SVs. Conflicting models have been proposed regarding the mechanisms of SV endocytosis, most notably clathrin/ AP-2-mediated endocytosis and clathrin-independent ultrafast endocytosis. Partitioning between these pathways has been suggested to be controlled by temperature and stimulus paradigm. We report on the comprehensive survey of six major SV proteins to show that SV endocytosis in hippocampal neurons at physiological temperature occurs independent of clathrin while the endocytic retrieval of a subset of SV proteins including the vesicular transporters for glutamate and GABA depend on sorting by the clathrin adaptor AP-2. Our findings highlight a clathrin-independent role of the clathrin adaptor AP-2 in the endocytic retrieval of select SV cargos from the presynaptic cell surface and suggest a unified model for the endocytosis of SV membranes at mammalian central synapses.

neuroscience↗

bioRxiv2021

Sulfonated rhodamines as impermeable labelling substrates for cell surface protein visualization

Sulfonated rhodamines that endow xanthene dyes with cellular impermeability are presented. We fuse charged sulfonates to red and far-red dyes to obtain Sulfo549 and Sulfo646, respectively, and further link these to SNAP- and Halo-tag substrates for protein self-labelling. Cellular impermeability is validated in live cell imaging experiments in transfected HEK cells and neurons derived from induced pluripotent stem cells (iPSCs). Lastly, we show that Sulfo646 is amenable to STED nanoscopy by recording membranes of SNAP/Halo-surface-labelled human iPSC-derived neuronal axons. We therefore provide an avenue for rendering dyes impermeable for exclusive extracellular visualization via self-labelling protein tags.

biochemistry↗

bioRxiv2020

Neuronal autophagy regulates presynaptic neurotransmission by controlling the axonal endoplasmic reticulum

Information processing in the brain is encoded as electrical impulses in neurons that are relayed from the presynaptic compartment to postsynaptic neurons by regulated neurotransmitter release. Neurons are known to rely on autophagy for the removal of defective proteins or organelles to maintain synaptic neurotransmission and to counteract neurodegeneration. In spite of its importance for neuronal health, the physiological substrates of neuronal autophagy in the absence of proteotoxic challenge have remained largely elusive. We use knockout mice conditionally lacking the essential autophagy protein ATG5 and quantitative proteomics to demonstrate that loss of neuronal autophagy causes the selective accumulation of tubular endoplasmic reticulum (ER) in axons, resulting in increased excitatory neurotransmission and compromised postnatal viability in vivo. The gain in excitatory neurotransmission is shown to be a consequence of elevated calcium release from ER stores via ryanodine receptors accumulated in axons and at presynaptic sites. We propose a model in which neuronal autophagy controls axonal ER calcium stores to regulate neurotransmission in healthy neurons and in the brain.

neuroscience↗

bioRxiv2019

Chr21 protein-protein interactions: enrichment in products involved in intellectual disabilities, autism and Late Onset Alzheimer Disease

Intellectual disability (ID) found in Down syndrome (DS), which is characterized by an extra copy of 234 genes on Chr21 is poorly understood. We first used two DS mouse models that either display an extra copy of the Dyrk1A gene or of the mouse Chr16 syntenic region. Exome sequencing of transcripts deregulated in embryonic hippocampus uncovers enrichment in genes involved in chromatin and synapse respectively. Using large-scale yeast two-hybrid screen (154 distinct screens) of human brain library containing at least 107 independent fragments, we identified 3,636 novel protein-protein interactions with an enrichment of direct interactors of both Chromosome 21(Hsa21) baits and rebounds in ID-related genes. Using proximity ligation assays, we identified that Hsa21-encoded proteins are located at the dendritic spine postsynaptic density in a protein network located at the dendritic spine post synapse. We located Hsa21 DYRK1A and DSCAM that confers a [~] 20-fold increase in Autism Spectrum Disorders (ASDs), in this postsynaptic network. We found that a DSCAM intracellular domain binds either DYRK1A or DLGs that are multimeric scaffolds for the clustering of receptors, ion channels, and associated signaling proteins. The DYRK1A-DSCAM interaction is conserved from drosophila to humans. The identified postsynaptic.network is enriched in ARC-related synaptic plasticity, ASDs and Late-Onset Alzheimer Disease. Altogether, these results emphasize links between DS and brain diseases with complex genetics.

neuroscience↗

bioRxiv2018

Mutations In PIK3C2A Cause Syndromic Short Stature, Skeletal Abnormalities, and Cataracts Associated With Ciliary Dysfunction

PIK3C2A is a class II member of the phosphoinositide 3-kinase (PI3K) family that catalyzes the phosphorylation of phosphatidylinositol (PI) into PI(3)P and the phosphorylation of PI(4)P into PI(3,4)P2. We identified homozygous loss-of-function mutations in PIK3C2A in children from three independent consanguineous families with short stature, coarse facial features, cataracts with secondary glaucoma, multiple skeletal abnormalities, neurological manifestations, among other findings. Cellular studies of patient-derived fibroblasts found that they lacked PIK3C2A protein, had impaired cilia formation and function, and demonstrated reduced proliferative capacity. Collectively, the genetic and molecular data implicate mutations in PIK3C2A in a new Mendelian disorder of PI metabolism, thereby shedding light on the critical role of a class II PI3K in growth, vision, skeletal formation and neurological development. This discovery expands what is known about disorders of PI metabolism and helps unravel the role of PIK3C2A and class II PI3Ks in health and disease.

genetics↗