Biology subjects

Klingauf, J.

Publications and source records attributed to Klingauf, J..

8 recordsLinked to original sources

bioRxiv2025

Optical recordings of synaptic vesicle fusion reveal diffusional dispersion

The primary unit of exocytosis, the synaptic vesicle, is replete with proteins which enable vesicle fusion. For maintaining synaptic transmission the vesicle proteins have to be rapidly cleared from the active zone. The immediate fate of the cargo post-fusion and the precise mechanism of its resorting into a readily retrievable pool (RRetP) for endocytosis remains unclear. To address this, we developed a purely presynaptic preparation, the xenapse, presynaptic boutons formed en face directly onto a glass coverslip enabling optical single vesicle recording by total internal reflection microscopy (TIRFM). Electron microscopy showed xenapses contain a few hundred synaptic vesicles (SVs), of which [~]40 SVs are primed (readily releasable pool, RRP), as revealed by single vesicle recordings using the pH-sensitive fluorescent protein pHluorin. Single fusion events synchronous with action potentials could be localized with [~]20 nm precision and rapid post-fusion diffusional dispersion of vesicular proteins observed with diffusion constants in the order of 0.1 {micro}m2/s. Unroofing of xenapses revealed numerous Clathrin-coated structures, enriched for vesicle cargo and numerically equivalent to about twice the measured RRP size. In this way SV proteins are rapidly cleared from the release site into this RRetP of pre-assembled pits to allow for rapid re-docking and re-priming at vacated release sites in synapses tuned for high-fidelity fast signaling.

neuroscience↗

bioRxiv2025

A slow and clathrin-dependent mode of compensatory endocytosis in hippocampal boutons at near-physiological temperature

The mode of presynaptic compensatory endocytosis has been controversial for decades. Recently, an ultrastructural study on synaptic membrane retrieval following synaptic vesicle fusion revealed an ultrafast mechanism of endocytosis in mouse hippocampal synapses at physiological temperature. Using live cell imaging of single-vesicle exo- and endocytosis in mouse hippocampal synapses, combined with accelerated optogenetic acidification of synaptic vesicles, we found that compensatory endocytosis occurs mostly through a slow clathrin-dependent and actin-independent mode at both room and physiological temperatures.

neuroscience↗

bioRxiv2025

Non-canonical role for ATPase HSC70 in driving Clathrin remodeling during compensatory endocytosis in synapse

Neurons use Clathrin-mediated endocytosis to retrieve synaptic vesicle (SV) proteins during compensatory endocytosis after presynaptic SV fusion. We have shown SV cargo to be re-sorted and pre-assembled outside the active zone into Clathrin-coated structures (CCS) of variable size and curvature, constituting a readily retrievable pool. During compensatory endocytosis CCS of the readily retrievable pool must be remodeled swiftly into spherical vesicles within 10 seconds at physiological temperature. How this is achieved remains elusive. Here we performed live-cell imaging on intact as well as scanning electron microscopy on unroofed hippocampal Xenapses, TIRFM-amenable presynaptic boutons formed en-face on microstructured and functionalized coverslips. While CCS can slowly remodel into spherical pits in unroofed Xenapses within tens of minutes, this process is highly accelerated in intact Xenapses, as evidenced by the rapid (< 10 seconds) exchange of EGFP-labelled Clathrin and AP2 adaptor after photo-bleaching. This fast remodeling of CCS was observed in the absence of stimulation and cannot be explained by constitutive endocytosis. Hence, this process must be driven by cytosolic factors which are lost during unroofing. Using membrane-permeant interfering peptides we identify Hsc70, the ATPase which along with Auxilin drives uncoating of endocytosed Clathrin-coated vesicles, to have an additional role in driving curvature of invaginating CCS.

neuroscience↗

bioRxiv2025

Slow scission of single synaptic vesicles by Dynamin at physiological temperature

Compensatory endocytosis is crucial for the presynapse to maintain a functional pool of fusion-competent vesicles. Slow Clathrin-mediated endocytosis has been widely regarded as the primary mechanism of retrieval but this has been challenged by competing Clathrin-independent endocytic models, most notably sub-second ultra-fast endocytosis, reported to be predominant at physiological temperature. Here, we sought to resolve the salience of the respective endocytic modes by using a purely presynaptic preparation, the Xenapse, amenable to total internal reflection fluorescence microscopy (TIRFM). While the role of Clathrin is in dispute, Dynamin is widely acknowledged to figure as the scission protein at the invaginated vesicle neck. Hence, we labelled the endogenous Dynamin I with EGFP by CRISPR-Cas9 techniques and visualized single synaptic Dynamin-mediated scission events at very high temporal resolution. This revealed only a single slow mode of Dynamin-dependent retrieval with a half time of [~] 9 seconds at physiological temperature. Cross-correlational analysis with fluorescently labelled Clathrin confirmed these Dynamin events to be Clathrin-dependent. We thereby affirm Clathrin-mediated endocytosis as the primary mode of compensatory retrieval.

neuroscience↗

bioRxiv2024

Primary Human Neutrophils and Monocytes/Macrophages Migrate along Endothelial Cell Boundaries to Optimize Search Efficiency

Neutrophils and monocytes/macrophages are sentinels of inflammatory signals. To reach the sites of action, both cell types attach to and then transmigrate the endothelial cell layer that lines the luminal side of blood vessels. While it has been reported that neutrophils and monocytes/macrophages actively migrate along the surface of the vasculature, it remains elusive if and how these motion pattern augment the efficiency of the immune system. Here, we conducted co-culture experiments of primary human monocytes and neutrophils, respectively, with human umbilical vein endothelial cells (HUVECs). Combining classical biomedical approaches with quantitative image analysis and numerical models, we find that immune cells simultaneously increase the number of sampled cells vs. traveled distance and sensitivity to chemokines by migrating along endothelial cell-cell boundaries. Collectively, these findings establish search optimization of neutrophils and monocytes/macrophages through limitation of motion pattern to cell-cell boundaries.

cell biology↗

bioRxiv2022

Tetraspanner-based nanodomains modulate BAR domain-induced membrane curvature

Topography is a critical feature driving formation and dynamics of protein and lipid domains within biological membranes. The yeast plasma membrane (PM) has provided a powerful model system to study lateral domain formation, including characteristic BAR domain-induced PM furrows. Currently, it is not clear how the components involved in the establishment of these furrows cooperate to precisely regulate local PM topography. Here we report opposing functions for the Sur7 and Nce102 families of tetraspanner proteins in modulating membrane curvature and domain topography. Using STED nanoscopy and freeze-fracture EM we found that Sur7 tetraspanners form multimeric strands at the upper edges of PM furrows, which counteract the forces exerted by BAR domain proteins and prevent membrane tubulation. In contrast, Nce102 tetraspanners are located basal to the Sur7 proteins and promote BAR domain-induced curvature. The segregation of the two tetraspanner-based nanodomains is further supported by differential distribution of ergosterol to the upper edge of furrows and PIP2 lipids at the furrow base. These findings suggest a general role of tetraspanner proteins in sculpting local membrane domains.

cell biology↗

bioRxiv2022

Early endosomes act as local exocytosis hubs to repair endothelial membrane damage

The plasma membrane of a cell is subject to stresses causing ruptures that must be repaired immediately to preserve membrane integrity and ensure cell survival. Yet, the spatio-temporal membrane dynamics at the wound site and the source of membrane required for wound repair are poorly understood. Here, we show that early endosomes, previously only known to function in the uptake of extracellular material and its endocytic transport, are involved in plasma membrane repair in human endothelial cells. Using live-cell imaging and correlative light and electron microscopy, we demonstrate that membrane injury triggers a previously unknown exocytosis of early endosomes that is induced by Ca2+ entering through the wound. This exocytosis is restricted to the vicinity of the wound site and mediated by the endosomal SNARE VAMP2, which is crucial for efficient membrane repair. Thus, the here identified Ca2+-evoked and localized exocytosis of early endosomes supplies the membrane material required for rapid resealing of a damaged plasma membrane, thereby providing the first line of defense against damage in mechanically challenged endothelial cells.

cell biology↗

bioRxiv2022

Dynamic interplay of protrusive microtubule and contractile actomyosin forces drives tissue extension

In order to shape a tissue, cell-based mechanical forces have to be integrated into global force patterns. Over the last decades, the importance of actomyosin contractile arrays, which are the key constituents of various morphogenetic processes, has been established for many tissues. Intriguingly, recent studies demonstrate that the microtubule cytoskeleton mediates folding and elongation of the epithelial sheet during Drosophila morphogenesis, placing microtubule mechanics en par with actin-based processes. While these studies establish the importance of both cytoskeletal systems during cell and tissue rearrangements, a mechanistic explanation of their functional hierarchy is currently missing. Here, we dissect the individual roles of these two key generators of mechanical forces during epithelium elongation. We demonstrate that microtubules dictate cell shape changes and actomyosin refines them. Combining experimental and numerical approaches, we find that altering the microtubule and actomyosin functions results in predictable changes in tissue shape. We further show that planar polarized microtubule patterning is independent of cell geometry and actomyosin-based mechanics. These results support a hierarchical mechanism, whereby microtubule-based forces in some epithelial systems prime actomyosin-generated forces.

developmental biology↗