Martin Kahms

A common origin of synaptic vesicles undergoing evoked and spontaneous fusion

There is a longstanding controversy on the identity of synaptic vesicles undergoing spontaneous versus evoked release. A recent study, introducing a new genetic probe, suggested that spontaneous release is driven by a resting pool of synaptic vesicles refractory to stimulation. We found that cross-depletion of spontaneously or actively recycling synaptic vesicle pools occurred on stimulation in rat hippocampal neurons and identified the recycling pool as a major source of spontaneous release.

Blocking endocytosis enhances short-term synaptic depression under conditions of normal availability of vesicles.

It is commonly thought that clathrin-mediated endocytosis is the rate-limiting step of synaptic transmission in small CNS boutons with limited capacity for synaptic vesicles, causing short-term depression during high rates of synaptic transmission. Here, we show by analyzing synaptopHluorin fluorescence that 200 action potentials evoke the same cumulative amount of vesicle fusion, irrespective of the frequency of stimulation (5-40 Hz), implying the absence of vesicle reuse, since the method used (alkaline-trapping) measures only first-round exocytosis. After blocking all slow or specifically clathrin-mediated endocytosis, however, the same stimulation patterns cause a rapid stimulation-frequency-dependent release depression. This form of depression does not reflect insufficient vesicle supply, but appears to be the result of slow clearance of vesicular components from the release site. Our findings uncover an important yet overlooked role of endocytic proteins for release site clearance in addition to their well-characterized role in endocytosis itself.

4Pi Microscopy of the Nuclear Pore Complex

To explore whether super-resolution fluorescence microscopy is able to resolve topographic features of single cellular protein complexes, a two-photon 4Pi microscope was used to study the nuclear pore complex (NPC). The microscope had an axial resolution of 110-130 nm and a two-color localization accuracy of 5-10 nm. In immune-labeled HeLa cells, NPCs could be resolved much better by 4Pi than by confocal microscopy. When two epitopes of the NPC, one localized at the tip of the cytoplasmic filaments and the other at the ring of the nuclear basket, were immune-labeled, they could be clearly resolved in single NPCs, with the distance between them determined to be 152 +/- 30 nm. In cells expressing a green fluorescent protein construct localized at the NPC center, the distances between the ring of the nuclear filaments and the NPC center was 76 +/- 12 (Potorous tridactylus cells) or 91 +/- 21 nm (normal rat kidney cells), whereas the distance between the NPC center and the tips of the cytoplasmic filaments was 84 +/- 18 nm, all values in good agreement with previous electron or single-molecule fluorescence estimates. We conclude that super-resolution fluorescence microscopy is a powerful method for analyzing single protein complexes and the cellular nanomachinery in general.

Binding Site Distribution of Nuclear Transport Receptors and Transport Complexes in Single Nuclear Pore Complexes

Transport through the nuclear pore complex (NPC) involves a large channel and an abundance of binding sites for nuclear transport receptors (NTRs). However, the mechanistically important distribution of NTR‐binding sites along the channel is vividly debated. In this study, we visualized binding site distributions directly by two complementary optical super‐resolution methods, single‐molecule microscopy and 4Pi microscopy. First, we analyzed the distribution of RanGDP because this important nuclear transport substrate has two types of binding sites at the NPC, direct and indirect, NTR‐mediated sites. We found that the direct binding sites had a maximum at approximately −30 nm with regard to the NPC center, whereas the indirect transport‐relevant binding sites peaked at approximately −10 nm. The 20 nm‐shift could be only resolved by 4Pi microscopy because of a two to threefold improved localization precision as compared with single‐molecule microscopy. Then we analyzed the distribution of the NTR Kapβ1 and a Kapβ1‐based transport complex and found them to have also binding maxima at approximately −10 nm. These observations support transport models in which NTR binding sites are distributed all along the transport channel and argue against models in which the cytoplasmic entrance of the channel is surrounded by a large cloud of binding sites.

Lighting up the nuclear pore complex

It is generally accepted that transport through the nuclear pore complex (NPC) involves an abundance of phenylalanine-glycine rich protein domains (FG-domains) that serve as docking sites for soluble nuclear transport receptors (NTRs) and their cargo complexes. But the precise mechanism of translocation through the NPC allowing for high speed and selectivity is still vividly debated. To ultimately decipher the underlying gating mechanism it is indispensable to shed more light on the molecular arrangement of FG-domains and the distribution of NTR-binding sites within the central channel of the NPC. In this review we revisit current transport models, summarize recent results regarding translocation through the NPC obtained by super-resolution microscopy and finally discuss the status and potential of optical methods in the analysis of the NPC.

Restoring synaptic vesicles during compensatory endocytosis

In the CNS (central nervous system), nerve cells communicate by transmitting signals from one to the next across chemical synapses. Electrical signals trigger controlled secretion of neurotransmitter by exocytosis of SV (synaptic vesicles) at the presynaptic site. Neurotransmitters diffuse across the synaptic cleft, activate receptor channels in the receiving neuron at the postsynaptic site, and thereby elicit a new electrical signal. Repetitive stimulation should result in fast depletion of fusion-competent SVs, given their limited number in the presynaptic bouton. Therefore, to support repeated rounds of release, a fast trafficking cycle is required that couples exocytosis and compensatory endocytosis. During this exo-endocytic cycle, a defined stoichiometry of SV proteins has to be preserved, that is, membrane proteins have to be sorted precisely. However, how this sorting is accomplished on a molecular level is poorly understood. In the present chapter we review recent findings regarding the molecular composition of SVs and the mechanisms that sort SV proteins during compensatory endocytosis. We identify self-assembly of SV components and individual cargo recognition by sorting adaptors as major mechanisms for maintenance of the SV protein complement.

The Presynaptic v-ATPase Reversibly Disassembles and Thereby Modulates Exocytosis but Is Not Part of the Fusion Machinery

Vacuolar H+-ATPase (v-ATPase) is a multi-subunit complex comprising two domains: the cytosolic V1 domain catalyzing ATP hydrolysis and the membranous V0 sector translocating protons across membranes. In addition to proton pumping, a direct function of the V0 proteolipid ring in membrane fusion has been proposed for yeast vacuolar fusion and synaptic vesicle exocytosis in Drosophila. Here, we show in cultured hippocampal neurons that in recycling synaptic vesicles, v-ATPases are only transiently assembled in a pH-dependent fashion during the tightly coupled cycle of exo-endocytosis. Upon locking v-ATPase in an assembled state by saliphenylhalamide, we observed use- and time-dependent release depression for stimuli exceeding release of primed vesicles but no abrogation of exocytosis. Thus, the membranous V0 sector is not part of the exocytotic fusion machinery. Instead, v-ATPase modulates release upstream of docking to favor fusion of fully filled synaptic vesicles.

Novel pH-Sensitive Lipid Based Exo-Endocytosis Tracers Reveal Fast Intermixing of Synaptic Vesicle Pools

Styryl dyes and genetically encoded pH-sensitive fluorescent proteins like pHluorin are well-established tools for the optical analysis of synaptic vesicle (SV) recycling at presynaptic boutons. Here, we describe the development of a new class of fluorescent probes based on pH-sensitive organic dyes covalently bound to lipids, providing a promising complementary assay to genetically encoded fluorescent probes. These new optical tracers allow a pure read out of membrane turnover during synaptic activity and visualization of multiple rounds of stimulation-dependent SV recycling without genetic perturbation. Measuring the incorporation efficacy of different dye-labeled lipids into budding SVs, we did not observe an enrichment of lipids with affinity for liquid ordered membrane domains. But most importantly, we found no evidence for a static segregation of SVs into recycling and resting pools. A small but significant fraction of SVs that is reluctant to release during a first round of evoked activity can be exocytosed during a second bout of stimulation, showing fast intermixing of SV pools within seconds. Furthermore, we found that SVs recycling spontaneously have a higher chance to re-occupy release sites than SVs recycling during high-frequency evoked activity. In summary, our data provide strong evidence for a highly dynamic and use-dependent control of the fractions of releasable or resting SVs.

Intrinsic refractive index matched 3D dSTORM with two objectives: Comparison of detection techniques

We have built a setup for 3D single molecule localisation microscopy (SMLM) where a very high resolution is achieved by, firstly, the use of two objectives instead of one and, secondly, minimizing optical aberrations by refractive index matching with a glycerol-water mixture as immersion medium in conjunction with glycerol-immersion objectives. Multiple optical paths of the microscope allow to switch between astigmatic and interferometric localisation along the optical axis, thus enabling a direct comparison of the performance of these localisation methods.

pH-Sensitive Fluorescent Lipids as Novel Probes to Monitor Vesicle Recycling

During synaptic transmission, neurotransmitters stored in presynaptic vesicles are released by excocytosis through fusion of vesicles with the plasma membrane. In a subsequent step, membranes and proteins at the synapse are reinternalized by a reverse process, endocytosis. In the analysis of this synaptic vesicle cycle genetically encoded pH-sensitive fluorescent proteins like the GFP-derivative pHluorin have become indispensable tools. These probes are capable of detecting changes in pH that accompany excocytosis and subsequent reacidification of endocytosed vesicles. Here we describe a new class of fluorescent probes, based on pH-sensitive organic dyes coupled to phospholipids, as promising alternative to genetically encoded fluorescent proteins like pHluorin. Moreover the pH-dependent fluorescence properties of these dyes are opposite to those of pHluorin.In hippocampal neurons, cell membranes can be stained in a pH dependent manner, and upon quenching of the fluorescence at the plasma membrane by a slightly basic pH, vesicle recycling can be monitored yielding fluorescence transients with kinetics mirroring those of the well characterized pHluorin signal. Furthermore, this approach can be used to study vesicle recycling in acute preparations like bipolar cells of the retina, where application of genetically encoded probes was not possible so far.This experimental approach using pH-dependent fluorescent lipids has not only the potential of being used in a variety of cellular and slice preparations, but in addition will shed light on an important presynaptic mechanism neglected so far, namely lipid recycling. Comparison of vesicle incorporation of different dye-labeled lipid moieties will bring new insights into lipid organisation and trafficking at the synapse.