Biology subjects

Preobraschenski, J.

Publications and source records attributed to Preobraschenski, J..

3 recordsLinked to original sources

bioRxiv2022

Regulation of the mammalian-brain V-ATPase via ultra-slow mode-switching

Summary paragraphVacuolar-type adenosine triphosphatases (V-ATPases)1-3 are electrogenic rotary mechanoenzymes structurally related to F-type ATP synthases4,5. They hydrolyze ATP to establish electrochemical proton gradients for a plethora of cellular processes1,3. In neurons, the loading of all neurotransmitters into synaptic vesicles is energized by ~1 V-ATPase molecule per synaptic vesicle6,7. To shed light into this bona fide single-molecule biological process, we investigated electrogenic proton pumping by single mammalian-brain V-ATPases, using individual synaptic vesicles fused with immobilized liposomes. We show V-ATPases do not pump continuously in time, as hypothesized by observing the rotation of bacterial homologs8 and assuming strict ATP/proton coupling. Instead, they stochastically switch between three novel ultra-long-lived proton-pumping, inactive, and proton-leaky modes. Upending conventional wisdom, direct observation of pumping revealed that physiologically relevant concentrations of ATP do not regulate the intrinsic pumping rate. Instead, ATP regulates V-ATPase activity via the switching probability of the proton-pumping mode. In contrast, electrochemical proton gradients regulate the pumping rate and the switching of the pumping and inactive modes. This work reveals and emphasises the mechanistic and biological importance of mode-switching in protein regulation.

biophysics↗

bioRxiv2022

Expansion microscopy at one nanometer resolution

Fluorescence imaging is one of the most versatile and widely-used tools in biology1. Although techniques to overcome the diffraction barrier were introduced more than two decades ago, and the nominal attainable resolution kept improving2, 3, fluorescence microscopy still fails to image the morphology of single proteins or small molecular complexes, either purified or in a cellular context4, 5. Here we report a solution to this problem, in the form of one-step nanoscale expansion (ONE) microscopy. We combined the 10-fold axial expansion of the specimen (1000-fold by volume) with a fluorescence fluctuation analysis6, 7 to enable the description of cultured cells, tissues, viral particles, molecular complexes and single proteins. At the cellular level, using immunostaining, our technology revealed detailed nanoscale arrangements of synaptic proteins, including a quasi-regular organisation of PSD95 clusters. At the single molecule level, upon main chain fluorescent labelling, we could visualise the shape of individual membrane and soluble proteins. Moreover, conformational changes undergone by the [~]17 kDa protein calmodulin upon Ca2+ binding were readily observable. We also imaged and classified molecular aggregates in cerebrospinal fluid samples from Parkinsons Disease (PD) patients, which represents a promising new development towards improved PD diagnosis. ONE microscopy is compatible with conventional microscopes and can be performed with the software we provide here as a free, open-source package. This technology bridges the gap between high-resolution structural biology techniques and light microscopy, and provides a new avenue for discoveries in biology and medicine.

biophysics↗

bioRxiv2021

Co-localization of different Neurotransmitter Transporters on the same Synaptic Vesicle is Bona-fide yet Sparse

Vesicular transporters (VTs) define the type of neurotransmitter that synaptic vesicles (SVs) store and release. While certain neurons in mammalian brain release multiple transmitters, the prevalence, physiology of such pluralism and if the release occurs from same or distinct vesicle pools is not clear. Using quantitative single vesicle imaging, we show that a small population of neuronal SVs indeed contain different VTs to accomplish corelease. Surprisingly, this population is highly diverse (27 types), expressing distinct dual transporters suggesting corelease of various combinations of neurotransmitters. Using glutamatergic vesicles as an example, we demonstrate that transporter colocalization not only determine the transmitter type but also influences the SV content and synaptic quantal size. Thus, presence of diverse transporters on the same vesicle is bona-fide and, depending on the VT types, this may act as one mechanism to regulate neurotransmitter type, content and release in space and time.

neuroscience↗