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Aleksander A. Rebane

Other affiliations: ETH Zurich
Bio: Aleksander A. Rebane is an academic researcher from Yale University. The author has contributed to research in topics: Vesicle & Exocytosis. The author has an hindex of 13, co-authored 19 publications receiving 483 citations. Previous affiliations of Aleksander A. Rebane include ETH Zurich.

Papers
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Journal ArticleDOI
TL;DR: It is suggested here that S-palmitoylation of anterograde cargo at the Golgi membrane interface is an anterogsrade signal and that it results in concentration in curved regions at the golgi rims by simple physical chemistry.

83 citations

Journal ArticleDOI
01 Sep 2014-eLife
TL;DR: In this article, the authors characterized the folding energy and kinetics of four representative N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) at a single-molecule level using high resolution optical tweezers.
Abstract: Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are evolutionarily conserved machines that couple their folding/assembly to membrane fusion. However, it is unclear how these processes are regulated and function. To determine these mechanisms, we characterized the folding energy and kinetics of four representative SNARE complexes at a single-molecule level using high-resolution optical tweezers. We found that all SNARE complexes assemble by the same step-wise zippering mechanism: slow N-terminal domain (NTD) association, a pause in a force-dependent half-zippered intermediate, and fast C-terminal domain (CTD) zippering. The energy release from CTD zippering differs for yeast (13 kBT) and neuronal SNARE complexes (27 kBT), and is concentrated at the C-terminal part of CTD zippering. Thus, SNARE complexes share a conserved zippering pathway and polarized energy release to efficiently drive membrane fusion, but generate different amounts of zippering energy to regulate fusion kinetics.

76 citations

Journal ArticleDOI
23 Dec 2015-eLife
TL;DR: Three distinct functions of SNARE domains whose assembly is intimately chaperoned by Munc18-1 are demonstrated, including t-SNARE C-terminus independent of syntaxin N-terminal regulatory domain (NRD), which is dependent upon the NRD.
Abstract: Plants, animals and other eukaryotes transport many large molecules within their cells inside membrane-bound packages called vesicles. These vesicles can fuse with the membrane of a target compartment in the cell to deliver their contents inside, or fuse with the cell’s membrane to release the contents outside of the cell. Membrane fusion is carried out by a group of proteins called SNAREs. These proteins are embedded on the membranes of both the vesicle and its target, and they bind to each other to form a tight complex. This complex docks the vesicle to the target and then acts like a “zipper” to pull the two membranes close enough to fuse. The best-studied SNARE proteins act in nerve cells and fuse vesicles to the cell’s membrane in order to release molecules called neurotransmitters. This process is essential for communication between nerve cells, and relies on a protein called Munc18-1. However, it is not well understood how SNARE proteins assemble into the complex and how Munc18-1 regulates this process. Ma et al. have now used a tool called “optical tweezers” to pull an assembled SNARE complex apart in the laboratory and then observe how it folds and assembles in a step-by-step process. These experiments showed that the complex assembled in four stages and not three as has been reported in previous work. SNARE proteins are made up of four parts called domains, and Ma et al. observed that the N-terminal domains were the first to bind to each other. Next, the binding progressed to the middle domain, then to the C-terminal domain and finally to the linker domain. An intermediate, half-zippered form was also observed. Ma et al. next analysed each domain in more detail and found that the N-terminal and C-terminal domains drive the docking of vesicles to the target membrane, the middle domain is crucial for assembling the SNARE complex correctly, and all three domains regulate the fusing of the membranes. Further experiments showed that Munc18-1 promoted the assembly of new SNARE complexes and stabilized the half-zippered form, rather than stabilizing the complex after it had fully assembled. This study will provide a new tool to examine many other proteins that regulate SNARE assembly, and a basis to understand the role of SNARE proteins in brain activity.

71 citations

Journal ArticleDOI
26 May 2015-PLOS ONE
TL;DR: This study quantitatively compares the image quality achieved when imaging Alexa Fluor 647-immunolabeled microtubules over an extended range of laser intensities and camera speeds using three criteria – localization precision, density of localized molecules, and resolution of reconstructed images based on Fourier Ring Correlation.
Abstract: Single-molecule switching nanoscopy overcomes the diffraction limit of light by stochastically switching single fluorescent molecules on and off, and then localizing their positions individually. Recent advances in this technique have greatly accelerated the data acquisition speed and improved the temporal resolution of super-resolution imaging. However, it has not been quantified whether this speed increase comes at the cost of compromised image quality. The spatial and temporal resolution depends on many factors, among which laser intensity and camera speed are the two most critical parameters. Here we quantitatively compare the image quality achieved when imaging Alexa Fluor 647-immunolabeled microtubules over an extended range of laser intensities and camera speeds using three criteria – localization precision, density of localized molecules, and resolution of reconstructed images based on Fourier Ring Correlation. We found that, with optimized parameters, single-molecule switching nanoscopy at high speeds can achieve the same image quality as imaging at conventional speeds in a 5–25 times shorter time period. Furthermore, we measured the photoswitching kinetics of Alexa Fluor 647 from single-molecule experiments, and, based on this kinetic data, we developed algorithms to simulate single-molecule switching nanoscopy images. We used this software tool to demonstrate how laser intensity and camera speed affect the density of active fluorophores and influence the achievable resolution. Our study provides guidelines for choosing appropriate laser intensities for imaging Alexa Fluor 647 at different speeds and a quantification protocol for future evaluations of other probes and imaging parameters.

70 citations

Journal ArticleDOI
Paul V. Ruijgrok1, R. Wüest1, Aleksander A. Rebane1, Alois Renn1, Vahid Sandoghdar1 
TL;DR: The observed lengthening of the lifetimes of nitrogen-vacancy color centers in individual diamond nanocrystals was attributed to the reduction of the local density of states, which is very promising for exploring the strong threedimensional localization of light directly on the microscopic scale.
Abstract: Fluorescence lifetimes of nitrogen-vacancy color centers in individual diamond nanocrystals were measured at the interface between a glass substrate and a strongly scattering medium. Comparison of the results with values recorded from the same nanocrystals at the glass-air interface revealed fluctuations of fluorescence lifetimes in the scattering medium. After discussing a range of possible systematic effects, we attribute the observed lengthening of the lifetimes to the reduction of the local density of states. Our approach is very promising for exploring the strong three-dimensional localization of light directly on the microscopic scale.

42 citations


Cited by
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Journal ArticleDOI
TL;DR: A framework for the study of condensate functions across these cellular length scales is provided, offering to bring new understanding of biological processes.
Abstract: Biomolecular condensates are found throughout eukaryotic cells, including in the nucleus, in the cytoplasm and on membranes. They are also implicated in a wide range of cellular functions, organizing molecules that act in processes ranging from RNA metabolism to signalling to gene regulation. Early work in the field focused on identifying condensates and understanding how their physical properties and regulation arise from molecular constituents. Recent years have brought a focus on understanding condensate functions. Studies have revealed functions that span different length scales: from molecular (modulating the rates of chemical reactions) to mesoscale (organizing large structures within cells) to cellular (facilitating localization of cellular materials and homeostatic responses). In this Roadmap, we discuss representative examples of biochemical and cellular functions of biomolecular condensates from the recent literature and organize these functions into a series of non-exclusive classes across the different length scales. We conclude with a discussion of areas of current interest and challenges in the field, and thoughts about how progress may be made to further our understanding of the widespread roles of condensates in cell biology.

355 citations

Journal ArticleDOI
11 Aug 2016-Cell
TL;DR: The development of whole-cell 4Pi single-molecule switching nanoscopy (W-4PiSMSN), an optical nanoscope that allows imaging of three-dimensional structures at 10- to 20-nm resolution throughout entire mammalian cells, is presented.

247 citations

Journal ArticleDOI
03 Jun 2021
TL;DR: This Primer explains the central concepts of single-molecule localization microscopy before discussing experimental considerations regarding fluorophores, optics and data acquisition, processing and analysis, and describes recent high-impact discoveries made by SMLM techniques.
Abstract: Single-molecule localization microscopy (SMLM) describes a family of powerful imaging techniques that dramatically improve spatial resolution over standard, diffraction-limited microscopy techniques and can image biological structures at the molecular scale. In SMLM, individual fluorescent molecules are computationally localized from diffraction-limited image sequences and the localizations are used to generate a super-resolution image or a time course of super-resolution images, or to define molecular trajectories. In this Primer, we introduce the basic principles of SMLM techniques before describing the main experimental considerations when performing SMLM, including fluorescent labelling, sample preparation, hardware requirements and image acquisition in fixed and live cells. We then explain how low-resolution image sequences are computationally processed to reconstruct super-resolution images and/or extract quantitative information, and highlight a selection of biological discoveries enabled by SMLM and closely related methods. We discuss some of the main limitations and potential artefacts of SMLM, as well as ways to alleviate them. Finally, we present an outlook on advanced techniques and promising new developments in the fast-evolving field of SMLM. We hope that this Primer will be a useful reference for both newcomers and practitioners of SMLM. This Primer explains the central concepts of single-molecule localization microscopy (SMLM) before discussing experimental considerations regarding fluorophores, optics and data acquisition, processing and analysis. The Primer further describes recent high-impact discoveries made by SMLM techniques and concludes by discussing emerging methodologies.

246 citations

Journal ArticleDOI
05 Feb 2015-Nature
TL;DR: Structures of ATP- and ADP-bound NSF, and the NSF/SNAP/SNARE (20S) supercomplex determined by single-particle electron cryomicroscopy at near-atomic to sub-nanometre resolution without imposing symmetry are reported.
Abstract: Evolutionarily conserved SNARE (soluble N-ethylmaleimide sensitive factor attachment protein receptors) proteins form a complex that drives membrane fusion in eukaryotes. The ATPase NSF (N-ethylmaleimide sensitive factor), together with SNAPs (soluble NSF attachment protein), disassembles the SNARE complex into its protein components, making individual SNAREs available for subsequent rounds of fusion. Here we report structures of ATP- and ADP-bound NSF, and the NSF/SNAP/SNARE (20S) supercomplex determined by single-particle electron cryomicroscopy at near-atomic to sub-nanometre resolution without imposing symmetry. Large, potentially force-generating, conformational differences exist between ATP- and ADP-bound NSF. The 20S supercomplex exhibits broken symmetry, transitioning from six-fold symmetry of the NSF ATPase domains to pseudo four-fold symmetry of the SNARE complex. SNAPs interact with the SNARE complex with an opposite structural twist, suggesting an unwinding mechanism. The interfaces between NSF, SNAPs, and SNAREs exhibit characteristic electrostatic patterns, suggesting how one NSF/SNAP species can act on many different SNARE complexes.

224 citations

Journal ArticleDOI
14 Jul 2020-Immunity
TL;DR: The structural and mechanistic insights into the roles of cGAS and STING in immunity and diseases revealed by recent studies are focused on.

217 citations