Spatacsin regulates directionality of lysosome trafficking by promoting the degradation of its partner AP5Z1.

The endoplasmic reticulum (ER) forms contacts with the lysosomal compartment, regulating lysosome positioning and motility. The movements of lysosomes are controlled by the attachment of molecular motors to their surface. However, the molecular mechanisms by which ER controls lysosome dynamics are still elusive. Here, using mouse brain extracts and mouse embryonic fibroblasts, we demonstrate that spatacsin is an ER-resident protein regulating the formation of tubular lysosomes, which are highly dynamic. Screening for spatacsin partners required for tubular lysosome formation showed spatacsin to act by regulating protein degradation. We demonstrate that spatacsin promotes the degradation of its partner AP5Z1, which regulates the relative amount of spastizin and AP5Z1 at lysosomes. Spastizin and AP5Z1 contribute to regulate tubular lysosome formation, as well as their trafficking by interacting with anterograde and retrograde motor proteins, kinesin KIF13A and dynein/dynactin subunit p150Glued, respectively. Ultimately, investigations in polarized mouse cortical neurons in culture demonstrated that spatacsin-regulated degradation of AP5Z1 controls the directionality of lysosomes trafficking. Collectively, our results identify spatacsin as a protein regulating the directionality of lysosome trafficking.

VAMP7 controls T cell activation by regulating the recruitment and phosphorylation of vesicular Lat at TCR-activation sites.

The mechanisms by which Lat (a key adaptor in the T cell antigen receptor (TCR) signaling pathway) and the TCR come together after TCR triggering are not well understood. We investigate here the role of SNARE proteins, which are part of protein complexes involved in the docking, priming and fusion of vesicles with opposing membranes, in this process. Here we found, by silencing approaches and genetically modified mice, that the vesicular SNARE VAMP7 was required for the recruitment of Lat-containing vesicles to TCR-activation sites. Our results indicated that this did not involve fusion of Lat-containing vesicles with the plasma membrane. VAMP7, which localized together with Lat on the subsynaptic vesicles, controlled the phosphorylation of Lat, formation of the TCR-Lat-signaling complex and, ultimately, activation of T cells. Our findings suggest that the transport and docking of Lat-containing vesicles with target membranes containing TCRs regulates TCR-induced signaling.

Targeted Photoconvertible BODIPYs Based on Directed Photooxidation-Induced Conversion for Applications in Photoconversion and Live Super-Resolution Imaging.

Photomodulable fluorescent probes are drawing increasing attention due to their applications in advanced bioimaging. Whereas photoconvertible probes can be advantageously used in tracking, photoswitchable probes constitute key tools for single-molecule localization microscopy to perform super-resolution imaging. Herein, we shed light on a red and far-red BODIPY, namely, BDP-576 and BDP-650, which possess both properties of conversion and switching. Our study demonstrates that these pyrrolyl-BODIPYs convert into typical green- and red-emitting BODIPYs that are perfectly adapted to microscopy. We also showed that this pyrrolyl-BODIPYs undergo Directed Photooxidation Induced Conversion, a photoconversion mechanism that we recently introduced, where the pyrrole moiety plays a central role. These unique features were used to develop targeted photoconvertible probes toward different organelles or subcellular units (plasma membrane, mitochondria, nucleus, actin, Golgi apparatus, etc.) using chemical targeting moieties and a Halo tag. We notably showed that BDP-650 could be used to track intracellular vesicles over more than 20 min in two-color imagings with laser scanning confocal microscopy, demonstrating its robustness. The switching properties of these photoconverters were studied at the single-molecule level and were then successfully used in live single-molecule localization microscopy in epithelial cells and neurons. Both membrane- and mitochondria- targeted probes could be used to decipher membrane 3D architecture and mitochondrial dynamics at the nanoscale. This study builds a bridge between the photoconversion and photoswitching properties of probes undergoing directed photooxidation and shows the versatility and efficacy of this mechanism in advanced live imaging.

Dual-Color Photoconvertible Fluorescent Probes Based on Directed Photooxidation Induced Conversion for Bioimaging.

We herein present a new concept to produce dual-color photoconvertible probes based on a mechanism called Directed Photooxidation Induced Conversion (DPIC). As a support of this mechanism, styryl-coumarins (SCs) bearing Aromatic Singlet Oxygen Reactive Moieties (ASORMs) like furan and pyrrole have been synthesized. SCs are bright fluorophores, which undergo a hypsochromic conversion upon visible light irradiation due to directed photooxidation of the ASORM that leads to the disruption of conjugation. SC-P, a yellow emitting probe bearing a pyrrole moiety, converts to a stable blue emitting coumarin with a 68 nm shift allowing the photoconversion and tracking of lipid droplet in live cells. This new approach might pave the way to a new generation of photoconvertible dyes for advanced bioimaging applications.

Role of the Sec22b-E-Syt complex in neurite growth and ramification.

Axons and dendrites are long and often ramified neurites that need particularly intense plasma membrane (PM) expansion during the development of the nervous system. Neurite growth depends on non-fusogenic Sec22b-Stx1 SNARE complexes at endoplasmic reticulum (ER)-PM contacts. Here, we show that Sec22b interacts with members of the extended synaptotagmin (E-Syt) family of ER lipid transfer proteins (LTPs), and this interaction depends on the longin domain of Sec22b. Overexpression of E-Syts stabilizes Sec22b-Stx1 association, whereas silencing of E-Syts has the opposite effect. Overexpression of wild-type E-Syt2, but not mutants unable to transfer lipids or attach to the ER, increase the formation of axonal filopodia and ramification of neurites in developing neurons. This effect is inhibited by a clostridial neurotoxin cleaving Stx1, and expression of the Sec22b longin domain and a Sec22b mutant with an extended linker between the SNARE and transmembrane domains. We conclude that Sec22b-Stx1 ER-PM contact sites contribute to PM expansion by interacting with LTPs, such as E-Syts.This article has an associated First Person interview with the first author of the paper.

Imaging and Measuring Vesicular Acidification with a Plasma Membrane-Targeted Ratiometric pH Probe.

Tracking the pH variation of intracellular vesicles throughout the endocytosis pathway is of prior importance to better assess the cell trafficking and metabolism of cells. Small molecular fluorescent pH probes are valuable tools in bioimaging but are generally not targeted to intracellular vesicles or are directly targeted to acidic lysosomes, thus not allowing the dynamic observation of the vesicular acidification. Herein, we designed Mem-pH, a fluorogenic ratiometric pH probe based on chromenoquinoline with appealing photophysical properties, which targets the plasma membrane (PM) of cells and further accumulates in the intracellular vesicles by endocytosis. The exposition of Mem-pH toward the vesicle's lumen allowed to monitor the acidification of the vesicles throughout the endocytic pathway and enabled the measurement of their pH via ratiometric imaging.

Molecular Tuning of Styryl Dyes Leads to Versatile and Efficient Plasma Membrane Probes for Cell and Tissue Imaging.

The plasma membrane (PM) plays a major role in many biological processes; therefore, its proper fluorescence staining is required in bioimaging. Among the commercially available PM probes, styryl dye FM1-43 is one of the most widely used. In this work, we demonstrated that fine chemical modifications of FM1-43 can dramatically improve the PM staining. The newly developed probes, SP-468 and SQ-535, were found to display enhanced photophysical properties (reduced cross-talk, higher brightness, improved photostability) and, unlike FM1-43, provided excellent and immediate PM staining in 5 different mammalian cell types including neurons (primary culture and tissue imaging). Taking advantage of these features, we successfully used SP-468 in STED super resolution neuronal imaging. Additionally, we showed that the new probes displayed differences in their internalization pathways compared to their parent FM1-43. Finally, we showed that the new probes kept the ability to stain the PM of plant cells. Overall, this work presents new useful probes for PM imaging in cells and tissues and provides insights on the molecular design of new PM targeting molecules.

Probing Polarity and Heterogeneity of Lipid Droplets in Live Cells Using a Push-Pull Fluorophore.

Lipid droplets (LDs) are organelles composed of a lipid core surrounded by a phospholipid monolayer. Lately, LDs have attracted considerable attention due to recent studies demonstrating their role in a variety of physiological processes as well as diseases. Herein we synthesized a push-pull molecule named DAF (Dimethyl Aniline Furaldehyde) that possesses a strong positive solvatochromism in emission of 119 nm from toluene to methanol. Its impressive fluorogenic properties from water to oil (2000-fold) as well as its high quantum yields (up to 0.97) led us to investigate its ability to sense the distribution of polarity in live cells by fluorescence ratiometric imaging. When added to live cells and excited at 405 nm, DAF immediately and brightly stain lipid droplets using a blue channel (410-500 nm) and cytoplasm in a red channel (500-600 nm). DAF also proved to be compatible with fixation thus allowing 3D imaging of LDs in their cytoplasm environment. Taking advantage of DAF emission in two distinct channels, ratiometric imaging was successfully performed and led to the polarity mapping of the cell unraveling some heterogeneity in polarity within LDs of the same cell.

New insights into the tetraspanin Tspan5 using novel monoclonal antibodies.

Tspan5 is a member of a subgroup of tetraspanins referred to as TspanC8. These tetraspanins directly interact with the metalloprotease ADAM10, regulate its exit from the endoplasmic reticulum and subsequent trafficking, and differentially regulate its ability to cleave various substrates and activate Notch signaling. The study of Tspan5 has been limited by the lack of good antibodies. This study provides new insights into Tspan5 using new monoclonal antibodies (mAbs), including two mAbs recognizing both Tspan5 and the highly similar tetraspanin Tspan17. Using these mAbs, we show that endogenous Tspan5 associates with ADAM10 in human cell lines and in mouse tissues where it is the most abundant, such as the brain, the lung, the kidney, or the intestine. We also uncover two TspanC8-specific motifs in the large extracellular domain of Tspan5 that are important for ADAM10 interaction and exit from the endoplasmic reticulum. One of the anti-Tspan5 mAbs does not recognize Tspan5 associated with ADAM10, providing a convenient way to measure the fraction of Tspan5 not associated with ADAM10. This fraction is minor in the cell lines tested, and it increases upon transfection of cells with TspanC8 tetraspanins such as Tspan15 or Tspan33 that inhibit Notch signaling. Finally, two antibodies inhibit ligand-induced Notch signaling, and this effect is stronger in cells depleted of the TspanC8 tetraspanin Tspan14, further indicating that Tspan5 and Tspan14 can compensate for each other in Notch signaling.

Ultrabright and Fluorogenic Probes for Multicolor Imaging and Tracking of Lipid Droplets in Cells and Tissues.

Lipid droplets (LDs) are intracellular lipid-rich organelles that regulate the storage of neutral lipids and were recently found to be involved in many physiological processes, metabolic disorders, and diseases including obesity, diabetes, and cancers. Herein we present a family of new fluorogenic merocyanine fluorophores based on an indolenine moiety and a dioxaborine barbiturate derivative. These so-called StatoMerocyanines (SMCy) fluoresce from yellow to the near-infrared (NIR) in oil with an impressive fluorescence enhancement compared to aqueous media. Additionally, SMCy display remarkably high molar extinction coefficients (up to 390 000 M-1 cm-1) and high quantum yield values (up to 100%). All the members of this new family specifically stain the LDs in live cells with very low background noise. Unlike Nile Red, a well-known lipid droplet marker, SMCy dyes possess narrow absorption and emission bands in the visible, thus allowing multicolor imaging. SMCy proved to be compatible with fixation and led to high-quality 3D images of lipid droplets in cells and tissues. Their high brightness allowed efficient tissue imaging of adipocytes and circulating LDs. Moreover their remarkably high two-photon absorption cross-section, especially SMCy5.5 (up to 13 300 GM), as well as their capacity to efficiently fluoresce in the NIR region led to two-photon multicolor tissue imaging (liver). Taking advantage of the available color palette, lipid droplet exchange between cells was tracked and imaged, thus demonstrating intercellular communication.

Evidence for altered dendritic spine compartmentalization in Alzheimer's disease and functional effects in a mouse model.

Alzheimer's disease (AD) is associated with a progressive loss of synapses and neurons. Studies in animal models indicate that morphological alterations of dendritic spines precede synapse loss, increasing the proportion of large and short ("stubby") spines. Whether similar alterations occur in human patients, and what their functional consequences could be, is not known. We analyzed biopsies from AD patients and APP x presenilin 1 knock-in mice that were previously shown to present a loss of pyramidal neurons in the CA1 area of the hippocampus. We observed that the proportion of stubby spines and the width of spine necks are inversely correlated with synapse density in frontal cortical biopsies from non-AD and AD patients. In mice, the reduction in the density of synapses in the stratum radiatum was preceded by an alteration of spine morphology, with a reduction of their length and an enlargement of their neck. Serial sectioning examined with electron microscopy allowed us to precisely measure spine parameters. Mathematical modeling indicated that the shortening and widening of the necks should alter the electrical compartmentalization of the spines, leading to reduced postsynaptic potentials in spine heads, but not in soma. Accordingly, there was no alteration in basal synaptic transmission, but long-term potentiation and spatial memory were impaired. These results indicate that an alteration of spine morphology could be involved in the early cognitive deficits associated with AD.

VAMP-7 links granule exocytosis to actin reorganization during platelet activation.

Platelet activation results in profound morphologic changes accompanied by release of granule contents. Recent evidence indicates that fusion of granules with the plasma membrane during activation provides auxiliary membrane to cover growing actin structures. Yet little is known about how membrane fusion is coupled with actin reorganization. Vesicle-associated membrane protein (VAMP)-7 is found on platelet vesicles and possesses an N-terminal longin domain capable of linking exocytosis to cytoskeletal remodeling. We have evaluated platelets from VAMP-7(-/-) mice to determine whether this VAMP isoform contributes to granule release and platelet spreading. VAMP-7(-/-) platelets demonstrated a partial defect in dense granule exocytosis and impaired aggregation. α Granule exocytosis from VAMP-7(-/-) platelets was diminished both in vitro and in vivo during thrombus formation. Consistent with a role of VAMP-7 in cytoskeletal remodeling, spreading on matrices was decreased in VAMP-7(-/-) platelets compared to wild-type controls. Immunoprecipitation of VAMP-7 revealed an association with VPS9-domain ankyrin repeat protein (VARP), an adaptor protein that interacts with both membrane-bound and cytoskeleton proteins and with Arp2/3. VAMP-7, VARP, and Arp2/3 localized to the platelet periphery during spreading. These studies demonstrate that VAMP-7 participates in both platelet granule secretion and spreading and suggest a mechanism whereby VAMP-7 links granule exocytosis with actin reorganization.

Statistical analysis of molecule colocalization in bioimaging.

The quantitative analysis of molecule interactions in bioimaging is key for understanding the molecular orchestration of cellular processes and is generally achieved through the study of the spatial colocalization between the different populations of molecules. Colocalization methods are traditionally divided into pixel-based methods that measure global correlation coefficients from the overlap between pixel intensities in different color channels, and object-based methods that first segment molecule spots and then analyze their spatial distributions with second-order statistics. Here, we present a review of such colocalization methods and give a quantitative comparison of their relative merits in different types of biological applications and contexts. We show on synthetic and biological images that object-based methods are more robust statistically than pixel-based methods, and allow moreover to quantify accurately the number of colocalized molecules.

Molecular mapping of neuronal architecture using STORM microscopy and new fluorescent probes for SMLM imaging.

Imaging neuronal architecture has been a recurrent challenge over the years, and the localization of synaptic proteins is a frequent challenge in neuroscience. To quantitatively detect and analyze the structure of synapses, we recently developed free SODA software to detect the association of pre and postsynaptic proteins. To fully take advantage of spatial distribution analysis in complex cells, such as neurons, we also selected some new dyes for plasma membrane labeling. Using Icy SODA plugin, we could detect and analyze synaptic association in both conventional and single molecule localization microscopy, giving access to a molecular map at the nanoscale level. To replace those molecular distributions within the neuronal three-dimensional (3D) shape, we used MemBright probes and 3D STORM analysis to decipher the entire 3D shape of various dendritic spine types at the single-molecule resolution level. We report here the example of synaptic proteins within neuronal mask, but these tools have a broader spectrum of interest since they can be used whatever the proteins or the cellular type. Altogether with SODA plugin, MemBright probes thus provide the perfect toolkit to decipher a nanometric molecular map of proteins within a 3D cellular context.

Spaced training activates Miro/Milton-dependent mitochondrial dynamics in neuronal axons to sustain long-term memory.

Neurons have differential and fluctuating energy needs across distinct cellular compartments, shaped by brain electrochemical activity associated with cognition. In vitro studies show that mitochondria transport from soma to axons is key to maintaining neuronal energy homeostasis. Nevertheless, whether the spatial distribution of neuronal mitochondria is dynamically adjusted in vivo in an experience-dependent manner remains unknown. In Drosophila, associative long-term memory (LTM) formation is initiated by an early and persistent upregulation of mitochondrial pyruvate flux in the axonal compartment of neurons in the mushroom body (MB). Through behavior experiments, super-resolution analysis of mitochondria morphology in the neuronal soma and in vivo mitochondrial fluorescence recovery after photobleaching (FRAP) measurements in the axons, we show that LTM induction, contrary to shorter-lived memories, is sustained by the departure of some mitochondria from MB neuronal soma and increased mitochondrial dynamics in the axonal compartment. Accordingly, impairing mitochondrial dynamics abolished the increased pyruvate consumption, specifically after spaced training and in the MB axonal compartment, thereby preventing LTM formation. Our results thus promote reorganization of the mitochondrial network in neurons as an integral step in elaborating high-order cognitive processes.

A polarized cell system amenable to subcellular resolution imaging of influenza virus infection.

The life cycle of influenza A viruses (IAV), and notably intracellular trafficking of the viral genome, depends on multiple interactions with the cellular cytoskeleton and endomembrane system. A limitation of the conventional cellular models used for mechanistic study and subcellular imaging of IAV infection is that they are cultured in two dimensions (2D) under non-polarizing conditions, and therefore they do not recapitulate the intracellular organization of the polarized respiratory epithelial cells naturally targeted by IAVs. To overcome this limitation, we developed an IAV-infection assay in a 3D cell culture system which allows imaging along the baso-lateral axis of polarized cells, with subcellular resolution. Here we describe a protocol to grow polarized monolayers of Caco2-TC7 cells on static Cytodex-3 microcarrier beads, infect them with IAV, and subsequently perform immunostaining and confocal imaging, or electron microscopy, on polarized IAV-infected cells. This method can be extended to other pathogens that infect human polarized epithelial cells.

A modular framework for multi-scale tissue imaging and neuronal segmentation.

The development of robust tools for segmenting cellular and sub-cellular neuronal structures lags behind the massive production of high-resolution 3D images of neurons in brain tissue. The challenges are principally related to high neuronal density and low signal-to-noise characteristics in thick samples, as well as the heterogeneity of data acquired with different imaging methods. To address this issue, we design a framework which includes sample preparation for high resolution imaging and image analysis. Specifically, we set up a method for labeling thick samples and develop SENPAI, a scalable algorithm for segmenting neurons at cellular and sub-cellular scales in conventional and super-resolution STimulated Emission Depletion (STED) microscopy images of brain tissues. Further, we propose a validation paradigm for testing segmentation performance when a manual ground-truth may not exhaustively describe neuronal arborization. We show that SENPAI provides accurate multi-scale segmentation, from entire neurons down to spines, outperforming state-of-the-art tools. The framework will empower image processing of complex neuronal circuitries.

Protein aggregates containing wild-type and mutant NOTCH3 are major drivers of arterial pathology in CADASIL.

Loss of arterial smooth muscle cells (SMCs) and abnormal accumulation of the extracellular domain of the NOTCH3 receptor (Notch3ECD) are the 2 core features of CADASIL, a common cerebral small vessel disease caused by highly stereotyped dominant mutations in NOTCH3. Yet the relationship between NOTCH3 receptor activity, Notch3ECD accumulation, and arterial SMC loss has remained elusive, hampering the development of disease-modifying therapies. Using dedicated histopathological and multiscale imaging modalities, we could detect and quantify previously undetectable CADASIL-driven arterial SMC loss in the CNS of mice expressing the archetypal Arg169Cys mutation. We found that arterial pathology was more severe and Notch3ECD accumulation greater in transgenic mice overexpressing the mutation on a wild-type Notch3 background (TgNotch3R169C) than in knockin Notch3R170C/R170C mice expressing this mutation without a wild-type Notch3 copy. Notably, expression of Notch3-regulated genes was essentially unchanged in TgNotch3R169C arteries. We further showed that wild-type Notch3ECD coaggregated with mutant Notch3ECD and that elimination of 1 copy of wild-type Notch3 in TgNotch3R169C was sufficient to attenuate Notch3ECD accumulation and arterial pathology. These findings suggest that Notch3ECD accumulation, involving mutant and wild-type NOTCH3, is a major driver of arterial SMC loss in CADASIL, paving the way for NOTCH3-lowering therapeutic strategies.

Understanding the nervous system: lessons from Frontiers in Neurophotonics.

The Frontiers in Neurophotonics Symposium is a biennial event that brings together neurobiologists and physicists/engineers who share interest in the development of leading-edge photonics-based approaches to understand and manipulate the nervous system, from its individual molecular components to complex networks in the intact brain. In this Community paper, we highlight several topics that have been featured at the symposium that took place in October 2022 in Québec City, Canada.

Glutamate controls tPA recycling by astrocytes, which in turn influences glutamatergic signals.

Tissue-type plasminogen activator (tPA) regulates physiological processes in the brain, such as learning and memory, and plays a critical role in neuronal survival and neuroinflammation in pathological conditions. Here we demonstrate, by combining mouse in vitro and in vivo data, that tPA is an important element of the cross talk between neurons and astrocytes. The data show that tPA released by neurons is constitutively endocytosed by astrocytes via the low-density lipoprotein-related protein receptor, and is then exocytosed in a regulated manner. The exocytotic recycling of tPA by astrocytes is inhibited in the presence of extracellular glutamate. Kainate receptors of astrocytes act as sensors of extracellular glutamate and, via a signaling pathway involving protein kinase C, modulate the exocytosis of tPA. Further, by thus capturing extracellular tPA, astrocytes serve to reduce NMDA-mediated responses potentiated by tPA. Overall, this work provides the first demonstration that the neuromodulator, tPA, may also be considered as a gliotransmitter.