Although intracellular Ca
2+
signals of oligodendroglia, the myelin-forming cells of the central nervous system, regulate vital cellular processes including myelination, few studies on ...oligodendroglia Ca
2+
signal dynamics have been carried out and existing software solutions are not adapted to the analysis of the complex Ca
2+
signal characteristics of these cells. Here, we provide a comprehensive solution to analyze oligodendroglia Ca
2+
imaging data at the population and single-cell levels. We describe a new analytical pipeline containing two free, open source and cross-platform software programs, Occam and post-prOccam, that enable the fully automated analysis of one- and two-photon Ca
2+
imaging datasets from oligodendroglia obtained by either ex vivo or in vivo Ca
2+
imaging techniques. Easily configurable, our software solution is optimized to obtain unbiased results from large datasets acquired with different imaging techniques. Compared to other recent software, our solution proved to be fast, low memory demanding and faithful in the analysis of oligodendroglial Ca
2+
signals in all tested imaging conditions. Our versatile and accessible Ca
2+
imaging data analysis tool will facilitate the elucidation of Ca
2+
-mediated mechanisms in oligodendroglia. Its configurability should also ensure its suitability with new use cases such as other glial cell types or even cells outside the CNS.
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.
Display omitted
•Miro-Milton-dependent mitochondria dynamics in mushroom body neurons sustain LTM•Small mitochondria are partially depleted from neuronal soma upon LTM formation•At the same time, mitochondria dynamics are increased in axons•This increase sustains LTM through axonal upregulation of mitochondria metabolism
Pavlowsky et al. investigate in vivo the role of mitochondria dynamics in associative olfactory memory. They demonstrate that Miro-Milton mitochondria dynamics are required to sustain the upregulation of axonal mitochondria metabolism specifically needed for long-term memory formation.
In vivo and in vitro assays, particularly reconstitution using artificial membranes, have established the role of synaptic soluble N-Ethylmaleimide–sensitive attachment protein receptors (SNAREs) ...VAMP2, Syntaxin-1A, and SNAP-25 in membrane fusion. However, using artificial membranes requires challenging protein purifications that could be avoided in a cell-based assay. Here, we developed a synthetic biological approach based on the generation of membrane cisternae by the integral membrane protein Caveolin in Escherichia coli and coexpression of SNAREs. Syntaxin-1A/SNAP-25/VAMP-2 complexes were formed and regulated by SNARE partner protein Munc-18a in the presence of Caveolin. Additionally, Syntaxin-1A/SNAP-25/VAMP-2 synthesis provoked increased length of E. coli only in the presence of Caveolin. We found that cell elongation required SNAP-25 and was inhibited by tetanus neurotoxin. This elongation was not a result of cell division arrest. Furthermore, electron and super-resolution microscopies showed that synaptic SNAREs and Caveolin coexpression led to the partial loss of the cisternae, suggesting their fusion with the plasma membrane. In summary, we propose that this assay reconstitutes membrane fusion in a simple organism with an easy-to-observe phenotype and is amenable to structure-function studies of SNAREs.
Activation of the JAK-STAT pathway by type I interferons (IFNs) requires clathrin-dependent endocytosis of the IFN-α and -β receptor (IFNAR), indicating a role for endosomal sorting in this process. ...The molecular machinery that brings the selective activation of IFN-α/β-induced JAK-STAT signalling on endosomes remains unknown. Here we show that the constitutive association of STAM with IFNAR1 and TYK2 kinase at the plasma membrane prevents TYK2 activation by type I IFNs. IFN-α-stimulated IFNAR endocytosis delivers the STAM-IFNAR complex to early endosomes where it interacts with Hrs, thereby relieving TYK2 inhibition by STAM and triggering signalling of IFNAR at the endosome. In contrast, when stimulated by IFN-β, IFNAR signalling occurs independently of Hrs as IFNAR is sorted to a distinct endosomal subdomain. Our results identify the molecular machinery that controls the spatiotemporal activation of IFNAR by IFN-α and establish the central role of endosomal sorting in the differential regulation of JAK-STAT signalling by IFN-α and IFN-β.
VAMP7 is involved in autophagy and in exocytosis-mediated neurite growth, two yet unconnected cellular pathways. Here, we find that nutrient restriction and activation of autophagy stimulate axonal ...growth, while autophagy inhibition leads to loss of neuronal polarity. VAMP7 knockout (KO) neuronal cells show impaired neurite growth, whereas this process is increased in autophagy-null ATG5 KO cells. We find that endoplasmic reticulum (ER)-phagy-related LC3-interacting-region-containing proteins Atlastin 3 and Reticulon 3 (RTN3) are more abundant in autophagy-related protein ATG5 KO and less abundant in VAMP7 KO secretomes. Treatment of neuronal cells with ATG5 or VAMP7 KO conditioned medium does not recapitulate the effect of these KOs on neurite growth. A nanobody directed against VAMP7 inhibits axonal overgrowth induced by nutrient restriction. Furthermore, expression of the inhibitory Longin domain of VAMP7 impairs the subcellular localization of RTN3 in neurons. We propose that VAMP7-dependent secretion of RTN3 regulates neurite growth.
Display omitted
•Nutrient restriction and autophagy regulate axonal growth and neuronal polarity•VAMP7 mediates nutrient restriction/autophagy-dependent neurite growth•VAMP7 mediates the secretion of ER-phagy factor Reticulon 3
VAMP7 is involved in exocytosis-mediated neurite growth and degradative autophagy. Using secretomics, Wojnacki et al. show that VAMP7 mediates the release of tubular ER-phagy receptor Reticulon 3, a secretory pathway greatly enhanced in autophagy-null neuronal cells. ER-phagy and unconventional secretion regulate neurite growth and polarization during nutrient deprivation.
In this paper, we explore the 3D structure of light scattering from dark-field illuminated live 3T3 cells in the presence of 40 nm gold nanomarkers. For this purpose, we use a high resolution ...holographic microscope combining the off-axis heterodyne geometry and the phase-shifting acquisition of the digital holograms. A comparative study of the 3D reconstructions of the scattered fields allows us to locate the gold markers which yield, contrarily to the cell structures, well defined bright scattering patterns that are not angularly titled and clearly located along the optical axis (z). This characterization is an unambiguous signature of the presence of gold biological nanomarkers, and validates the capability of digital holographic microscopy to discriminate them from background signals in live cells.
Cell polarization is a fundamental biological process implicated in nearly every aspect of multicellular development. The role of cell-extracellular matrix contacts in the establishment and the ...orientation of cell polarity have been extensively studied. However, the respective contributions of substrate mechanics and biochemistry remain unclear. Here we propose a believed novel single-cell approach to assess the minimal polarization trigger. Using nonadhered round fibroblast cells, we show that stiffness sensing through single localized integrin-mediated cues are necessary and sufficient to trigger and direct a shape polarization. In addition, the traction force developed by cells has to reach a minimal threshold of 56 ± 1.6 pN for persistent polarization. The polarization kinetics increases with the stiffness of the cue. The polarized state is characterized by cortical actomyosin redistribution together with cell shape change. We develop a physical model supporting the idea that a local and persistent inhibition of actin polymerization and/or myosin activity is sufficient to trigger and sustain the polarized state. Finally, the cortical polarity propagates to an intracellular polarity, evidenced by the reorientation of the centrosome. Our results define the minimal adhesive requirements and quantify the mechanical checkpoint for persistent cell shape and organelle polarization, which are critical regulators of tissue and cell development.
PDF
