Oriented cell divisions are controlled by a conserved molecular cascade involving Gαi, LGN, and NuMA. Here, we show that NDP52 regulates spindle orientation via remodeling the polar cortical actin cytoskeleton. siRNA-mediated NDP52 suppression surprisingly revealed a ring-like compact subcortical F-actin architecture surrounding the spindle in prophase/prometaphase cells, which resulted in severe defects of astral microtubule growth and an aberrant spindle orientation. Remarkably, NDP52 recruited the actin assembly factor N-WASP and regulated the dynamics of the subcortical F-actin ring in mitotic cells. Mechanistically, NDP52 was found to bind to phosphatidic acid-containing vesicles, which absorbed cytoplasmic N-WASP to regulate local filamentous actin growth at the polar cortex. Our TIRFM analyses revealed that NDP52-containing vesicles anchored N-WASP and shortened the length of actin filaments in vitro. Based on these results we propose that NDP52-containing vesicles regulate cortical actin dynamics through N-WASP to accomplish a spatiotemporal regulation between astral microtubules and the actin network for proper spindle orientation and precise chromosome segregation. In this way, intracellular vesicles cooperate with microtubules and actin filaments to regulate proper mitotic progression. Since NDP52 is absent from yeast, we reason that metazoans have evolved an elaborate spindle positioning machinery to ensure accurate chromosome segregation in mitosis.

Metabolic coordination between neurons and astrocytes is critical for the health of the brain. However, neuron-astrocyte coupling of lipid metabolism, particularly in response to neural activity, remains largely uncharacterized. Here, we demonstrate that toxic fatty acids (FAs) produced in hyperactive neurons are transferred to astrocytic lipid droplets by ApoE-positive lipid particles. Astrocytes consume the FAs stored in lipid droplets via mitochondrial β-oxidation in response to neuronal activity and turn on a detoxification gene expression program. Our findings reveal that FA metabolism is coupled in neurons and astrocytes to protect neurons from FA toxicity during periods of enhanced activity. This coordinated mechanism for metabolizing FAs could underlie both homeostasis and a variety of disease states of the brain.

How nicotine exposure produces long-lasting changes that remodel neural circuits with addiction is unknown. Here, we report that long-term nicotine exposure alters the trafficking of α4β2-type nicotinic acetylcholine receptors (α4β2Rs) by dispersing and redistributing the Golgi apparatus. In cultured neurons, dispersed Golgi membranes were distributed throughout somata, dendrites and axons. Small, mobile vesicles in dendrites and axons lacked standard Golgi markers and were identified by other Golgi enzymes that modify glycans. Nicotine exposure increased levels of dispersed Golgi membranes, which required α4β2R expression. Similar nicotine-induced changes occurred in vivo at dopaminergic neurons at mouse nucleus accumbens terminals, consistent with these events contributing to nicotine’s addictive effects. Characterization in vitro demonstrated that dispersal was reversible, that dispersed Golgi membranes were functional, and that membranes were heterogenous in size, with smaller vesicles emerging from larger “ministacks”, similar to Golgi dispersal induced by nocadazole. Protocols that increased cultured neuronal synaptic excitability also increased Golgi dispersal, without the requirement of α4β2R expression. Our findings reveal novel activity- and nicotine-dependent changes in neuronal intracellular morphology. These changes regulate levels and location of dispersed Golgi membranes at dendrites and axons, which function in local trafficking at subdomains.

Type I collagen is the main component of bone matrix and other connective tissues. Rerouting of its procollagen precursor to a degradative pathway is crucial for osteoblast survival in pathologies involving excessive intracellular buildup of procollagen that is improperly folded and/or trafficked. What cellular mechanisms underlie this rerouting remains unclear. To study these mechanisms, we employed live-cell imaging and correlative light and electron microscopy (CLEM) to examine procollagen trafficking both in wild-type mouse osteoblasts and osteoblasts expressing a bone pathology-causing mutant procollagen. We found that although most procollagen molecules successfully trafficked through the secretory pathway in these cells, a subpopulation did not. The latter molecules appeared in numerous dispersed puncta colocalizing with COPII subunits, autophagy markers and ubiquitin machinery, with more puncta seen in mutant procollagen-expressing cells. Blocking endoplasmic reticulum exit site (ERES) formation suppressed the number of these puncta, suggesting they formed after procollagen entry into ERESs. The punctate structures containing procollagen, COPII, and autophagic markers did not move toward the Golgi but instead were relatively immobile. They appeared to be quickly engulfed by nearby lysosomes through a bafilomycin-insensitive pathway. CLEM and fluorescence recovery after photobleaching experiments suggested engulfment occurred through a noncanonical form of autophagy resembling microautophagy of ERESs. Overall, our findings reveal that a subset of procollagen molecules is directed toward lysosomal degradation through an autophagic pathway originating at ERESs, providing a mechanism to remove excess procollagen from cells.

Neurons in the developing brain undergo extensive structural refinement as nascent circuits adopt their mature form. This physical transformation of neurons is facilitated by the engulfment and degradation of axonal branches and synapses by surrounding glial cells, including microglia and astrocytes. However, the small size of phagocytic organelles and the complex, highly ramified morphology of glia have made it difficult to define the contribution of these and other glial cell types to this crucial process. Here, we used large-scale, serial section transmission electron microscopy (TEM) with computational volume segmentation to reconstruct the complete 3D morphologies of distinct glial types in the mouse visual cortex, providing unprecedented resolution of their morphology and composition. Unexpectedly, we discovered that the fine processes of oligodendrocyte precursor cells (OPCs), a population of abundant, highly dynamic glial progenitors, frequently surrounded small branches of axons. Numerous phagosomes and phagolysosomes (PLs) containing fragments of axons and vesicular structures were present inside their processes, suggesting that OPCs engage in axon pruning. Single-nucleus RNA sequencing from the developing mouse cortex revealed that OPCs express key phagocytic genes at this stage, as well as neuronal transcripts, consistent with active axon engulfment. Although microglia are thought to be responsible for the majority of synaptic pruning and structural refinement, PLs were ten times more abundant in OPCs than in microglia at this stage, and these structures were markedly less abundant in newly generated oligodendrocytes, suggesting that OPCs contribute substantially to the refinement of neuronal circuits during cortical development.

Receptor tyrosine kinases (RTK) are important cell surface receptors that transduce extracellular signals across the plasma membrane. The traditional view of how these receptors function is that ligand binding to the extracellular domains acts as a master-switch that enables receptor monomers to dimerize and subsequently trans-phosphorylate each other on their intracellular domains. However, a growing body of evidence suggests that receptor oligomerization is not merely a consequence of ligand binding, but is instead part of a complex process responsible for regulation of receptor activation. Importantly, the oligomerization dynamics and subsequent activation of these receptors are affected by other cellular components, such as cytoskeletal machineries and cell membrane lipid characteristics. Thus receptor activation is not an isolated molecular event mediated by the ligand-receptor interaction, but instead involves orchestrated interactions between the receptors and other cellular components. Measuring receptor oligomerization dynamics on live cells can yield important insights into the characteristics of these interactions. Therefore, it is imperative to develop techniques that can probe receptor movements on the plasma membrane with optimal temporal and spatial resolutions. Various microscopic techniques have been used for this purpose. Optical techniques including single molecule tracking (SMT) and fluorescence correlation spectroscopy (FCS) measure receptor diffusion on live cells. Receptor-receptor interactions can also be assessed by detecting Förster resonance energy transfer (FRET) between fluorescently-labeled receptors situated in close proximity or by counting the number of receptors within a diffraction limited fluorescence spot (stepwise bleaching). This review will describe recent developments of optical techniques that have been used to study receptor oligomerization on living cells. This article is part of a Special Issue entitled: Interactions between membrane receptors in cellular membranes edited by Kalina Hristova.

The endoplasmic reticulum (ER) has a complex morphology comprised of stacked sheets, tubules, and three-way junctions, which together function as a platform for protein synthesis of membrane and secretory proteins. Specific ER subdomains are thought to be spatially organized to enable protein synthesis activity, but precisely where these domains are localized is unclear, especially relative to the plethora of organelle interactions taking place on the ER. Here, we use single-molecule tracking of ribosomes and mRNA in combination with simultaneous imaging of ER to assess the sites of membrane protein synthesis on the ER. We found that ribosomes were widely distributed throughout different ER morphologies, but the synthesis of membrane proteins (including Type I, II, and multi-spanning) and an ER luminal protein (Calreticulin) occurred primarily at three-way junctions. Lunapark played a key role in stabilizing transmembrane protein mRNA at three-way junctions. We additionally found that translating mRNAs coding for transmembrane proteins are in the vicinity of lysosomes and translate through a cap-independent but eIF2-dependent mechanism. These results support the idea that discrete ER subdomains co-exist with lysosomes to support specific types of protein synthesis activities, with ER-lysosome interactions playing an important role in the translation of secretome mRNAs.

Neuronal dendrites must relay synaptic inputs over long distances, but the mechanisms by which activity-evoked intracellular signals propagate over macroscopic distances remain unclear. Here, we discovered a system of periodically arranged endoplasmic reticulum-plasma membrane (ER-PM) junctions tiling the plasma membrane of dendrites at \~1 μm intervals, interlinked by a meshwork of ER tubules patterned in a ladder-like array. Populated with Junctophilin-linked plasma membrane voltage-gated Ca2+ channels and ER Ca2+-release channels (ryanodine receptors), ER-PM junctions are hubs for ER-PM crosstalk, fine-tuning of Ca2+ homeostasis, and local activation of the Ca2+/calmodulin-dependent protein kinase II. Local spine stimulation activates the Ca2+ modulatory machinery facilitating voltage-independent signal transmission and ryanodine receptor-dependent Ca2+ release at ER-PM junctions over 20 μm away. Thus, interconnected ER-PM junctions support signal propagation and Ca2+ release from the spine-adjacent ER. The capacity of this subcellular architecture to modify both local and distant membrane-proximal biochemistry potentially contributes to dendritic computations.HighlightsPeriodic ER-PM junctions tile neuronal dendritic plasma membrane in rodent and fly.ER-PM junctions are populated by ER tethering and Ca2+ release and influx machinery.ER-PM junctions act as sites for local activation of CaMKII.Local spine activation drives Ca2+ release from RyRs at ER-PM junctions over 20 μm.

Primary cilia on granule cell neuron progenitors in the developing cerebellum detect sonic hedgehog to facilitate proliferation. Following differentiation, cerebellar granule cells become the most abundant neuronal cell type in the brain. While essential during early developmental stages, the fate of granule cell cilia is unknown. Here, we provide nanoscopic resolution of ciliary dynamics by studying developmental changes in granule cell cilia using large-scale electron microscopy volumes and immunostaining of mouse cerebella. We found that many granule cell primary cilia were intracellular and concealed from the external environment. Cilia were disassembed in differentiating granule cell neurons in a process we call cilia deconstruction that was distinct from pre-mitotic cilia resorption in proliferating progenitors. In differentiating granule cells, ciliary loss involved unique disassembly intermediates, and, as maturation progressed, mother centriolar docking at the plasma membrane. Cilia did not reform from the docked centrioles, rather, in adult mice granule cell neurons remained unciliated. Many neurons in other brain regions require cilia to regulate function and connectivity. In contrast, our results show that granule cell progenitors had concealed cilia that underwent deconstruction potentially to prevent mitogenic hedgehog responsiveness. The ciliary deconstruction mechanism we describe could be paradigmatic of cilia removal during differentiation in other tissues.

The Hippo pathway was originally discovered to control tissue growth in Drosophila and includes the Hippo kinase (Hpo; MST1/2 in mammals), scaffold protein Salvador (Sav; SAV1 in mammals) and the Warts kinase (Wts; LATS1/2 in mammals). The Hpo kinase is activated by binding to Crumbs-Expanded (Crb-Ex) and/or Merlin-Kibra (Mer-Kib) proteins at the apical domain of epithelial cells. Here we show that activation of Hpo also involves the formation of supramolecular complexes with properties of a biomolecular condensate, including concentration dependence and sensitivity to starvation, macromolecular crowding, or 1,6-hexanediol treatment. Overexpressing Ex or Kib induces formation of micron-scale Hpo condensates in the cytoplasm, rather than at the apical membrane. Several Hippo pathway components contain unstructured low-complexity domains and purified Hpo-Sav complexes undergo phase separation in vitro. Formation of Hpo condensates is conserved in human cells. We propose that apical Hpo kinase activation occurs in phase separated "signalosomes" induced by clustering of upstream pathway components.