Ultra-High Resolution 3D Imaging of Whole Cells.

Fluorescence nanoscopy, or super-resolution microscopy, has become an important tool in cell biological research. However, because of its usually inferior resolution in the depth direction (50-80 nm) and rapidly deteriorating resolution in thick samples, its practical biological application has been effectively limited to two dimensions and thin samples. Here, we present the development of whole-cell 4Pi single-molecule switching nanoscopy (W-4PiSMSN), an optical nanoscope that allows imaging of three-dimensional (3D) structures at 10- to 20-nm resolution throughout entire mammalian cells. We demonstrate the wide applicability of W-4PiSMSN across diverse research fields by imaging complex molecular architectures ranging from bacteriophages to nuclear pores, cilia, and synaptonemal complexes in large 3D cellular volumes.

An open-access volume electron microscopy atlas of whole cells and tissues.

Understanding cellular architecture is essential for understanding biology. Electron microscopy (EM) uniquely visualizes cellular structures with nanometre resolution. However, traditional methods, such as thin-section EM or EM tomography, have limitations in that they visualize only a single slice or a relatively small volume of the cell, respectively. Focused ion beam-scanning electron microscopy (FIB-SEM) has demonstrated the ability to image small volumes of cellular samples with 4-nm isotropic voxels1. Owing to advances in the precision and stability of FIB milling, together with enhanced signal detection and faster SEM scanning, we have increased the volume that can be imaged with 4-nm voxels by two orders of magnitude. Here we present a volume EM atlas at such resolution comprising ten three-dimensional datasets for whole cells and tissues, including cancer cells, immune cells, mouse pancreatic islets and Drosophila neural tissues. These open access data (via OpenOrganelle2) represent the foundation of a field of high-resolution whole-cell volume EM and subsequent analyses, and we invite researchers to explore this atlas and pose questions.

Control of organelle size: the Golgi complex.

The Golgi complex processes secretory proteins and lipids, carries out protein sorting and signaling, and supports growth and composition of the plasma membrane. Golgi complex size likely is regulated to meet the demands of each function, and this may involve differential changes of its distinct subdomains. Nevertheless, the primary size change is elongation of the Golgi ribbon-like network as occurs during Golgi complex doubling for mitosis and during differentiation involving upregulated secretion. One hypothesis states that Golgi complex size is set by the abundance of secretory cargo and Golgi complex components that, through binding vesicle coat complexes, drive vesicle coat formation to alter Golgi complex influx and efflux. Regulation of transport factors controlling Golgi membrane traffic is also observed and may control Golgi complex size, but more work is needed to directly link these events to Golgi complex size regulation, especially during differentiation of specialized cell types.

The role of Atg proteins in autophagosome formation.

Macroautophagy is mediated by a unique organelle, the autophagosome, which encloses a portion of cytoplasm for delivery to the lysosome. Autophagosome formation is dynamically regulated by starvation and other stresses and involves complicated membrane reorganization. Since the discovery of yeast Atg-related proteins, autophagosome formation has been dissected at the molecular level. In this review we describe the molecular mechanism of autophagosome formation with particular focus on the function of Atg proteins and the long-standing discussion regarding the origin of the autophagosome membrane.

Upregulation of the secretory pathway Ca2+/Mn2+-ATPase isoform 1 in LPS-stimulated microglia and its involvement in Mn2+-induced Golgi fragmentation.

Microglia play an important protective role in the healthy nervous tissue, being able to react to a variety of stimuli that induce different intracellular cascades for specific tasks. Ca2+ signaling can modulate these pathways, and we recently reported that microglial functions depend on the endoplasmic reticulum as a Ca2+ store, which involves the Ca2+ transporter SERCA2b. Here, we investigated whether microglial functions may also rely on the Golgi, another intracellular Ca2+ store that depends on the secretory pathway Ca2+/Mn2+-transport ATPase isoform 1 (SPCA1). We found upregulation of SPCA1 upon lipopolysaccharide stimulation of microglia BV2 cells and primary microglia, where alterations of the Golgi ribbon were also observed. Silencing and overexpression experiments revealed that SPCA1 affects cell morphology, Golgi apparatus integrity, and phagocytic functions. Since SPCA1 is also an efficient Mn2+ transporter and considering that Mn2+ excess causes manganism in the brain, we addressed the role of microglial SPCA1 in Mn2+ toxicity. Our results revealed a clear effect of Mn2+ excess on the viability and morphology of microglia. Subcellular analysis showed Golgi fragmentation and subsequent alteration of SPCA1 distribution from early stages of toxicity. Removal of Mn2+ by washing improved the culture viability, although it did not effectively reverse Golgi fragmentation. Interestingly, pretreatment with curcumin maintained microglia cultures viable, prevented Mn2+-induced Golgi fragmentation, and preserved SPCA Ca2+-dependent activity, suggesting curcumin as a potential protective agent against Mn2+-induced Golgi alterations in microglia.

Cascade Delivery to Golgi Apparatus and On-Site Formation of Subcellular Drug Reservoir for Cancer Metastasis Suppression.

As the foremost cause of cancer-related death, metastasis consists of three steps: invasion, circulation, and colonization. Only targeting one single phase of the metastasis cascade may be insufficient since there are many alternative routes for tumor cells to disseminate. Here, to target the whole cascade of metastasis, hybrid erythrocyte and tumor cell membrane-coated nanoparticle (Hyb-NP) is designed with dual functions of increasing circulation time and recognizing primary, circulating, and colonized tumors. After loading with monensin, a recently reported metastasis inhibitor, the delivery system profoundly reduces spontaneous metastasis in an orthotopic breast cancer model. Underlying mechanism studies reveal that Hyb-NP can deliver monensin to its action site in the Golgi apparatus, and in return, monensin can block the exocytosis of Hyb-NP from the Golgi apparatus, forming a reservoir-like subcellular structure. Notably, the Golgi apparatus reservoir displays three vital functions for suppressing metastasis initialization, including enhanced subcellular drug retention, metastasis-related cytokine release inhibition, and directional migration inhibition. Collectively, based on metastasis cascade targeting at the tissue level, further formation of the Golgi apparatus drug reservoir at the subcellular level provides a potential therapeutic strategy for cancer metastasis suppression.

GPA5 Encodes a Rab5a Effector Required for Post-Golgi Trafficking of Rice Storage Proteins.

Dense vesicles (DVs) are vesicular carriers, unique to plants, that mediate post-Golgi trafficking of storage proteins to protein storage vacuoles (PSVs) in seeds. However, the molecular mechanisms regulating the directional targeting of DVs to PSVs remain elusive. Here, we show that the rice (Oryza sativa) glutelin precursor accumulation5 (gpa5) mutant is defective in directional targeting of DVs to PSVs, resulting in discharge of its cargo proteins into the extracellular space. Molecular cloning revealed that GPA5 encodes a plant-unique phox-homology domain-containing protein homologous to Arabidopsis (Arabidopsis thaliana) ENDOSOMAL RAB EFFECTOR WITH PX-DOMAIN. We show that GPA5 is a membrane-associated protein capable of forming homodimers and that it is specifically localized to DVs in developing endosperm. Colocalization, biochemical, and genetic evidence demonstrates that GPA5 acts in concert with Rab5a and VPS9a to regulate DV-mediated post-Golgi trafficking to PSVs. Furthermore, we demonstrated that GPA5 physically interacts with a class C core vacuole/endosome tethering complex and a seed plant-specific VAMP727-containing R-soluble N-ethylmaleimide sensitive factor attachment protein receptor complex. Collectively, our results suggest that GPA5 functions as a plant-specific effector of Rab5a required for mediating tethering and membrane fusion of DVs with PSVs in rice endosperm.

Transport through the Golgi apparatus by rapid partitioning within a two-phase membrane system.

The prevailing view of intra-Golgi transport is cisternal progression, which has a key prediction--that newly arrived cargo exhibits a lag or transit time before exiting the Golgi. Instead, we find that cargo molecules exit at an exponential rate proportional to their total Golgi abundance with no lag. Incoming cargo molecules rapidly mix with those already in the system and exit from partitioned domains with no cargo privileged for export based on its time of entry into the system. Given these results, we constructed a new model of intra-Golgi transport that involves rapid partitioning of enzymes and transmembrane cargo between two lipid phases combined with relatively rapid exchange among cisternae. Simulation and experimental testing of this rapid partitioning model reproduced all the key characteristics of the Golgi apparatus, including polarized lipid and protein gradients, exponential cargo export kinetics, and cargo waves.

Peptidylglycine α-amidating monooxygenase is required for atrial secretory granule formation.

The discovery of atrial secretory granules and the natriuretic peptides stored in them identified the atrium as an endocrine organ. Although neither atrial nor brain natriuretic peptide (ANP, BNP) is amidated, the major membrane protein in atrial granules is peptidylglycine α-amidating monooxygenase (PAM), an enzyme essential for amidated peptide biosynthesis. Mice lacking cardiomyocyte PAM (PamMyh6-cKO/cKO) are viable, but a gene dosage-dependent drop in atrial ANP and BNP content occurred. Ultrastructural analysis of adult PamMyh6-cKO/cKO atria revealed a 13-fold drop in the number of secretory granules. When primary cultures of Pam0-Cre-cKO/cKO atrial myocytes (no Cre recombinase, PAM floxed) were transduced with Cre-GFP lentivirus, PAM protein levels dropped, followed by a decline in ANP precursor (proANP) levels. Expression of exogenous PAM in PamMyh6-cKO/cKO atrial myocytes produced a dose-dependent rescue of proANP content; strikingly, this response did not require the monooxygenase activity of PAM. Unlike many prohormones, atrial proANP is stored intact. A threefold increase in the basal rate of proANP secretion by PamMyh6-cKO/cKO myocytes was a major contributor to its reduced levels. While proANP secretion was increased following treatment of control cultures with drugs that block the activation of Golgi-localized Arf proteins and COPI vesicle formation, proANP secretion by PamMyh6-cKO/cKO myocytes was unaffected. In cells lacking secretory granules, expression of exogenous PAM led to the accumulation of fluorescently tagged proANP in the cis-Golgi region. Our data indicate that COPI vesicle-mediated recycling of PAM from the cis-Golgi to the endoplasmic reticulum plays an essential role in the biogenesis of proANP containing atrial granules.

Disorder of Golgi Apparatus Precedes Anoxia-Induced Pathology of Mitochondria.

Mitochondrial malfunction and morphologic disorganization have been observed in brain cells as part of complex pathological changes. However, it is unclear what may be the role of mitochondria in the initiation of pathologic processes or if mitochondrial disorders are consequences of earlier events. We analyzed the morphologic reorganization of organelles in an embryonic mouse brain during acute anoxia using an immunohistochemical identification of the disordered mitochondria, followed by electron microscopic three-dimensional (3D) reconstruction. We found swelling of the mitochondrial matrix after 3 h anoxia and probable dissociation of mitochondrial stomatin-like protein 2 (SLP2)-containing complexes after 4.5 h anoxia in the neocortex, hippocampus, and lateral ganglionic eminence. Surprisingly, deformation of the Golgi apparatus (GA) was detected already after 1 h of anoxia, when the mitochondria and other organelles still had a normal ultrastructure. The disordered GA showed concentrical swirling of the cisternae and formed spherical onion-like structures with the trans-cisterna in the center of the sphere. Such disturbance of the Golgi architecture likely interferes with its function for post-translational protein modification and secretory trafficking. Thus, the GA in embryonic mouse brain cells may be more vulnerable to anoxic conditions than the other organelles, including mitochondria.

Ultrastructural Reorganization of Endotheliocytes of Pulmonary Blood Capillaries in COVID-19.

The ultrastructural organization endotheliocytes of pulmonary blood capillaries in COVID-19 was studied on autopsy material using electron microscopy. Swelling of the cytoplasm and mitochondria with destruction of the cristae, dilation of the Golgi complex cisternae, a decrease in the volume density of the luminal and basal caveolae and free transport vesicles, an increase of the rough endoplasmic reticulum, as well as the presence of elements of coronavirus replication (reticulovesicular structures, zippered endoplasmic reticulum, electron-dense particles in the Golgi cisternae, and vacuoles with viral particles) were revealed. Further studies of the intracellular mechanisms used by the virus to replicate could help to develop antiviral drugs for the treatment of the new coronavirus infection.

A fluorescent reporter system enables spatiotemporal analysis of host cell modification during herpes simplex virus-1 replication.

Herpesviruses are large and complex viruses that have a long history of coevolution with their host species. One important factor in the virus-host interaction is the alteration of intracellular morphology during viral replication with critical implications for viral assembly. However, the details of this remodeling event are not well understood, in part because insufficient tools are available to deconstruct this highly heterogeneous process. To provide an accurate and reliable method of investigating the spatiotemporal dynamics of virus-induced changes to cellular architecture, we constructed a dual-fluorescent reporter virus that enabled us to classify four distinct stages in the infection cycle of herpes simplex virus-1 at the single cell level. This timestamping method can accurately track the infection cycle across a wide range of multiplicities of infection. We used high-resolution fluorescence microscopy analysis of cellular structures in live and fixed cells in concert with our reporter virus to generate a detailed and chronological overview of the spatial and temporal reorganization during viral replication. The highly orchestrated and striking relocation of many organelles around the compartments of secondary envelopment during transition from early to late gene expression suggests that the reshaping of these compartments is essential for virus assembly. We furthermore find that accumulation of HSV-1 capsids in the cytoplasm is accompanied by fragmentation of the Golgi apparatus with potential impact on the late steps of viral assembly. We anticipate that in the future similar tools can be systematically applied for the systems-level analysis of intracellular morphology during replication of other viruses.

The SARS-CoV-2 envelope and membrane proteins modulate maturation and retention of the spike protein, allowing assembly of virus-like particles.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a ß-coronavirus, is the causative agent of the COVID-19 pandemic. Like for other coronaviruses, its particles are composed of four structural proteins: spike (S), envelope (E), membrane (M), and nucleoprotein (N) proteins. The involvement of each of these proteins and their interactions are critical for assembly and production of ß-coronavirus particles. Here, we sought to characterize the interplay of SARS-CoV-2 structural proteins during the viral assembly process. By combining biochemical and imaging assays in infected versus transfected cells, we show that E and M regulate intracellular trafficking of S as well as its intracellular processing. Indeed, the imaging data reveal that S is relocalized at endoplasmic reticulum (ER)-Golgi intermediate compartment (ERGIC) or Golgi compartments upon coexpression of E or M, as observed in SARS-CoV-2-infected cells, which prevents syncytia formation. We show that a C-terminal retrieval motif in the cytoplasmic tail of S is required for its M-mediated retention in the ERGIC, whereas E induces S retention by modulating the cell secretory pathway. We also highlight that E and M induce a specific maturation of N-glycosylation of S, independently of the regulation of its localization, with a profile that is observed both in infected cells and in purified viral particles. Finally, we show that E, M, and N are required for optimal production of virus-like-particles. Altogether, these results highlight how E and M proteins may influence the properties of S proteins and promote the assembly of SARS-CoV-2 viral particles.

A Self-Assembling Probe for Imaging the States of Golgi Apparatus in Live Single Cells.

Despite the enormous progress in genomics and proteomics, it is still challenging to assess the states of organelles in living cells with high spatiotemporal resolution. Based on our recent finding of enzyme-instructed self-assembly of a thiophosphopeptide that targets the Golgi Apparatus (GA) instantly, we use the thiophosphopeptide, which is enzymatically responsive and redox active, as an integrative probe for revealing the state of the GA of live cells at the single cell level. By imaging the probe in the GA of live cells over time, our results show that the accumulation of the probe at the GA depends on cell types. By comparison to a conventional Golgi probe, this self-assembling probe accumulates at the GA much faster and are sensitive to the expression of alkaline phosphatases. In addition, subtle changes of the fluorophore results in slightly different GA responses. This work illustrates a novel class of active molecular probes that combine enzyme-instructed self-assembly and redox reaction for high-resolution imaging of the states of subcellular organelles over a large area and extended times.

Separating Golgi Proteins from Cis to Trans Reveals Underlying Properties of Cisternal Localization.

The order of enzymatic activity across Golgi cisternae is essential for complex molecule biosynthesis. However, an inability to separate Golgi cisternae has meant that the cisternal distribution of most resident proteins, and their underlying localization mechanisms, are unknown. Here, we exploit differences in surface charge of intact cisternae to perform separation of early to late Golgi subcompartments. We determine protein and glycan abundance profiles across the Golgi; over 390 resident proteins are identified, including 136 new additions, with over 180 cisternal assignments. These assignments provide a means to better understand the functional roles of Golgi proteins and how they operate sequentially. Protein and glycan distributions are validated in vivo using high-resolution microscopy. Results reveal distinct functional compartmentalization among resident Golgi proteins. Analysis of transmembrane proteins shows several sequence-based characteristics relating to pI, hydrophobicity, Ser abundance, and Phe bilayer asymmetry that change across the Golgi. Overall, our results suggest that a continuum of transmembrane features, rather than discrete rules, guide proteins to earlier or later locations within the Golgi stack.

Arabinosyl Deacetylase Modulates the Arabinoxylan Acetylation Profile and Secondary Wall Formation.

Acetylation, a prevalent modification of cell-wall polymers, is a tightly controlled regulatory process that orchestrates plant growth and environmental adaptation. However, due to limited characterization of the enzymes involved, it is unclear how plants establish and dynamically regulate the acetylation pattern in response to growth requirements. In this study, we identified a rice (Oryza sativa) GDSL esterase that deacetylates the side chain of the major rice hemicellulose, arabinoxylan. Acetyl esterases involved in arabinoxylan modification were screened using enzymatic assays combined with mass spectrometry analysis. One candidate, DEACETYLASE ON ARABINOSYL SIDECHAIN OF XYLAN1 (DARX1), is specific for arabinosyl residues. Disruption of DARX1 via Tos17 insertion and CRISPR/Cas9 approaches resulted in the accumulation of acetates on the xylan arabinosyl side chains. Recombinant DARX1 abolished the excess acetyl groups on arabinoxylan-derived oligosaccharides of the darx1 mutants in vitro. Moreover, DARX1 is localized to the Golgi apparatus. Two-dimensional 13C-13C correlation spectroscopy and atomic force microscopy further revealed that the abnormal acetylation pattern observed in darx1 interrupts arabinoxylan conformation and cellulose microfibril orientation, resulting in compromised secondary wall patterning and reduced mechanical strength. This study provides insight into the mechanism controlling the acetylation pattern on arabinoxylan side chains and suggests a strategy to breed robust elite crops.

Glycome and Proteome Components of Golgi Membranes Are Common between Two Angiosperms with Distinct Cell-Wall Structures.

The plant endoplasmic reticulum-Golgi apparatus is the site of synthesis, assembly, and trafficking of all noncellulosic polysaccharides, proteoglycans, and proteins destined for the cell wall. As grass species make cell walls distinct from those of dicots and noncommelinid monocots, it has been assumed that the differences in cell-wall composition stem from differences in biosynthetic capacities of their respective Golgi. However, immunosorbence-based screens and carbohydrate linkage analysis of polysaccharides in Golgi membranes, enriched by flotation centrifugation from etiolated coleoptiles of maize (Zea mays) and leaves of Arabidopsis (Arabidopsis thaliana), showed that arabinogalactan-proteins and arabinans represent substantial portions of the Golgi-resident polysaccharides not typically found in high abundance in cell walls of either species. Further, hemicelluloses accumulated in Golgi at levels that contrasted with those found in their respective cell walls, with xyloglucans enriched in maize Golgi, and xylans enriched in Arabidopsis. Consistent with this finding, maize Golgi membranes isolated by flotation centrifugation and enriched further by free-flow electrophoresis, yielded >200 proteins known to function in the biosynthesis and metabolism of cell-wall polysaccharides common to all angiosperms, and not just those specific to cell-wall type. We propose that the distinctive compositions of grass primary cell walls compared with other angiosperms result from differential gating or metabolism of secreted polysaccharides post-Golgi by an as-yet unknown mechanism, and not necessarily by differential expression of genes encoding specific synthase complexes.

Organelle membrane-specific chemical labeling and dynamic imaging in living cells.

Lipids play crucial roles as structural elements, signaling molecules and material transporters in cells. However, the functions and dynamics of lipids within cells remain unclear because of a lack of methods to selectively label lipids in specific organelles and trace their movement by live-cell imaging. We describe here a technology for the selective labeling and fluorescence imaging (microscopic or nanoscopic) of phosphatidylcholine in target organelles. This approach involves the metabolic incorporation of azido-choline, followed by a spatially limited bioorthogonal reaction that enables the visualization and quantitative analysis of interorganelle lipid transport in live cells. More importantly, with live-cell imaging, we obtained direct evidence that the autophagosomal membrane originates from the endoplasmic reticulum. This method is simple and robust and is thus powerful for real-time tracing of interorganelle lipid trafficking.

Sec17 (α-SNAP) and Sec18 (NSF) restrict membrane fusion to R-SNAREs, Q-SNAREs, and SM proteins from identical compartments.

Membrane fusion at each organelle requires conserved proteins: Rab-GTPases, effector tethering complexes, Sec1/Munc18 (SM)-family SNARE chaperones, SNAREs of the R, Qa, Qb, and Qc families, and the Sec17/α-SNAP and ATP-dependent Sec18/NSF SNARE chaperone system. The basis of organelle-specific fusion, which is essential for accurate protein compartmentation, has been elusive. Rab family GTPases, SM proteins, and R- and Q-SNAREs may contribute to this specificity. We now report that the fusion supported by SNAREs alone is both inefficient and promiscuous with respect to organelle identity and to stimulation by SM family proteins or complexes. SNARE-only fusion is abolished by the disassembly chaperones Sec17 and Sec18. Efficient fusion in the presence of Sec17 and Sec18 requires a tripartite match between the organellar identities of the R-SNARE, the Q-SNAREs, and the SM protein or complex. The functions of Sec17 and Sec18 are not simply negative regulation; they stimulate fusion with either vacuolar SNAREs and their SM protein complex HOPS or endoplasmic reticulum/cis-Golgi SNAREs and their SM protein Sly1. The fusion complex of each organelle is assembled from its own functionally matching pieces to engage Sec17/Sec18 for fusion stimulation rather than inhibition.

Loss of MUNC18-1 leads to retrograde transport defects in neurons.

Loss of the exocytic Sec1/MUNC18 protein MUNC18-1 or its target-SNARE partners SNAP25 and syntaxin-1 results in rapid, cell-autonomous and unexplained neurodegeneration, which is independent of their known role in synaptic vesicle exocytosis. cis-Golgi abnormalities are the earliest cellular phenotypes before degeneration occurs. Here, we investigated whether loss of MUNC18-1 causes defects in intracellular membrane transport pathways in primary murine neurons that may explain neurodegeneration. Electron, confocal and super resolution microscopy confirmed that loss of MUNC18-1 expression results in a smaller cis-Golgi. In addition, we now show that medial-Golgi and the trans-Golgi Network are also affected. However, stacking and cisternae ultrastructure of the Golgi were normal. Overall, ultrastructure of null mutant neurons was remarkably normal just hours before cell death occurred. By synchronizing protein trafficking by conditional cargo retention in the endoplasmic reticulum using selective hooks (RUSH) and immunocytochemistry, we show that anterograde Endoplasmic Reticulum-to-Golgi and Golgi exit of endogenous and exogenous proteins were normal. In contrast, loss of MUNC18-1 caused reduced retrograde Cholera Toxin B-subunit transport from the plasma membrane to the Golgi. In addition, MUNC18-1-deficiency resulted in abnormalities in retrograde TrkB trafficking in an antibody uptake assay. We conclude that MUNC18-1 deficient neurons have normal anterograde but reduced retrograde transport to the Golgi. The impairments in retrograde pathways suggest a role of MUNC18-1 in endosomal SNARE-dependent fusion and provide a plausible explanation for the observed Golgi abnormalities and cell death in MUNC18-1 deficient neurons.