Integrated multimodality microscope for accurate and efficient target-guided cryo-lamellae preparation.

Cryo-electron tomography (cryo-ET) is a revolutionary technique for resolving the structure of subcellular organelles and macromolecular complexes in their cellular context. However, the application of the cryo-ET is hampered by the sample preparation step. Performing cryo-focused ion beam milling at an arbitrary position on the sample is inefficient, and the target of interest is not guaranteed to be preserved when thinning the cell from several micrometers to less than 300 nm thick. Here, we report a cryogenic correlated light, ion and electron microscopy (cryo-CLIEM) technique that is capable of preparing cryo-lamellae under the guidance of three-dimensional confocal imaging. Moreover, we demonstrate a workflow to preselect and preserve nanoscale target regions inside the finished cryo-lamellae. By successfully preparing cryo-lamellae that contain a single centriole or contact sites between subcellular organelles, we show that this approach is generally applicable, and shall help in innovating more applications of cryo-ET.

Molecular-scale axial localization by repetitive optical selective exposure.

We introduce an axial localization with repetitive optical selective exposure (ROSE-Z) method for super-resolution imaging. By using an asymmetric optical scheme to generate interference fringes, a <2 nm axial localization precision was achieved with only ~3,000 photons, which is an approximately sixfold improvement compared to previous astigmatism methods. Nanoscale three-dimensional and two-color imaging was demonstrated, illustrating how this method achieves superior performance and facilitates the investigation of cellular nanostructures.

RNA-binding protein DDX1 is responsible for fatty acid-mediated repression of insulin translation.

The molecular mechanism in pancreatic ß cells underlying hyperlipidemia and insulin insufficiency remains unclear. Here, we find that the fatty acid-induced decrease in insulin levels occurs due to a decrease in insulin translation. Since regulation at the translational level is generally mediated through RNA-binding proteins, using RNA antisense purification coupled with mass spectrometry, we identify a novel insulin mRNA-binding protein, namely, DDX1, that is sensitive to palmitate treatment. Notably, the knockdown or overexpression of DDX1 affects insulin translation, and the knockdown of DDX1 eliminates the palmitate-induced repression of insulin translation. Molecular mechanism studies show that palmitate treatment causes DDX1 phosphorylation at S295 and dissociates DDX1 from insulin mRNA, thereby leading to the suppression of insulin translation. In addition, DDX1 may interact with the translation initiation factors eIF3A and eIF4B to regulate translation. In high-fat diet mice, the inhibition of insulin translation happens at an early prediabetic stage before the elevation of glucose levels. We speculate that the DDX1-mediated repression of insulin translation worsens the situation of insulin resistance and contributes to the elevation of blood glucose levels in obese animals.

DDX1 regulates alternative splicing and insulin secretion in pancreatic ß cells.

DEAD-box helicase 1 (DDX1) is a multifunction protein involved in diverse cellular processes including transcription, viral replication, mRNA/miRNA processing, and tRNA splicing. Here, we report a novel function of DDX1 in mRNA alternative splicing in pancreatic ß cells. By performing integrated data analysis of high-throughput RNA sequencing (RNA-Seq), and cross-linking and immunoprecipitation coupled with deep sequencing (CLIP-Seq), we identify hundreds of alternative splicing genes that are targeted by DDX1. These DDX1-targeted alternative splicing genes are mainly associated with calcium ion binding, high voltage-gated calcium channel, and transmembrane transporter. Functionally, silencing DDX1 impairs calcium influx and insulin secretion in the pancreatic ß cells. These results reveal an important role for DDX1 in the regulation of gene alternative splicing and insulin secretion in pancreatic ß cells.

PKG and NHR-49 signalling co-ordinately regulate short-term fasting-induced lysosomal lipid accumulation in C. elegans.

Lysosomes act as terminal degradation organelles to hydrolyse macromolecules derived from both the extracellular space and the cytoplasm. In Caenorhabditis elegans fasting induces the lysosomal compartment to expand. However, the molecular and cellular mechanisms for this stress response remain largely unclear. In the present study, we find that short-term fasting leads to increased accumulation of polar lipids in lysosomes. The fasting response is co-ordinately regulated by EGL-4, the C. elegans PKG (protein kinase G) orthologue, and nuclear hormone receptor NHR-49. Further results demonstrate that EGL-4 acts in sensory neurons to enhance lysosomal lipid accumulation through inhibiting the DAF-3/SMAD pathway, whereas NHR-49 acts in intestine to inhibit lipids accumulation via activation of IPLA-2 (intracellular membrane-associated calcium-independent phospholipase A2) in cytoplasm and other hydrolases in lysosomes. Remarkably, the lysosomal lipid accumulation is independent of autophagy and RAB-7-mediated endocytosis. Taken together, our results reveal a new mechanism for lysosomal lipid metabolism during the stress response, which may provide new clues for investigations of lysosome function in energy homoeostasis.

A proximity labeling strategy enables proteomic analysis of inter-organelle membrane contacts.

Inter-organelle membrane contacts are highly dynamic and act as central hubs for many biological processes, but the protein compositions remain largely unknown due to the lack of efficient tools. Here, we developed BiFCPL to analyze the contact proteome in living cells by a bimolecular fluorescence complementation (BiFC)-based proximity labeling (PL) strategy. BiFCPL was applied to study mitochondria-endoplasmic reticulum contacts (MERCs) and mitochondria-lipid droplet (LD) contacts. We identified 403 highly confident MERC proteins, including many transiently resident proteins and potential tethers. Moreover, we demonstrated that mitochondria-LD contacts are sensitive to nutrient status. A comparative proteomic analysis revealed that 60 proteins are up- or downregulated at contact sites under metabolic challenge. We verified that SQLE, an enzyme for cholesterol synthesis, accumulates at mitochondria-LD contact sites probably to utilize local ATP for cholesterol synthesis. This work provides an efficient method to identify key proteins at inter-organelle membrane contacts in living cells.

Cryogenic superresolution correlative light and electron microscopy of vitreous sections.

Fluorescence microscopy and electron microscopy complement each other as the former provides labelling and localisation of specific molecules and target structures while the latter possesses excellent revolving power of fine structure in context. These two techniques can combine as correlative light and electron microscopy (CLEM) to reveal the organisation of materials within the cell. Frozen hydrated sections allow microscopic observations of cellular components in situ in a near-native state and are compatible with superresolution fluorescence microscopy and electron tomography if sufficient hardware and software support is available and a well-designed protocol is followed. The development of superresolution fluorescence microscopy greatly increases the precision of fluorescence annotation of electron tomograms. Here, we provide detailed instructions on how to perform cryogenic superresolution CLEM on vitreous sections. From fluorescence-labelled cells to high pressure freezing, cryo-ultramicrotomy, cryogenic single-molecule localisation microscopy, cryogenic electron tomography and image registration, electron tomograms with features of interest highlighted by superresolution fluorescence signals are expected to be obtained.

Proteomic analysis of insulin secretory granules in INS-1 cells by protein correlation profiling.

ABSTRACT: Insulin secretory granules (ISGs), a group of distinguishing organelles in pancreatic ß cells, are responsible for the storage and secretion of insulin to maintain blood glucose homeostasis. The molecular mechanisms of ISG biogenesis, maturation, transportation, and exocytosis are still largely unknown because the proteins involved in these distinct steps have not been fully identified. Subcellular fractionation by density gradient centrifugation has been successfully employed to analyze the proteomes of numerous organelles. However, use of this method to elucidate the ISG proteome is limited by co-fractionated contaminants because ISGs are very dynamic and have abundant exchanges or contacts with other organelles, such as the Golgi apparatus, lysosomes, and endosomes. In this study, we developed a new strategy for identifying ISG proteins by protein correlation profiling (PCP)-based proteomics, which included ISG purification by OptiPrep density gradient centrifugation, label-free quantitative proteome, and identification of ISG proteins by correlating fractionation profiles between candidates and known ISG markers. Using this approach, we were able to identify 81 ISG proteins. Among them, TM9SF3, a nine-transmembrane protein, was considered a high confidence ISG candidate protein highlighted in the PCP network. Further biochemical and immunofluorescence assays indicated that TM9SF3 localized in ISGs, suggesting that it is a potential new ISG marker.

HID-1 is required for homotypic fusion of immature secretory granules during maturation.

Secretory granules, also known as dense core vesicles, are generated at the trans-Golgi network and undergo several maturation steps, including homotypic fusion of immature secretory granules (ISGs) and processing of prehormones to yield active peptides. The molecular mechanisms governing secretory granule maturation are largely unknown. Here, we investigate a highly conserved protein named HID-1 in a mouse model. A conditional knockout of HID-1 in pancreatic ß cells leads to glucose intolerance and a remarkable increase in the serum proinsulin/insulin ratio caused by defective proinsulin processing. Large volume three-dimensional electron microscopy and immunofluorescence imaging reveal that ISGs are much more abundant in the absence of HID-1. We further demonstrate that HID-1 deficiency prevented secretory granule maturation by blocking homotypic fusion of immature secretory granules. Our data identify a novel player during the early maturation of immature secretory granules.