Interactomic study on interaction between lipid droplets and mitochondria

An increasing body of evidence shows that the lipid droplet, a neutral lipid storage organelle, plays a role in lipid metabolism and energy homeostasis through its interaction with mitochondria. However, the cellular functions and molecular mechanisms of the interaction remain ambiguous. Here we present data from transmission electron microscopy, fluorescence imaging, and reconstitution assays, demonstrating that lipid droplets physically contact mitochondria in vivo and in vitro. Using a bimolecular fluorescence complementation assay in Saccharomyces cerevisiae, we generated an interactomic map of protein-protein contacts of lipid droplets with mitochondria and peroxisomes. The lipid droplet proteins Erg6 and Pet10 were found to be involved in 75% of the interactions detected. Interestingly, interactions between 3 pairs of lipid metabolic enzymes were detected. Collectively, these data demonstrate that lipid droplets make physical contacts with mitochondria and peroxisomes, and reveal specific molecular interactions that suggest active participation of lipid droplets in lipid metabolism in yeast.     

Single organelle dynamics linked to 3D structure by correlative live‐cell imaging and 3D electron microscopy

Live‐cell correlative light‐electron microscopy (live‐cell‐CLEM) integrates live movies with the corresponding electron microscopy (EM) image, but a major challenge is to relate the dynamic characteristics of single organelles to their 3‐dimensional (3D) ultrastructure. Here, we introduce focused ion beam scanning electron microscopy (FIB‐SEM) in a modular live‐cell‐CLEM pipeline for a single organelle CLEM. We transfected cells with lysosomal‐associated membrane protein 1‐green fluorescent protein (LAMP‐1‐GFP), analyzed the dynamics of individual GFP‐positive spots, and correlated these to their corresponding fine‐architecture and immediate cellular environment. By FIB‐SEM we quantitatively assessed morphological characteristics, like number of intraluminal vesicles and contact sites with endoplasmic reticulum and mitochondria. Hence, we present a novel way to integrate multiple parameters of subcellular dynamics and architecture onto a single organelle, which is relevant to address biological questions related to membrane trafficking, organelle biogenesis and positioning. Furthermore, by using CLEM to select regions of interest, our method allows for targeted FIB‐SEM, which significantly reduces time required for image acquisition and data processing.      

Maintaining social contacts: The physiological relevance of organelle interactions

Membrane-bound organelles in eukaryotic cells form an interactive network to coordinate and facilitate cellular functions. The formation of close contacts, termed “membrane contact sites” (MCSs), represents an intriguing strategy for organelle interaction and coordinated interplay. Emerging research is rapidly revealing new details of MCSs. They represent ubiquitous and diverse structures, which are important for many aspects of cell physiology and homeostasis. Here, we provide a comprehensive overview of the physiological relevance of organelle contacts. We focus on mitochondria, peroxisomes, the Golgi complex and the plasma membrane, and discuss the most recent findings on their interactions with other subcellular organelles and their multiple functions, including membrane contacts with the ER, lipid droplets and the endosomal/lysosomal compartment.     

Ultrastructural relationship of the phagophore with surrounding organelles

Phagophore nucleates from a subdomain of the endoplasmic reticulum (ER) termed the omegasome and also makes contact with other organelles such as mitochondria, Golgi complex, plasma membrane and recycling endosomes during its formation. We have used serial block face scanning electron microscopy (SB-EM) and electron tomography (ET) to image phagophore biogenesis in 3 dimensions and to determine the relationship between the phagophore and surrounding organelles at high resolution. ET was performed to confirm whether membrane contact sites (MCSs) are evident between the phagophore and those surrounding organelles. In addition to the known contacts with the ER, we identified MCSs between the phagophore and membranes from putative ER exit sites, late endosomes or lysosomes, the Golgi complex and mitochondria. We also show that one phagophore can have simultaneous MCSs with more than one organelle. Future membrane flux experiments are needed to determine whether membrane contacts also signify lipid translocation.     

Interactions between the endoplasmic reticulum, mitochondria, plasma membrane and other subcellular organelles

Several recent works show structurally and functionally dynamic contacts between mitochondria, the plasma membrane, the endoplasmic reticulum, and other subcellular organelles. Many cellular processes require proper cooperation between the plasma membrane, the nucleus and subcellular vesicular/tubular networks such as mitochondria and the endoplasmic reticulum. It has been suggested that such contacts are crucial for the synthesis and intracellular transport of phospholipids as well as for intracellular Ca²⁺ homeostasis, controlling fundamental processes like motility and contraction, secretion, cell growth, proliferation and apoptosis. Close contacts between smooth sub-domains of the endoplasmic reticulum and mitochondria have been shown to be required also for maintaining mitochondrial structure. The overall distance between the associating organelle membranes as quantified by electron microscopy is small enough to allow contact formation by proteins present on their surfaces, allowing and regulating their interactions. In this review we give a historical overview of studies on organelle interactions, and summarize the present knowledge and hypotheses concerning their regulation and (patho)physiological consequences.     

Calcium-Induced Ultrastructural Interactions in Adrenocorticocytes

Steroidogenesis in adrenocorticocytes is closely related to intracellular [Ca²⁺]. To detect ultrastructural changes induced by growth in cytosolic [Ca²⁺], we used a rat adrenocortical cell culture, which was examined with electron microscopy and morphometric analysis. We established that either KCl-induced membrane depolarization evoking Ca²⁺ influx into the cell via voltage-operated Ca channels and Ca²⁺ ionophore, A23187, induced remarkable ultrastructural interactions between several cytosolic organelles. Lipid droplets known as key elements for Ca²⁺-induced steroidogenesis directly contacted with organelles containing the enzymes providing steroidogenic reactions (mitochondria, smooth and rough endoplasmic reticulum, nucleus, peroxisomes, and lysosomes). In most cases, the lipid droplets formed a specialized morphological structure at the sites of contact with the partner organelles. These structures are interpreted as a specialized transporting system, which provides contacts between organelles and exchange of intermediate products of the steroidogenesis process between the droplet and organelles.     

Lipid droplets interact with mitochondria using SNAP23

Triglyceride-containing lipid droplets (LD) are dynamic organelles stored on demand in all cells. These droplets grow through a fusion process mediated by SNARE proteins, including SNAP23. The droplets have also been shown to be highly motile and interact with other cell organelles, including peroxisomes and the endoplasmic reticulum. We have used electron and confocal microscopy to demonstrate that LD form complexes with mitochondria in NIH 3T3 fibroblasts. Using an in vitro system of purified LD and mitochondria, we also show the formation of the LD-mitochondria complex, in which cytosolic factors are involved. Moreover, the presence of LD markers in mitochondria isolated by subcellular fractionations is demonstrated. Finally, ablation of SNAP23 using siRNA reduced complex formation and beta oxidation, which suggests that the LD-mitochondria complex is functional in the cell.     

Interorganellar communication and membrane contact sites in protozoan parasites

A key characteristic of eukaryotic cells is the presence of organelles with discrete boundaries and functions. Such subcellular compartmentalization into organelles necessitates platforms for communication and material exchange between each other which often involves vesicular trafficking and associated processes. Another way is via the close apposition between organellar membranes, called membrane contact sites (MCSs). Apart from lipid transfer, MCSs have been implicated to mediate in various cellular processes including ion transport, apoptosis, and organelle dynamics. In mammalian and yeast cells, contact sites have been reported between the membranes of the following: the endoplasmic reticulum (ER) and the plasma membrane (PM), ER and the Golgi apparatus, ER and endosomes (i.e., vacuoles, lysosomes), ER and lipid droplets (LD), the mitochondria and vacuoles, the nucleus and vacuoles, and the mitochondria and lipid droplets, whereas knowledge of MCSs in non-model organisms such as protozoan parasites is extremely limited. Growing evidence suggests that MCSs play more general and conserved roles in cell physiology. In this mini review, we summarize and discuss representative MCSs in divergent parasitic protozoa, and highlight the universality, diversity, and the contribution of MCSs to parasitism.     

Are endoplasmic reticulum subdomains shaped by asymmetric distribution of phospholipids? Evidence from a C. elegans model system

Physical contact between organelles are widespread, in part to facilitate the shuttling of protein and lipid cargoes for cellular homeostasis. How do protein‐protein and protein‐lipid interactions shape organelle subdomains that constitute contact sites? The endoplasmic reticulum (ER) forms extensive contacts with multiple organelles, including lipid droplets (LDs) that are central to cellular fat storage and mobilization. Here, we focus on ER‐LD contacts that are highlighted by the conserved protein seipin, which promotes LD biogenesis and expansion. Seipin is enriched in ER tubules that form cage‐like structures around a subset of LDs. Such enrichment is strongly dependent on polyunsaturated and cyclopropane fatty acids. Based on these findings, we speculate on molecular events that lead to the formation of seipin‐positive peri‐LD cages in which protein movement is restricted. We hypothesize that asymmetric distribution of specific phospholipids distinguishes cage membrane tubules from the bulk ER.     

Phospholipid Synthesis and Transport in Mammalian Cells

Membranes of mammalian subcellular organelles contain defined amounts of specific phospholipids that are required for normal functioning of proteins in the membrane. Despite the wide distribution of most phospholipid classes throughout organelle membranes, the site of synthesis of each phospholipid class is usually restricted to one organelle, commonly the endoplasmic reticulum (ER). Thus, phospholipids must be transported from their sites of synthesis to the membranes of other organelles. In this article, pathways and subcellular sites of phospholipid synthesis in mammalian cells are summarized. A single, unifying mechanism does not explain the inter‐organelle transport of all phospholipids. Thus, mechanisms of phospholipid transport between organelles of mammalian cells via spontaneous membrane diffusion, via cytosolic phospholipid transfer proteins, via vesicles and via membrane contact sites are discussed. As an example of the latter mechanism, phosphatidylserine (PS) is synthesized on a region of the ER (mitochondria‐associated membranes, MAM) and decarboxylated to phosphatidylethanolamine in mitochondria. Some evidence is presented suggesting that PS import into mitochondria occurs via membrane contact sites between MAM and mitochondria. Recent studies suggest that protein complexes can form tethers that link two types of organelles thereby promoting lipid transfer. However, many questions remain about mechanisms of inter‐organelle phospholipid transport in mammalian cells.     

Subcellular visualization of light microscopic specimens by laser scanning microscopy and computer analysis: a new application of image analysis.

To identify subcellular organelles or to observe their pathological changes in sections prepared for light microscopy, immuno- and/or enzyme histochemical staining for the marker substances or enzymes of those subcellular organelles are frequently employed. With conventional light microscopes (CLM), however, it is hardly possible to determine whether or not the target organelles are properly stained and to confirm their fine structure. In the present study, the laser scanning microscope (LSM) was employed to obtain highly contrasted images of histochemically stained subcellular organelles at the limit of resolution in light microscopy. To refine or characterize those images, images built up as electronic signals in LSM were further processed in the Image Analysis System (IAS) with pipeline. Thus, the approximate figures of subcellular organelles such as microtubules, endoplasmic reticula, secretory granules, and mitochondria were visualized in brightfield on sections prepared for light microscopy (paraffin, frozen sections and cultured living cells). The validity of the images obtained by LSM or LSM-IAS was confirmed by immunoelectron microscopy when possible. The LSM images of histochemically stained suborganelles of various cells were definitely improved (refined and/or strengthened) by processing them with IAS.     

Lipid droplets lighting up: Insights from live microscopy

Lipid droplets emerge as important intracellular organelles relevant for lipid homeostasis and the pathophysiology of metabolic diseases. Here, we present a personal view on the current knowledge about the biogenesis of mammalian cytoplasmic lipid droplets, with a focus on microscopy and especially live imaging. We also discuss difficulties related to the lipid droplet proteome, contentious views on lipid droplet growth, and last but not least the evidence for the heterogeneity of lipid droplets within a single cell. We conclude with an outline of the most important future challenges.     

Nonperturbative chemical imaging of organelle transport in living cells with coherent anti-stokes Raman scattering microscopy

Nonperturbative monitoring of intracellular organelle transport in unstained living cells was achieved with coherent anti-Stokes Raman scattering (CARS) microscopy. To avoid possible interference with the organelle transport introduced by laser radiation, we first examined different illumination conditions. Using a new photodamage criterion based on morphological changes of the cells, we determined the threshold values of both pulse energy and average power at relevant wavelengths. Under excitation conditions much milder than the threshold levels, we were able to monitor the motions of lipid droplet (LD) organelles in steroidogenic mouse adrenal cortical (Y-1) cells with CARS microscopy in real time without perturbations to the cells. Particle tracking analyses revealed subdiffusion as well as active transport of LDs along microtubules. Interestingly, LD active transport is only present in Y-1 cells that rounded up in culture, a morphological change associated with steroidogenesis, suggesting possible involvements of LD active transport in the latter. Simultaneous imaging of LDs and mitochondria with CARS and two-photon fluorescence microscopy clearly showed that interactions between the two organelles could be facilitated by high LD motility. These observations demonstrate CARS microscopy as a powerful noninvasive imaging tool for studying dynamic processes in living cells.     

Fluorescence Microscopy Gets Faster and Clearer: Roles of Photochemistry and Selective Illumination

Significant advances in fluorescence microscopy tend be a balance between two competing qualities wherein improvements in resolution and low light detection are typically accompanied by losses in acquisition rate and signal-to-noise, respectively. These trade-offs are becoming less of a barrier to biomedical research as recent advances in optoelectronic microscopy and developments in fluorophore chemistry have enabled scientists to see beyond the diffraction barrier, image deeper into live specimens, and acquire images at unprecedented speed. Selective plane illumination microscopy has provided significant gains in the spatial and temporal acquisition of fluorescence specimens several mm in thickness. With commercial systems now available, this method promises to expand on recent advances in 2-photon deep-tissue imaging with improved speed and reduced photobleaching compared to laser scanning confocal microscopy. Superresolution microscopes are also available in several modalities and can be coupled with selective plane illumination techniques. The combination of methods to increase resolution, acquisition speed, and depth of collection are now being married to common microscope systems, enabling scientists to make significant advances in live cell and in situ imaging in real time. We show that light sheet microscopy provides significant advantages for imaging live zebrafish embryos compared to laser scanning confocal microscopy.     

Fluorescence Tracing of Intracellular Proteins

Developments in fluorescence microscopy and the availability of fluorescently labeled antibodies and probes for localization of molecules and organelles have made the microscope an indispensable tool with which one can map specific molecules to subcellular loci allowing deep insight into cell and organelle biology. Furthermore, confocal microscopy permits analysis of the three dimensional architecture of cells that could not be accomplished by conventional light microscopy. The goal of fluorescence protein tracing by microscopy is to visualize cellular constituents and general cytoarchitecture as close to native organization as possible. To achieve this, and to preserve cellular structure in the best possible manner, the specimen is usually fixed chemically. Here I review several standard fixation, permeabilization and labeling schemes followed by examples of several standard imaging techniques.     

脂滴与线粒体相互作用的研究进展

肥胖和多种代谢类疾病的发生有着密切的关系,而导致肥胖的脂肪多以中性脂的形式储存于细胞的一种细胞器——脂滴中。越来越多的研究表明,脂滴能够和其它细胞器发生相互作用,而它和线粒体的相互作用可能与Ⅱ型糖尿病的形成密切相关:非正常的脂滴和线粒体的相互作用有可能是导致细胞胰岛素抵抗的重要原因。我们通过对脂滴表面蛋白质组学、脂滴与线粒体的空间位置,以及相关蛋白等研究的总结,结合本实验室的研究结果,对脂滴与线粒体相互作用的物质基础及可能方式、受骨骼肌有氧运动的影响,及其与骨骼肌胰岛素抵抗发生的关系等,进行了讨论。     

Correlative 3D imaging of whole mammalian cells with light and electron microscopy

We report methodological advances that extend the current capabilities of ion-abrasion scanning electron microscopy (IA-SEM), also known as focused ion beam scanning electron microscopy, a newly emerging technology for high resolution imaging of large biological specimens in 3D. We establish protocols that enable the routine generation of 3D image stacks of entire plastic-embedded mammalian cells by IA-SEM at resolutions of ∼10–20 nm at high contrast and with minimal artifacts from the focused ion beam. We build on these advances by describing a detailed approach for carrying out correlative live confocal microscopy and IA-SEM on the same cells. Finally, we demonstrate that by combining correlative imaging with newly developed tools for automated image processing, small 100 nm-sized entities such as HIV-1 or gold beads can be localized in SEM image stacks of whole mammalian cells. We anticipate that these methods will add to the arsenal of tools available for investigating mechanisms underlying host-pathogen interactions, and more generally, the 3D subcellular architecture of mammalian cells and tissues.     

Imaging of subcellular structures by scanning ion microscopy and mass spectrometry. Advantage of cryofixation and freeze substitution procedure over chemical preparation

With the IMS 4F, a scanning ion microscope and mass spectrometer (SIMS), it is possible to map chemical elements with a lateral resolution of about 250 nm over a field of view of 50 × 50 μm². Such conditions should enable the imaging of subcellular structures with constitutive ionic species such as CN^?, P^?, S^?. The study was performed on heart and renal tissues prepared either by chemical procedure or cryofixation-freeze substitution (CF-FS) prior to embedding. Heart tissue was chosen because cardiocytes display a simple structural organization whereas the structural organization of kidney tubular cells is more complex. Whatever the preparation procedure, nuclei were easily identified due to their high P^? content. The CN^?, P^?, and S^? ion images obtained on heart and renal tissues prepared by chemical procedure showed weak contrasts inside the cytoplasm so that it was difficult to recognize the organelles. After CF-FS, enhanced contrasted images allow organelle (mitochondria, myofibrils, lysosomes, vacuoles, basal lamina, etc) characterization. This work demonstrated that CF-FS is a more suitable preparation procedure than chemical method to reveal organelle structures by their chemical composition. The improvements in the imaging of these structures is an essential step to establish the correlation between the localization of a trace element (or a molecule tagged with isotopes or particular atoms) and its subcellular targets.     

基于抗坏血酸过氧化酶的电镜成像和活细胞邻近标记方法的应用进展

Alice Ting实验室开发的抗坏血酸过氧化物酶(engineered ascorbate peroxidase,APEX),相对于经典的辣根过氧化物酶(horse radish peroxidase,HRP),其酶活性不再受细胞内蛋白质定位的影响,可以在几乎所有的亚细胞区域保持活性,这使其在研究亚细胞尺度以及活细胞水平生物学问题时极具优势.目前,基于APEX的二氨基联苯胺(diaminobenzidine,DAB)染色标记技术已经成功地实现对全细胞、亚细胞器和蛋白质水平的电镜成像.同时,与质谱技术结合,基于APEX的活细胞生物素邻近标记方法也极大地推动了亚细胞器蛋白质组学,以及目标蛋白在特定时空条件下邻近蛋白质组学的研究发展.本文将从以上两个方面阐述APEX技术的基本原理及最新应用进展,并讨论和展望其在实际应用中存在的局限性和挑战.     

Lipid droplets: a unified view of a dynamic organelle

Lipid droplets form the main lipid store in eukaryotic cells. Although all cells seem to be able to generate lipid droplets, their biogenesis, regulatory mechanisms and interactions with other organelles remain largely elusive. In this article, we outline some of the recent developments in lipid droplet cell biology. We show the mobile and dynamic nature of this organelle, and advocate the adoption of a unified nomenclature to consolidate terminology in this emerging field.