Contact (Thousand Oaks (Ventura County, Calif.))最新文献

Contact sites are areas of close apposition between two membranes that coordinate nonvesicular communication between organelles. Such interactions serve a wide range of cellular functions from regulating metabolic pathways to executing stress responses and coordinating organelle inheritance. The past decade has seen a dramatic increase in information on certain contact sites, mostly those involving the endoplasmic reticulum. However, despite its central role in the secretory pathway, the Golgi apparatus and its contact sites remain largely unexplored. In this review, we discuss the current knowledge of Golgi contact sites and share our thoughts as to why Golgi contact sites are understudied. We also highlight what exciting future directions may exist in this emerging field.

Milk-secreting epithelial cells of the mammary gland are functionally specialized for the synthesis and secretion of large quantities of neutral lipids, a major macronutrient in milk from most mammals. Milk lipid synthesis and secretion are hormonally regulated and secretion occurs by a unique apocrine mechanism. Neutral lipids are synthesized and packaged into perilipin-2 (PLIN2) coated cytoplasmic lipid droplets within specialized cisternal domains of rough endoplasmic reticulum (ER). Continued lipid synthesis by ER membrane enzymes and lipid droplet fusion contribute to the large size of these cytoplasmic lipid droplets (5-15 μm in diameter). Lipid droplets are directionally trafficked within the epithelial cell to the apical plasma membrane. Upon contact, a molecular docking complex assembles to tether the droplet to the plasma membrane and facilitate its membrane envelopment. This docking complex consists of the transmembrane protein, butyrophilin, the cytoplasmic housekeeping protein, xanthine dehydrogenase/oxidoreductase, the lipid droplet coat proteins, PLIN2, and cell death-inducing DFFA-like effector A. Interactions of mitochondria, Golgi, and secretory vesicles with docked lipid droplets have also been reported and may supply membrane phospholipids, energy, or scaffold cytoskeleton for apocrine secretion of the lipid droplet. Final secretion of lipid droplets into the milk occurs in response to oxytocin-stimulated contraction of myoepithelial cells that surround milk-secreting epithelial cells. The mechanistic details of lipid droplet release are unknown at this time. The final secreted milk fat globule consists of a triglyceride core coated with a phospholipid monolayer and various coat proteins, fully encased in a membrane bilayer.

Lipid droplets (LDs) are dynamic fat-storage organelles that interact readily with numerous cellular structures and organelles. A prominent LD contact site is with degradative vesicles such as autophagosomes, lysosomes, autolysosomes, and late endosomes. These contacts support lipid catabolism through the selective autophagy of LDs (i.e., lipophagy) or the recruitment of cytosolic lipases to the LD surface (i.e., lipolysis). However, LD-autophagosome contacts serve additional functions beyond lipid catabolism, including the supply of lipids for autophagosome biogenesis. In this review, we discuss the molecular mediators of LD contacts with autophagosomes and other degradative organelles as well as the diverse cellular functions of these contact sites in health and disease.

Peroxisomes and the endoplasmic reticulum (ER) cooperate extensively in lipid-related metabolic pathways, and the ER also provides phospholipids to enable the peroxisomal membrane to expand prior to division. Recently, we identified peroxisomal proteins ACBD5 and ACBD4, and the ER protein VAPB as tethering components which physically interact to foster peroxisome-ER associations at membrane contact sites. Overexpression or loss of these tether proteins alters the extent of peroxisome-ER interactions, impacting on lipid exchange between these two compartments. To facilitate further studies into peroxisome-ER associations at the level of membrane contact sites, their role, composition and regulation, we have developed two fluorescence-based systems to monitor peroxisome-ER interactions. We modified a proximity ligation assay and a split-fluorescence reporter system using split superfolder green fluorescent protein. Using the proximity ligation assay we were able to measure changes in peroxisome-ER interactions whilst the split-fluorescence reporter was more limited and only allowed us to label ER-peroxisome contacts. We show that both techniques can be useful additions to the toolkit of methods to study peroxisome-ER associations and explore the relative merits of each.

Loss of the endoplasmic reticulum (ER)-mitochondria encounter structure (ERMES) complex that resides in contact sites between the yeast ER and mitochondria leads to impaired respiration; however, the reason for that is not clear. We find that in ERMES null mutants, there is an increase in the level of mRNAs encoding for biosynthetic enzymes of coenzyme Q₆ (CoQ₆), an essential electron carrier of the mitochondrial respiratory chain. We show that the mega complexes involved in CoQ₆ biosynthesis (CoQ synthomes) are destabilized in ERMES mutants. This, in turn, affects the level and distribution of CoQ₆ within the cell, resulting in reduced mitochondrial CoQ₆. We suggest that these outcomes contribute to the reduced respiration observed in ERMES mutants. Fluorescence microscopy experiments demonstrate close proximity between the CoQ synthome and ERMES, suggesting a spatial coordination. The involvement of the ER-mitochondria contact site in regulation of CoQ₆ biogenesis highlights an additional level of communication between these two organelles.

Lipid droplets (LDs) are fat storage organelles integral to energy homeostasis and a wide range of cellular processes. LDs physically and functionally interact with many partner organelles, including the ER, mitochondria, lysosomes, and peroxisomes. Recent findings suggest that the dynamics of LD inter-organelle contacts is in part controlled by LD intracellular motility. LDs can be transported directly by motor proteins along either actin filaments or microtubules, via Kinesin-1, Cytoplasmic Dynein, and type V Myosins. LDs can also be propelled indirectly, by hitchhiking on other organelles, cytoplasmic flows, and potentially actin polymerization. Although the anchors that attach motors to LDs remain elusive, other regulators of LD motility have been identified, ranging from modification of the tracks to motor co-factors to members of the perilipin family of LD proteins. Manipulating these regulatory pathways provides a tool to probe whether altered motility affects organelle contacts and has revealed that LD motility can promote interactions with numerous partners, with profound consequences for metabolism. LD motility can cause dramatic redistribution of LDs between a clustered and a dispersed state, resulting in altered organelle contacts and LD turnover. We propose that LD motility can thus promote switches in the metabolic state of a cell. Finally, LD motility is also important for LD allocation during cell division. In a number of animal embryos, uneven allocation results in a large difference in LD content in distinct daughter cells, suggesting cell-type specific LD needs.

Delivery of low-density lipoprotein-derived cholesterol to the endoplasmic reticulum (ER) is essential for cholesterol homeostasis, yet the mechanism of this transport has largely remained elusive. Two recent reports shed some light on this process, uncovering a role for Niemann Pick type-C1 protein (NPC1) in the formation of membrane contact sites (MCS) between late endosomes (LE)/lysosomes (Lys) and the ER. Both studies identified a loss of MCS in cells lacking functional NPC1, where cholesterol accumulates in late endocytic organelles. Remarkably, and taking different approaches, both studies have made a striking observation that expansion of LE/Lys-ER MCS can rescue the cholesterol accumulation phenotype in NPC1 mutant or deficient cells. In both cases, the cholesterol was shown to be transported to the ER, demonstrating the importance of ER-LE/Lys contact sites in the direct transport of low-density lipoprotein-derived cholesterol to the ER.

Mitochondria undergo spontaneous transient elevations in matrix pH associated with drops in mitochondrial membrane potential. These mitopHlashes require a functional respiratory chain and the profusion protein optic atrophy 1, but their mechanistic basis is unclear. To gain insight on the origin of these dynamic events, we resolved the ultrastructure of flashing mitochondria by correlative light and electron microscopy. HeLa cells expressing the matrix-targeted pH probe mitoSypHer were screened for mitopHlashes and fixed immediately after the occurrence of a flashing event. The cells were then processed for imaging by serial block face scanning electron microscopy using a focused ion beam to generate ~1,200 slices of 10 nm thickness from a 28 μm × 15 μm cellular volume. Correlation of live/fixed fluorescence and electron microscopy images allowed the unambiguous identification of flashing and nonflashing mitochondria. Three-dimensional reconstruction and surface mapping revealed that each tomogram contained two flashing mitochondria of unequal sizes, one being much larger than the average mitochondrial volume. Flashing mitochondria were 10-fold larger than silent mitochondria but with a surface to volume ratio and a cristae volume similar to nonflashing mitochondria. Flashing mitochondria were connected by tubular structures, formed more membrane contact sites, and a constriction was observed at a junction between a flashing mitochondrion and a nonflashing mitochondrion. These data indicate that flashing mitochondria are structurally preserved and bioenergetically competent but form numerous membrane contact sites and are connected by tubular structures, consistent with our earlier suggestion that mitopHlashes might be triggered by the opening of fusion pores between contiguous mitochondria.

In naïve cells, the endoplasmic reticulum (ER) and the ER-resident Vesicle-associated membrane protein-Associated Proteins (VAP) are common components of sites of membrane contacts that mediate the nonvesicular transfer of lipids between organelles. There is increasing recognition that the hijacking of VAP by intracellular pathogens is a novel mechanism of host-pathogen interaction. Here, we summarize our recent findings showing that the Chlamydia inclusion membrane protein IncV tethers the ER to the inclusion membrane by binding to VAP via the molecular mimicry of two eukaryotic FFAT motifs. We extend the discussion to other microorganisms that have evolved similar mechanisms.