Author response: Syncytin-mediated open-ended membrane tubular connections facilitate the intercellular transfer of cargos including Cas9 protein

Identification of Lipid Droplets in Gut Bacteria

Syncytin-mediated open-ended membrane tubular connections facilitate the intercellular transfer of cargos including Cas9 protein

AbstractMuch attention has been focused on the possibility that cytoplasmic proteins and RNA may be conveyed between cells in extracellular vesicles (EVs) and tunneling nanotube (TNT) structures. Here, we set up two quantitative delivery reporters to study cargo transfer between cells. We found that EVs are internalized by reporter cells but do not efficiently deliver functional Cas9 protein to the nucleus. In contrast, donor and acceptor cells co-cultured to permit cell contact resulted in a highly effective transfer. Among our tested donor and acceptor cell pairs, HEK293T and MDA-MB-231 recorded optimal intercellular transfer. Depolymerization of F-actin greatly decreased Cas9 transfer whereas inhibitors of endocytosis or knock-down of genes implicated in this process had little effect on transfer. Imaging results suggest that intercellular transfer of cargos occurred through open-ended membrane tubular connections. In contrast, cultures consisting only of HEK293T cells form close-ended tubular connections ineffective in cargo transfer. Depletion of human endogenous fusogens, syncytins, especially syncytin-2 in MDA-MB-231 cells, significantly reduced Cas9 transfer. Full-length mouse syncytin, but not truncated mutants, rescued the effect of depletion of human syncytins on Cas9 transfer. Mouse syncytin overexpression in HEK293T cells partially facilitated Cas9 transfer among HEK293T cells. These findings suggest that syncytin may serve as the fusogen responsible for the formation of an open-ended connection between cells.

Lipid Droplet Is an Ancient and Inheritable Organelle in Bacteria

AbstractLipid droplet (LD) is a monolayer phospholipid membrane-bound organelle found in all eukaryotes and several prokaryotes which plays key roles in cellular lipid homeostasis and human health. The origin and evolution of the organelle remains unknown. Here, we report that through screening over 660 bacteria using biophysical and biochemical methods, plus LD isolation and proteomic tool, LDs were identified in most of these microbes, affiliated with five main bacterial phyla. Moreover, LDs were also identified in E. coli overexpressing lipid synthesis enzymes, indicating that bacteria without detectable LDs possessed the ability of LD biogenesis. The similarity of isolated LDs from representative strains and evolutionary analysis of LD major protein PspA demonstrate that LDs were conserved in bacteria. Furthermore, time-lapse imaging revealed that LDs were inheritable accompanying with bacterial growth and division. Finally, a common ancestor of LD-containing bacteria was predicted to originate 3.19 billion years ago by a phylogenetic analysis. Our findings suggest that LD is a widespread and inheritable organelle from an ancient common ancestor.

The New Face of the Lipid Droplet: Lipid Droplet Proteins

AbstractThe lipid droplet (LD) is an organelle with vital functions found in nearly all organisms. LD proteomic research has provided fundamentally important insights into this organelle's functions. The review provides a summary of LD proteomic studies conducted across diverse organisms and cell and tissue types. The accumulated proteomic data are reviewed for evidence of a protein targeting mechanism for the organelle. The hypotheses for several specific localization mechanisms based on what is known about targeting mechanisms for other organelles and vesicles are provided. Although the nature of the mechanism is not known, the functional data demonstrate that the targeting mechanism and, indeed, the organelle itself, is conserved from prokaryotes to eukaryotes. It is hoped that the review will help inspire further research leading to novel discoveries in the field.

Hydroxysteroid dehydrogenase family proteins on lipid droplets through bacteria, C. elegans, and mammals

The lipid droplet: A conserved cellular organelle

Bacterial lipid droplets bind to DNA via an intermediary protein that enhances survival under stress

AbstractLipid droplets (LDs) are multi-functional organelles consisting of a neutral lipid core surrounded by a phospholipid monolayer, and exist in organisms ranging from bacteria to humans. Here we study the functions of LDs in the oleaginous bacterium Rhodococcus jostii. We show that these LDs bind to genomic DNA through the major LD protein, MLDS, which increases survival rate of the bacterial cells under nutritional and genotoxic stress. MLDS expression is regulated by a transcriptional regulator, MLDSR, that binds to the operator and promoter of the operon encoding both proteins. LDs sequester MLDSR, controlling its availability for transcriptional regulation. Our findings support the idea that bacterial LDs can regulate nucleic acid function and facilitate bacterial survival under stress.

Dynamics of the Lipid Droplet Proteome of the Oleaginous Yeast Rhodosporidium toruloides

ABSTRACT Lipid droplets (LDs) are ubiquitous organelles that serve as a neutral lipid reservoir and a hub for lipid metabolism. Manipulating LD formation, evolution, and mobilization in oleaginous species may lead to the production of fatty acid-derived biofuels and chemicals. However, key factors regulating LD dynamics remain poorly characterized. Here we purified the LDs and identified LD-associated proteins from cells of the lipid-producing yeast Rhodosporidium toruloides cultured under nutrient-rich, nitrogen-limited, and phosphorus-limited conditions. The LD proteome consisted of 226 proteins, many of which are involved in lipid metabolism and LD formation and evolution. Further analysis of our previous comparative transcriptome and proteome data sets indicated that the transcription level of 85 genes and protein abundance of 77 proteins changed under nutrient-limited conditions. Such changes were highly relevant to lipid accumulation and partially confirmed by reverse transcription-quantitative PCR. We demonstrated that the major LD structure protein Ldp1 is an LD marker protein being upregulated in lipid-rich cells. When overexpressed in Saccharomyces cerevisiae , Ldp1 localized on the LD surface and facilitated giant LD formation, suggesting that Ldp1 plays an important role in controlling LD dynamics. Our results significantly advance the understanding of the molecular basis of lipid overproduction and storage in oleaginous yeasts and will be valuable for the development of superior lipid producers.

Microorganism lipid droplets and biofuel development

Tracing Evolutionary Footprints to Identify Novel Gene Functional Linkages