The hereditary spastic paraplegia gene, spastin, regulates microtubule stability to modulate synaptic structure and function

BACKGROUND: Hereditary Spastic Paraplegia (HSP) is a devastating neurological disease causing spastic weakness of the lower extremities and eventual axonal degeneration. Over 20 genes have been linked to HSP in humans; however, mutations in one gene, spastin (SPG4), are the cause of >40% of all cases. Spastin is a member of the ATPases associated with diverse cellular activities (AAA) protein family, and contains a microtubule interacting and organelle transport (MIT) domain. Previous work in cell culture has proposed a role for Spastin in regulating microtubules. RESULTS: Employing Drosophila transgenic methods for overexpression and RNA interference (RNAi), we have investigated the role of Spastin in vivo. We show that Drosophila Spastin (D-Spastin) is enriched in axons and synaptic connections. At neuromuscular junctions (NMJ), Dspastin RNAi causes morphological undergrowth and reduced synaptic area. Moreover, Dspastin overexpression reduces synaptic strength, whereas Dspastin RNAi elevates synaptic currents. By using antibodies against posttranslationally modified alpha-Tubulin, we find that Dspastin regulates microtubule stability. Functional synaptic defects caused by Dspastin RNAi and overexpression were pharmacologically alleviated by agents that destabilize and stabilize microtubules, respectively. CONCLUSIONS: Loss of Dspastin in Drosophila causes an aberrantly stabilized microtubule cytoskeleton in neurons and defects in synaptic growth and neurotransmission. These in vivo data strongly support previous reports, providing a probable cause for the neuronal dysfunction in spastin-linked HSP disease. The role of Spastin in regulating neuronal microtubule stability suggests therapeutic targets for HSP treatment and may provide insight into neurological disorders linked to microtubule dysfunction.     

Fat-specific Protein 27 Undergoes Ubiquitin-dependent Degradation Regulated by Triacylglycerol Synthesis and Lipid Droplet Formation

The fat-specific protein 27 (Fsp27), a protein localized to lipid droplets (LDs), plays an important role in controlling lipid storage and mitochondrial activity in adipocytes. Fsp27-null mice display increased energy expenditure and are resistant to high fat diet-induced obesity and diabetes. However, little is known about how the Fsp27 protein is regulated. Here, we show that Fsp27 stability is controlled by the ubiquitin-dependent proteasomal degradation pathway in adipocytes. The ubiquitination of Fsp27 is regulated by three lysine residues located in the C-terminal region. Substitution of these lysine residues with alanines greatly increased Fsp27 stability and enhanced lipid storage in adipocytes. Furthermore, Fsp27 was stabilized and rapidly accumulated following treatment with β-agonists that induce lipolysis and fatty acid re-esterification in adipocytes. More importantly, Fsp27 stabilization was dependent on triacylglycerol synthesis and LD formation, because knockdown of diacylglycerol acyltransferase in adipocytes significantly reduced Fsp27 accumulation in adipocytes. Finally, we observed that increased Fsp27 during β-agonist treatment preferentially associated with LDs. Taken together, our data revealed that Fsp27 can be stabilized by free fatty acid availability, triacylglycerol synthesis, and LD formation. The stabilization of Fsp27 when free fatty acids are abundant further enhances lipid storage, providing positive feedback to regulate lipid storage in adipocytes.     

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

Lipid droplets (LDs) are the main fat storing sites in almost all species from bacteria to humans. The perilipin family has been found as LD proteins in mammals, Drosophila, and a couple of slime molds, but no bacterial LD proteins containing sequence conservation were identified. In this study, we reported that the hydroxysteroid dehydrogenase (HSD) family was found on LDs across all organisms by LD proteomic analysis. Imaging experiments confirmed LD targeting of three representative HSD proteins including ro01416 in RHA1, DHS-3 in C. elegans, and 17β-HSD11 in human cells. In C. elegans, 17β-HSD11 family proteins (DHS-3, DHS-4 and DHS-19) were localized on LDs in distinct tissues. In intestinal cells of C. elegans, DHS-3 targeted to cytoplasmic LDs, while DHS-9 labeled nuclear LDs. Furthermore, the N-terminal hydrophobic domains of 17β-HSD11 family were necessary for their targeting to LDs. Last, 17β-HSD11 family proteins induced LD aggregation, and deletion of DHS-3 in C. elegans caused lipid decrease. Independent of their presumptive catalytic sites, 17β-HSD11 family proteins regulated LD dynamics and lipid metabolism through affecting the LD-associated ATGL, which was conserved between C. elegans and humans. Together, these findings for HSDs provide a new insight not only into the mechanistic studies of the dynamics and functions of LDs in multiple organisms, but also into understanding the evolutionary history of the organelle.     

Whole-genome RNAi screen identifies methylation-related genes influencing lipid metabolism in Caenorhabditis elegans

Lipid droplets (LDs) are highly conserved multifunctional cellular organelles and aberrant lipid storage in LDs can lead to many metabolic diseases. However, the molecular mechanisms governing lipid dynamic changes remain elusive, and the high-throughput screen of genes influencing LD morphology was limited by lacking specific LD marker proteins in the powerful genetic tool Caenorhabditis elegans. In this study, we established a new method to conduct whole-genome RNAi screen using LD resident protein DHS-3 as a LD marker, and identified 78 genes involved in significant LD morphologic changes. Among them, mthf-1, as well as a series of methylation-related genes, was found dramatically influencing lipid metabolism. SREBP-1 and SCD1 homologs in C. elegans were involved in the lipid metabolic change of mthf-1(RNAi) worms, and the regulation of ATGL-1 also contributed to it by decreasing triacylglycerol (TAG) hydrolysis. Overall, this study not only identified important genes involved in LD dynamics, but also provided a new tool for LD study using C. elegans, with implications for the study of lipid metabolic diseases.     

Abnormal accumulation of lipid droplets in neurons induces the conversion of alpha-Synuclein to proteolytic resistant forms in a Drosophila model of Parkinson’s disease

Parkinson’s disease (PD) is a neurodegenerative disorder characterized by alpha-synuclein (αSyn) aggregation and associated with abnormalities in lipid metabolism. The accumulation of lipids in cytoplasmic organelles called lipid droplets (LDs) was observed in cellular models of PD. To investigate the pathophysiological consequences of interactions between αSyn and proteins that regulate the homeostasis of LDs, we used a transgenic Drosophila model of PD, in which human αSyn is specifically expressed in photoreceptor neurons. We first found that overexpression of the LD-coating proteins Perilipin 1 or 2 (dPlin1/2), which limit the access of lipases to LDs, markedly increased triacylglyclerol (TG) loaded LDs in neurons. However, dPlin-induced-LDs in neurons are independent of lipid anabolic (diacylglycerol acyltransferase 1/midway, fatty acid transport protein/dFatp) and catabolic (brummer TG lipase) enzymes, indicating that alternative mechanisms regulate neuronal LD homeostasis. Interestingly, the accumulation of LDs induced by various LD proteins (dPlin1, dPlin2, CG7900 or Klarsicht^LD-BD) was synergistically amplified by the co-expression of αSyn, which localized to LDs in both Drosophila photoreceptor neurons and in human neuroblastoma cells. Finally, the accumulation of LDs increased the resistance of αSyn to proteolytic digestion, a characteristic of αSyn aggregation in human neurons. We propose that αSyn cooperates with LD proteins to inhibit lipolysis and that binding of αSyn to LDs contributes to the pathogenic misfolding and aggregation of αSyn in neurons.     

Deficiency of a Lipid Droplet Protein,Perilipin 5, Suppresses Myocardial Lipid Accumulation,Thereby Preventing Type 1 Diabetes-Induced Heart Malfunction

Lipid droplet (LD) is a ubiquitous organelle that stores triacylglycerol and other neutral lipids. Perilipin 5 (Plin5), a member of the perilipin protein family that is abundantly expressed in the heart, is essential to protect LDs from attack by lipases, including adipose triglyceride lipase. Plin5 controls heart metabolism and performance by maintaining LDs under physiological conditions. Aberrant lipid accumulation in the heart leads to organ malfunction, or cardiomyopathy. To elucidate the role of Plin5 in a metabolically disordered state and the mechanism of lipid-induced cardiomyopathy, we studied the effects of streptozotocin-induced type 1 diabetes in Plin5-knockout (KO) mice. In contrast to diabetic wild-type mice, diabetic Plin5-KO mice lacked detectable LDs in the heart and did not exhibit aberrant lipid accumulation, excessive reactive oxygen species (ROS) generation, or heart malfunction. Moreover, diabetic Plin5-KO mice exhibited lower heart levels of lipotoxic molecules, such as diacylglycerol and ceramide, than wild-type mice. Membrane translocation of protein kinase C and the assembly of NADPH oxidase 2 complex on the membrane were also suppressed. The results suggest that diabetic Plin5-KO mice are resistant to type 1 diabetes-induced heart malfunction due to the suppression of the diacylglycerol/ceramide-protein kinase C pathway and of excessive ROS generation by NADPH oxidase.     

Regulation of TG accumulation and lipid droplet morphology by the novel TLDP1 in Aurantiochytrium limacinum F26-b

Thraustochytrids are marine single-cell protists that produce large amounts of PUFAs, such as DHA. They accumulate PUFAs in lipid droplets (LDs), mainly as constituent(s) of triacylglycerol (TG). We identified a novel protein in the LD fraction of Aurantiochytrium limacinum F26-b using 2D-difference gel electrophoresis. The protein clustered with orthologs of thraustochytrids; however, the cluster was evolutionally different from known PAT family proteins or plant LD protein; thus, we named it thraustochytrid-specific LD protein 1 (TLDP1). TLDP1 surrounded LDs when expressed as a GFP-tagged form. Disruption of the tldp1 gene decreased the content of TG and number of LDs per cell; however, irregular and unusually large LDs were generated in tldp1-deficient mutants. Although the level of TG synthesis was unchanged by the disruption of tldp1, the level of TG degradation was higher in tldp1-deficient mutants than in the WT. These phenotypic abnormalities in tldp1-deficient mutants were restored by the expression of tldp1. These results indicate that TLDP1 is a thraustochytrid-specific LD protein and regulates the TG accumulation and LD morphology in A. limacinum F26-b.     

A defect of the vacuolar putative lipase Atg15 accelerates degradation of lipid droplets through lipolysis

Lipid droplets (LDs) are the conserved organelles for the deposit of neutral lipids, and function as reservoirs of membrane and energy sources. To date, functional links between autophagy and LD dynamics have not been fully elucidated. Here, we report that a vacuolar putative lipase, Atg15, required for degradation of autophagic bodies, is crucial for the maintenance of LD amount in the yeast Saccharomyces cerevisiae in the stationary phase. Mutant analyses revealed that the putative lipase motif and vacuolar localization of Atg15 are important for the maintenance of LD amount. Loss of autophagosome formation by simultaneous deletion of core ATG genes cancelled the reduction in the LD amount in ATG15-deleted cells, indicating that degradation of autophagic bodies accounts for the functional involvement of Atg15 in LD dynamics. The reduced level of LDs in the mutant strain was dependent on Tgl3 and Tgl4, major lipases for lipolysis in S. cerevisiae. An altered phosphorylation status of Tgl3, higher accumulation of Tgl4, and closer associations of Tgl3 and Tgl4 with LDs were detected in the ATG15-deleted cells. Furthermore, increased levels of downstream metabolites of lipolysis in the mutant strain strongly suggested enhanced lipolytic activity caused by loss of ATG15. Our data provide evidence for a novel link between autophagic flux and LD dynamics integrated with Atg15 activity.     

Lipid droplets induced by secreted phospholipase A2 and unsaturated fatty acids protect breast cancer cells from nutrient and lipotoxic stress

Cancer cells driven by the Ras oncogene scavenge unsaturated fatty acids (FAs) from their environment to counter nutrient stress. The human group X secreted phospholipase A₂ (hGX sPLA₂) releases FAs from membrane phospholipids, stimulates lipid droplet (LD) biogenesis in Ras-driven triple-negative breast cancer (TNBC) cells and enables their survival during starvation. Here we examined the role of LDs, induced by hGX sPLA₂ and unsaturated FAs, in protection of TNBC cells against nutrient stress. We found that hGX sPLA₂ releases a mixture of unsaturated FAs, including ω-3 and ω-6 polyunsaturated FAs (PUFAs), from TNBC cells. Starvation-induced breakdown of LDs induced by low micromolar concentrations of unsaturated FAs, including PUFAs, was associated with protection from cell death. Interestingly, adipose triglyceride lipase (ATGL) contributed to LD breakdown during starvation, but it was not required for the pro-survival effects of hGX sPLA₂ and unsaturated FAs. High micromolar concentrations of PUFAs, but not OA, induced oxidative stress-dependent cell death in TNBC cells. Inhibition of triacylglycerol (TAG) synthesis suppressed LD biogenesis and potentiated PUFA-induced cell damage. On the contrary, stimulation of LD biogenesis by hGX sPLA₂ and suppression of LD breakdown by ATGL depletion reduced PUFA-induced oxidative stress and cell death. Finally, lipidomic analyses revealed that sequestration of PUFAs in LDs by sPLA₂-induced TAG remodelling and retention of PUFAs in LDs by inhibition of ATGL-mediated TAG lipolysis protect from PUFA lipotoxicity. LDs are thus antioxidant and pro-survival organelles that guard TNBC cells against nutrient and lipotoxic stress and emerge as attractive targets for novel therapeutic interventions.     

Lipid droplets in skeletal muscle during grass snake (Natrix natrix L.) development

Lipid droplets (LDs) are common organelles observed in Eucaryota. They are multifunctional organelles (involved in lipid storage, metabolism, and trafficking) that originate from endoplasmic reticulum (ER). LDs consist of a neutral lipid core, made up of diacyl- and triacylglycerols (DAGs and TAGs) and cholesterol esters (CEs), surrounded by a phospholipid monolayer and proteins, which are necessary for their structure and dynamics.Here, we report the protein and lipid composition as well as characterization and dynamics of grass snake (Natrix natrix) skeletal muscle LDs at different developmental stages. In the present study, we used detailed morphometric, LC-MS, quantitative lipidomic analyses of LDs isolated from the skeletal muscles of the snake embryos, immunofluorescence, and TEM.Our study also provides a valuable insight concerning the LDs' multifunctionality and ability to interact with a variety of organelles. These LD features are reflected in their proteome composition, which contains scaffold proteins, metabolic enzymes signalling polypeptides, proteins necessary for the formation of docking sites, and many others. We also provide insights into the biogenesis and growth of muscle LDs goes beyond the conventional mechanism based on the synthesis and incorporation of TAGs and LD fusion. We assume that the formation and functioning of grass snake muscle LDs are based on additional mechanisms that have not yet been identified, which could be related to the unique features of reptiles that are manifested in the after-hatching period of life, such as a reptile-specific strategy for energy saving during hibernation.     

Lipid droplets contribute myogenic differentiation in C2C12 by promoting the remodeling of the acstin-filament

Lipid droplet (LD), a multi-functional organelle, is found in most eukaryotic cells. LDs participate in the regulation of many cellular processes including proliferation, stress, and apoptosis. Previous studies showed the athlete’s paradox that trained athletes accumulate LDs in their skeletal muscle. However, the impact of LDs on skeletal muscle and myogenesis is not clear. We discovered that C2C12 myoblast cells containing more LDs formed more multinucleated muscle fibers. We also discovered that LDs promoted cell migration and fusion by promoting actin-filaments remodeling. Mechanistically, two LD-proteins, Acyl-CoA synthetase long chain family member 3 (ACSL3) and lysophosphatidylcholine acyltransferase 1 (LPCAT1), medicated the recruitment of actinin proteins which contributed to actin-filaments formation on the surface of LDs. During remodeling, the actinin proteins on LDs surface translocated to actin-filaments via ARF1/COPI vesicles. Our study demonstrate LDs contribute to cell differentiation, which lead to new insight into the LD function.Subject terms: Cell migration, Organelles     

Chlamydia trachomatis Infection Leads to Defined Alterations to the Lipid Droplet Proteome in Epithelial Cells

The obligate intracellular bacterium Chlamydia trachomatis is a major human pathogen and a main cause of genital and ocular diseases. During its intracellular cycle, C. trachomatis replicates inside a membrane-bound vacuole termed an “inclusion”. Acquisition of lipids (and other nutrients) from the host cell is a critical step in chlamydial replication. Lipid droplets (LD) are ubiquitous, ER-derived neutral lipid-rich storage organelles surrounded by a phospholipids monolayer and associated proteins. Previous studies have shown that LDs accumulate at the periphery of, and eventually translocate into, the chlamydial inclusion. These observations point out to Chlamydia-mediated manipulation of LDs in infected cells, which may impact the function and thereby the protein composition of these organelles. By means of a label-free quantitative mass spectrometry approach we found that the LD proteome is modified in the context of C. trachomatis infection. We determined that LDs isolated from C. trachomatis-infected cells were enriched in proteins related to lipid metabolism, biosynthesis and LD-specific functions. Interestingly, consistent with the observation that LDs intimately associate with the inclusion, a subset of inclusion membrane proteins co-purified with LD protein extracts. Finally, genetic ablation of LDs negatively affected generation of C. trachomatis infectious progeny, consistent with a role for LD biogenesis in optimal chlamydial growth.     

Lipid Accumulation, Lipid Body Formation, and Acyl Coenzyme A Oxidases of the Yeast Yarrowia lipolytica

Yarrowia lipolytica contains five acyl-coenzyme A oxidases (Aox), encoded by the POX1 to POX5 genes, that catalyze the limiting step of peroxisomal β-oxidation. In this study, we analyzed morphological changes of Y. lipolytica growing in an oleic acid medium and the effect of POX deletions on lipid accumulation. Protrusions involved in the uptake of lipid droplets (LDs) from the medium were seen in electron micrographs of the surfaces of wild-type cells grown on oleic acid. The number of protrusions and surface-bound LDs increased during growth, but the sizes of the LDs decreased. The sizes of intracellular lipid bodies (LBs) and their composition depended on the POX genotype. Only a few, small, intracellular LBs were observed in the mutant expressing only Aox4p (Δpox2 Δpox3 Δpox5), but strains expressing either Aox3p or both Aox3p and Aox4p had the same number of LBs as did the wild type. In contrast, strains expressing either Aox2p or both Aox2p and Aox4p formed fewer, but larger, LBs than did the wild type. The size of the LBs increased proportionately with the amount of triacylglycerols in the LBs of the mutants. In summary, Aox2p expression regulates the size of cellular triacylglycerol pools and the size and number of LBs in which these fatty acids accumulate.     

Triacylglycerol Synthesis Enzymes Mediate Lipid Droplet Growth by Relocalizing from the ER to Lipid Droplets

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Highlights? Triacylglyceride (TG) synthesis is coupled with lipid droplet (LD) growth ? Two LD populations exist: growing LDs, containing TG enzymes, and small LDs ? Specific TG synthesis enzymes move from the ER to LDs through membrane bridges ? LD localization of TG enzymes mediates expansion of a subset of LDs     

Composition and occurrence of lipid droplets in the cyanobacterium Nostoc punctiforme

Inclusions of neutral lipids termed lipid droplets (LDs) located throughout the cell were identified in the cyanobacterium Nostoc punctiforme by staining with lipophylic fluorescent dyes. LDs increased in number upon entry into stationary phase and addition of exogenous fructose indicating a role for carbon storage, whereas high-light stress did not increase LD numbers. LD accumulation increased when nitrate was used as the nitrogen source during exponential growth as compared to added ammonia or nitrogen-fixing conditions. Analysis of isolated LDs revealed enrichment of triacylglycerol (TAG), α-tocopherol, and C17 alkanes. LD TAG from exponential phase growth contained mainly saturated C16 and C18 fatty acids, whereas stationary phase LD TAG had additional unsaturated fatty acids characteristic of whole cells. This is the first characterization of cyanobacterial LD composition and conditions leading to their production. Based upon their abnormally large size and atypical location, these structures represent a novel sub-organelle in cyanobacteria.     

mmBCFA C17iso ensures endoplasmic reticulum integrity for lipid droplet growth

In eukaryote cells, lipid droplets (LDs) are key intracellular organelles that dynamically regulate cellular energy homeostasis. LDs originate from the ER and continuously contact the ER during their growth. How the ER affects LD growth is largely unknown. Here, we show that RNAi knockdown of acs-1, encoding an acyl-CoA synthetase required for the biosynthesis of monomethyl branched-chain fatty acids C15iso and C17iso, remarkably prevented LD growth in Caenorhabditis elegans. Dietary C17iso, or complex lipids with C17iso including phosphatidylcholine, phosphatidylethanolamine, and triacylglycerol, could fully restore the LD growth in the acs-1RNAi worms. Mechanistically, C17iso may incorporate into phospholipids to ensure the membrane integrity of the ER so as to maintain the function of ER-resident enzymes such as SCD/stearoyl-CoA desaturase and DGAT2/diacylglycerol acyltransferase for appropriate lipid synthesis and LD growth. Collectively, our work uncovers a unique fatty acid, C17iso, as the side chain of phospholipids for determining the ER homeostasis for LD growth in an intact organism, C. elegans.     

Identification and functional characterization of a lipid droplet protein CtLDP1 from an oleaginous yeast Candida tropicalis SY005

Proteins residing in lipid droplets (LDs) of organisms exhibit diverse physiological roles. Since the LD proteins of yeasts are largely unexplored, we have identified a putative LD protein gene, CtLDP1 in the oleaginous yeast Candida tropicalis SY005 and characterized its function. The increased lipid accumulation in SY005 could be correlated with enhanced (~2.67-fold) expression of the CtLDP1 after low-nitrogen stress. The N-terminal transmembrane domain similar to perilipin proteins and the amphipathic α-helices predicted in silico, presumably aid in targeting the CtLDP1 to LD membranes. Heterologous expression of CtLDP1-mCherry fusion in Saccharomyces cerevisiae revealed localization in LDs, yet the expression of CtLDP1 did not show significant effect on LD formation in transformed cells. Molecular docking showed favourable interactions of the protein with sterol class of molecules, but not with triacylglycerol (TAG); and this was further experimentally verified by co-localization of the mCherry-tagged protein in TAG-deficient (but steryl ester containing) LDs. While oleic acid supplementation caused coalescence of LDs into supersized ones (average diameter = 1.19 ± 0.12 μm; n = 160), this effect was suppressed due to CtLDP1 expression, and the cells mostly exhibited numerous smaller LDs (average diameter = 0.46 ± 0.05 μm; n = 160). Moreover, CtLDP1 expression in pet10Δ knockout strain of S. cerevisiae restored multiple LD formation, indicating functional complementation of the protein. Overall, this study documents functional characterization of an LD-stabilizing protein from an oleaginous strain of Candida genus for the first time, and provides insights on the characteristics of LD proteins in oleaginous yeasts for future metabolic engineering.