Phospholipid Scramblase 1 Localizes Proximal to Sphingomyelin Synthase Isoforms but Is Not Involved in Sphingomyelin Synthesis.

Ceramide (Cer) is synthesized de novo in the bilayer of the endoplasmic reticulum and transported to the cytosolic leaflet of the trans-Golgi apparatus for sphingomyelin (SM) synthesis. As the active site of SM synthase (SMS) is located on the luminal side of the Golgi membrane, Cer translocates to the lumen via transbilayer movement for SM synthesis. However, the mechanism of transbilayer movement is not fully understood. As the Cer-related translocases seem to localize near the SMS, the protein was identified using proximity-dependent biotin identification proteomics. Phospholipid scramblase 1 (PLSCR1), which is thought to act as a scramblase for phosphatidylserine and phosphatidylethanolamine, was identified as a protein proximal to the SMS isoforms SMS1 and SMS2. Although five isoforms of PLSCR have been reported in humans, only PLSCR1, PLSCR3, and PLSCR4 are expressed in HEK293T cells. Confocal microscopic analysis showed that PLSCR1 and PLSCR4 partially co-localized with p230, a trans-Golgi network marker, where SMS isoforms are localized. We established CRISPR/Cas9-mediated PLSCR1, PLSCR3, and PLSCR4 single-knockout cells and PLSCR1, 3, 4 triple knockout HEK293T cells. Liquid chromatography-tandem mass spectrometry revealed that the levels of species with distinct acyl chains in Cer and SM were not significantly different in single knockout cells or in the triple knockout cells compared to the wild-type cells. Our findings suggest that PLSCR1 is localized in the vicinity of SMS isoforms, however is not involved in the transbilayer movement of Cer for SM synthesis.

Development of a natural product optimization strategy for inhibitors against MraY, a promising antibacterial target.

MraY (phospho-N-acetylmuramoyl-pentapeptide-transferase) inhibitory natural products are attractive molecules as candidates for a new class of antibacterial agents to combat antimicrobial-resistant bacteria. Structural optimization of these natural products is required to improve their drug-like properties for therapeutic use. However, chemical modifications of these natural products are painstaking tasks due to complex synthetic processes, which is a bottleneck in advancing natural products to the clinic. Here, we develop a strategy for a comprehensive in situ evaluation of the build-up library, which enables us to streamline the preparation of the analogue library and directly assess its biological activities. We apply this approach to a series of MraY inhibitory natural products. Through construction and evaluation of the 686-compound library, we identify promising analogues that exhibit potent and broad-spectrum antibacterial activity against highly drug-resistant strains in vitro as well as in vivo in an acute thigh infection model. Structures of the MraY-analogue complexes reveal distinct interaction patterns, suggesting that these analogues represent MraY inhibitors with unique binding modes. We further demonstrate the generality of our strategy by applying it to tubulin-binding natural products to modulate their tubulin polymerization activities.

A facile method for monitoring sphingomyelin synthase activity in HeLa cells using liquid chromatography/mass spectrometry.

Sphingomyelin synthase (SMS) is a sphingolipid-metabolizing enzyme involved in the de novo synthesis of sphingomyelin (SM) from ceramide (Cer). Recent studies have indicated that SMS is a key therapeutic target for metabolic diseases such as fatty liver, type 2 diabetes, atherosclerosis, and colorectal cancer. However, very few SMS inhibitors have been identified because of the limited sensitivity and selectivity of the current fluorescence-based screening assay. In this study, we developed a simple cell-based assay coupled with liquid chromatography/tandem mass spectrometry (LC-MS/MS) to screen for SMS inhibitors. HeLa cells stably expressing SMS1 or SMS2 were used for the screening. A non-fluorescent unnatural C6-Cer was used as a substrate for SMS to produce C6-SM. C6-Cer and C6-SM levels in the cells were monitored and quantified using LC-MS/MS. The activity of ginkgolic acid C15:1 (GA), a known SMS inhibitor, was measured. GA had half-maximal inhibitory concentrations of 5.5 µM and 3.6 µM for SMS1 and SMS2, respectively. To validate these findings, hSMS1 and hSMS2 proteins were optimized for molecular docking studies. In silico analyses were conducted to assess the interaction of GA with SMS1 and SMS2, and its binding affinity. This study offers an analytical approach for screening novel SMS inhibitors and provides in silico support for the experimental findings.

Characterization of NMCR-3, NMCR-4 and NMCR-5, three novel non-mobile colistin resistance determinants: Implications for MCR-3, MCR-7, and MCR-5 progenitors, respectively.

In this study, the progenitors of MCR-3, MCR-7 and MCR-5, namely NMCR-3, NMCR-4 and NMCR-5, were firstly discovered and indicating Aeromonas was a natural reservoir for MCR-3 and MCR-7. Furthermore, different evolutionary models for MCR-3, MCR-7 and MCR-5 were proposed.

Disruption of sphingomyelin synthase 2 gene alleviates cognitive impairment in a mouse model of Alzheimer's disease.

The membrane raft accommodates the key enzymes synthesizing amyloid ß (Aß). One of the two characteristic components of the membrane raft, cholesterol, is well known to promote the key enzymes that produce amyloid-ß (Aß) and exacerbate Alzheimer's disease (AD) pathogenesis. Given that the raft is a physicochemical platform for the sound functioning of embedded bioactive proteins, the other major lipid component sphingomyelin may also be involved in AD. Here we knocked out the sphingomyelin synthase 2 gene (SMS2) in 3xTg AD model mice by hybridization, yielding SMS2KO mice (4S mice). The novel object recognition test in 9/10-month-old 4S mice showed that cognitive impairment in 3xTg mice was alleviated by SMS2KO, though performance in the Morris water maze (MWM) was not improved. The tail suspension test detected a depressive trait in 4S mice, which may have hindered the manifestation of performance in the wet, stressful environment of MWM. In the hippocampal CA1, hyperexcitability in 3xTg was also found alleviated by SMS2KO. In the hippocampal dentate gyrus of 4S mice, the number of neurons positive with intracellular Aß or its precursor proteins, the hallmark of young 3xTg mice, is reduced to one-third, suggesting an SMS2KO-led suppression of syntheses of those peptides in the dentate gyrus. Although we previously reported that large-conductance calcium-activated potassium (BK) channels are suppressed in 3xTg mice and their recovery relates to cognitive amelioration, no changes occurred by hybridization. Sphingomyelin in the membrane raft may serve as a novel target for AD drugs.

Two natural compounds as potential inhibitors against the Helicobacter pylori and Acinetobacter baumannii IspD enzymes.

In a vast majority of bacteria, protozoa and plants, the methylerythritol phosphate (MEP) pathway is utilized for the synthesis of isopentenyl diphosphate (IDP) and dimethylallyl diphosphate (DMADP), which are precursors for isoprenoids. Isoprenoids, such as cholesterol and coenzyme Q, play a variety of crucial roles in physiological activities, including cell-membrane formation, protein degradation, cell apoptosis, and transcription regulation. In contrast, humans employ the mevalonate (MVA) pathway for the production of IDP and DMADP, rendering proteins in the MEP pathway appealing targets for antimicrobial agents. This pathway consists of seven consecutive enzymatic reactions, of which 4-diphosphocytidyl-2C-methyl-D-erythritol synthase (IspD) and 2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (IspF) catalyze the third and fifth steps, respectively. In this study, we characterized the enzymatic activities and protein structures of Helicobacter pylori IspDF and Acinetobacter baumannii IspD. Then, using the direct interaction-based thermal shift assay, we conducted a compound screening of an approved drug library and identified 27 hit compounds potentially binding to AbIspD. Among them, two natural products, rosmarinic acid and tanshinone IIA sodium sulfonate, exhibited inhibitory activities against HpIspDF and AbIspD, by competing with one of the substrates, MEP. Moreover, tanshinone IIA sodium sulfonate also demonstrated certain antibacterial effects against H. pylori. In summary, we identified two IspD inhibitors from approved ingredients, broadening the scope for antibiotic discovery targeting the MEP pathway.

Inositol-Exchange Activity in Human Primordial Placenta.

Human placenta is an intensively growing tissue. Phosphatidylinositol (PI) and its derivatives are part of the signaling pathway in the regulation of trophoblast cell differentiation. There are two different enzymes that take part in the direct PI synthesis: phosphatidylinositol synthase (PIS) and inositol exchange enzyme (IE). The presence of PIS is known in the human placenta, but IE activity has not been documented before. In our study, we describe the physiological properties of the two enzymes in vitro. PIS and IE were studied in different Mn2+ and Mg2+ concentrations that enabled us to separate the individual enzyme activities. Enzyme activity was measured by incorporation of 3[H]inositol in human primordial placenta tissue or microsomes. Optimal PIS activity was achieved between 0.5 and 2.0 mM Mn2+ concentration, but higher concentrations inhibit enzyme activity. In the presence of Mg2+, the enzyme activity increases continuously up to a concentration of 100 mM. PIS was inhibited by nucleoside di- and tri-phosphates. PI production increases between 0.1 and 10 mM Mn2+ concentration. The incorporation of [3H]inositol into PI increased by 57% when adding stabile GTP analog. The described novel pathway of inositol synthesis may provide an additional therapeutic approach of inositol supplementation before and during pregnancy.

The unusual substrate specificity of Escherichia coli cardiolipin synthase C does not require the product of the transcriptionally engaged ymdB gene.

Polycistronic transcription and translation of ymdB-clsC have been thought to be required for full activity of ClsC. The authentic initiation codon of the clsC gene is present within the open reading frame of the upstream located ymdB gene. ClsC translated from authentic initiation codon drives cardiolipin (CL) synthesis without transcriptionally paired YmdB. YmdB is not necessary for the substrate specificity of ClsC utilizing phosphatidylethanolamine (PE) as a co-substrate.

Redirecting raltitrexed from cancer cell thymidylate synthase to Mycobacterium tuberculosis phosphopantetheinyl transferase.

There is a compelling need to find drugs active against Mycobacterium tuberculosis (Mtb). 4'-Phosphopantetheinyl transferase (PptT) is an essential enzyme in Mtb that has attracted interest as a potential drug target. We optimized a PptT assay, used it to screen 422,740 compounds, and identified raltitrexed, an antineoplastic antimetabolite, as the most potent PptT inhibitor yet reported. While trying unsuccessfully to improve raltitrexed's ability to kill Mtb and remove its ability to kill human cells, we learned three lessons that may help others developing antibiotics. First, binding of raltitrexed substantially changed the configuration of the PptT active site, complicating molecular modeling of analogs based on the unliganded crystal structure or the structure of cocrystals with inhibitors of another class. Second, minor changes in the raltitrexed molecule changed its target in Mtb from PptT to dihydrofolate reductase (DHFR). Third, the structure-activity relationship for over 800 raltitrexed analogs only became interpretable when we quantified and characterized the compounds' intrabacterial accumulation and transformation.

Longitudinal plasma proteomic analysis identifies biomarkers and combinational targets for anti-PD1-resistant cancer patients.

The response rate of anti-PD1 therapy is limited, and the influence of anti-PD1 therapy on cancer patients is unclear. To address these challenges, we conducted a longitudinal analysis of plasma proteomic changes with anti-PD1 therapy in non-small cell lung cancer (NSCLC), alveolar soft part sarcoma (ASPS), and lymphoma patients. We included 339 plasma samples before and after anti-PD1 therapy from 193 patients with NSCLC, ASPS, or lymphoma. The plasma proteins were detected using data-independent acquisition-mass spectrometry and customable antibody microarrays. Differential proteomic characteristics in responders (R) and non-responders (NR) before and after anti-PD1 therapy were elucidated. A total of 1019 proteins were detected using our in-depth proteomics platform and distributed across 10-12 orders of abundance. By comparing the differential plasma proteome expression between R and NR groups, 50, 206, and 268 proteins were identified in NSCLC, ASPS, and lymphoma patients, respectively. Th17, IL-17, and JAK-STAT signal pathways were identified upregulated in NR group, while cellular senescence and transcriptional misregulation pathways were activated in R group. Longitudinal proteomics analysis revealed the IL-17 signaling pathway was downregulated after treatment. Consistently, many proteins were identified as potential combinatorial therapeutic targets (e.g., IL-17A and CD22). Five noninvasive biomarkers (FLT4, SFTPB, GNPTG, F5, and IL-17A) were further validated in an independent lymphoma cohort (n = 39), and another three noninvasive biomarkers (KIT, CCL3, and TNFSF1) were validated in NSCLC cohort (n = 76). Our results provide molecular insights into the anti-PD1 therapy in cancer patients and identify new therapeutic strategies for anti-PD1-resistant patients.

Early diagnostic clues of mucolipidosis type II: Significance of radiological findings.

Mucolipidosis type-II (ML-II) is an ultra-rare disorder caused by deficiency of N-acetylglucosaminyl-1-phosphotransferase enzyme due to biallelic pathogenic variants in GNPTAB gene. There are a few known about the natural history of ML-II. In this study, we presented the natural course of 24 patients diagnosed with ML-II. Mean age at diagnosis was 9.3 ± 5.7 months. All patients had coarse face, developmental delay, and hypotonia. The mean survival time was 3.01 ± 1.4 years. The oldest patient was 6.5 years old. Twelve patients died due to lung infection and respiratory failure. We observed early and significant radiological findings of ML-II were different from typical dysostosis multiplex such as femoral cloaking, rickets-like changes, and talocalcaneal stippling. These are significant findings observed in the fetal or newborn period which is considered to be highly characteristic of ML-II and disappears in the first year. Cloaking, rickets-like changes, and stippling were not observed in patients older than three months of age and this suggests that these findings disappear within the first year. These radiological features can be used as important clues for diagnosis. We detected eight different pathogenic variants in GNPTAB gene, three of them were novel.

Sphingomyelin synthase 2 promotes the stemness of breast cancer cells via modulating NF-κB signaling pathway.

OBJECTIVES: Multi-drug resistance (MDR) to chemotherapy is the main obstacle influencing the anti-tumor effect in breast cancer, which might lead to the metastasis and recurrence of cancer. Until now, there are still no effective methods that can overcome MDR. In this study, we aimed to investigate the role of sphingomyelin synthase 2 (SMS2) in breast cancer resistance. METHODS: Quantitative RT-PCR analysis was performed to assess changes in mRNA expression. Western blot analysis was performed to detect protein expression. Inhibitory concentration value of adriamycin (ADR) was evaluated using CCK 8 assay. The stemness ability of breast cancer cells was assessed by spheroid-formation assay. Immunofluorescence staining was conducted to show the cellular distribution of proteins. Breast tumor masses were harvested from the xenograft tumor mouse model. RESULTS: SMS2 overexpression increased the IC50 values of breast cancer cells. SMS2 decreased the CD24 transcription level but increased the transcription levels of stemness-related genes including CD44, ALDH, OCT 4 and SOX2 in breast cancer cells. SMS2 overexpression promoted the nuclear translocation of phosphorylated NF-κB, while suppression of SMS2 could inhibit the NF-κB pathway. CONCLUSIONS: SMS2 increased the stemness of breast cancer cells via NF-κB signaling pathway, leading to resistance to the chemotherapeutic drug ADR. Thus, SMS2 might play a critical role in the development of breast cancer resistance, which is a previously unrecognized mechanism in breast cancer MDR development.

Mutation in mitral valve prolapse susceptible gene DCHS1 causes familial mitral annular disjunction.

BACKGROUND: Mitral annular disjunction (MAD) is an under-recognised phenotype associated with severe ventricular arrhythmias. Limited knowledge has been gained on its molecular genesis. METHODS: A total of 150 unrelated deceased Chinese were collected for whole-exome sequencing, with analysis focusing on a panel of 118 genes associated with 'abnormal mitral valve morphology'. Cases were prespecified as 'longitudinally extensive MAD (LE-MAD)' or 'longitudinally less-extensive MAD (LLE-MAD)' according to the gross disjunctional length with a cut-off of 4.0 mm. The pedigree investigation was conducted on a case carrying an ultra-rare (minor allele frequency <0.1%) deleterious variant in DCHS1. RESULTS: Seventy-seven ultra-rare deleterious variants were finally identified. Exclusively, 12 ultra-rare deleterious variants distributed in nine genes occurred in LE-MAD, which were ANK1, COL3A1, DCHS1, FBN2, GNPTAB, LZTR1, PLD1, RYR1 and VPS13B. Ultra-rare deleterious variants in those nine genes were predominantly distributed in LE-MAD compared with LLE-MAD (28% vs 5%, OR 7.30, 95% CI 2.33 to 23.38; p<0.001), and the only gene related to LE-MAD with borderline significance was DCHS1. LE-MAD was consistently observed in a sizeable Chinese family, in which LE-MAD independently co-segregated with an ultra-rare deleterious variant in DCHS1, rs145429962. CONCLUSION: This study initially proposed that isolated LE-MAD might be a particular phenotype of MAD with a complex genetic predisposition. Deleterious variants in DCHS1 might be associated with the morphogenesis of LE-MAD.

SGMS1-AS1/MicroRNA-106a-5p/CPT2 Axis as a Novel Target for Regulating Lactate Metabolism in Colon Cancer.

PURPOSE: The malignant transformation of cells can lead to aerobic glycolysis, an important form of metabolic reprogramming in colon cancer cells, which can cause the accumulation of lactate and accelerate the proliferation of tumor cells also enhance their chemotherapy drug resistance. The aim of this study was to investigate the possible molecular mechanisms responsible for the increased lactate expression in colon cancer. METHODS: Several bioinformatics methods, including differential analysis, gene ontology enrichment, univariate and multivariate Cox regression analysis were used to find the lactic acid-related gene carnitine palmitoyltransferase 2. We analyzed the relationship between carnitine palmitoyltransferase 2 and clinical features as well as immune microenvironment. To further explore the mechanism of carnitine palmitoyltransferase 2 in colon cancer, we performed methylation analysis and constructed a competitive endogenous RNA network, which was validated in cell lines and clinical specimens. RESULTS: We used bioinformatics to select the lactic acid-related gene carnitine palmitoyltransferase 2 and found low expression of carnitine palmitoyltransferase 2 was associated with poor prognosis in colon cancer. An inhibitory tumor microenvironment was created when carnitine palmitoyltransferase 2 expression was reduced, with decreased CD4 T cells, CD8 T cells, dendritic cells, and B cells but increased cancer-associated fibroblasts. Methylation analysis showed that the abnormal decrease in carnitine palmitoyltransferase 2 might be caused by hypermethylation. We constructed a network of SGMS1-AS1/microRNA-106a-5p/carnitine palmitoyltransferase 2 and verified their expression in cell lines and clinical specimens. CONCLUSION: Our work revealed the possible mechanism of lactate accumulation in colon cancer and explored a new potential treatment for colon cancer by cutting off aerobic glycolysis in tumor cells.

SGMS2 in primary osteoporosis with facial nerve palsy.

Pathogenic heterozygous variants in SGMS2 cause a rare monogenic form of osteoporosis known as calvarial doughnut lesions with bone fragility (CDL). The clinical presentations of SGMS2-related bone pathology range from childhood-onset osteoporosis with low bone mineral density and sclerotic doughnut-shaped lesions in the skull to a severe spondylometaphyseal dysplasia with neonatal fractures, long-bone deformities, and short stature. In addition, neurological manifestations occur in some patients. SGMS2 encodes sphingomyelin synthase 2 (SMS2), an enzyme involved in the production of sphingomyelin (SM). This review describes the biochemical structure of SM, SM metabolism, and their molecular actions in skeletal and neural tissue. We postulate how disrupted SM gradient can influence bone formation and how animal models may facilitate a better understanding of SGMS2-related osteoporosis.

Human sphingomyelin synthase 1 generates diacylglycerol in the presence and absence of ceramide via multiple enzymatic activities.

Sphingomyelin (SM) synthase 1 (SMS1), which is involved in lipodystrophy, deafness, and thrombasthenia, generates diacylglycerol (DG) and SM using phosphatidylcholine (PC) and ceramide as substrates. Here, we found that SMS1 possesses DG-generating activities via hydrolysis of PC and phosphatidylethanolamine (PE) in the absence of ceramide and ceramide phosphoethanolamine synthase (CPES) activity. In the presence of the same concentration (4.7 mol%) of PC and ceramide, the amounts of DG produced by SMS and PC-phospholipase C (PLC) activities of SMS1 were approximately 65% and 35% of total DG production, respectively. PC-PLC activity showed substrate selectivity for saturated and/or monounsaturated fatty acid-containing PC species. A PC-PLC/SMS inhibitor, D609, inhibited only SMS activity. Mn2+ inhibited only PC-PLC activity. Intriguingly, DG attenuated SMS/CPES activities. Our study indicates that SMS1 is a unique enzyme with PC-PLC/PE-PLC/SMS/CPES activities.

Unraveling mucolipidosis type III gamma through whole genome sequencing in late-onset retinitis pigmentosa: a case report.

BACKGROUND: We describe the case of a 47-year-old man referred to a retinal clinic and diagnosed with late-onset retinitis pigmentosa. Surprisingly, genetic testing revealed compound heterozygous pathogenic variants in GNPTG, leading to the diagnosis of the autosomal recessive lysosomal storage disorder mucolipidosis type III gamma. Mucolipidosis type III gamma is typically diagnosed during childhood due to symptoms relating to skeletal dysplasia. Retinal dystrophy is not a common phenotypic feature. CASE PRESENTATION: Ophthalmologic examination was consistent with a mild form of retinitis pigmentosa and included fundus photography, measurement of best-corrected visual acuity, optical coherence tomography, electroretinogram and visual field testing. Extraocular findings included joint restriction and pains from an early age leading to bilateral hip replacement by age 30, aortic insufficiency, and hypertension. Genetic analysis was performed by whole genome sequencing filtered for a gene panel of 325 genes associated with retinal disease. Two compound heterozygous pathogenic variants were identified in GNPTG, c.347_349del and c.607dup. The diagnosis of mucolipidosis type III gamma was confirmed biochemically by measurement of increased activities of specific lysosomal enzymes in plasma. CONCLUSION: To our knowledge this is the first description of retinitis pigmentosa caused by compound heterozygous variants in GNPTG, providing further indications that late-onset retinal dystrophy is part of the phenotypic spectrum of mucolipidosis type III gamma.

Sphingomyelin synthase-related protein SMSr is a phosphatidylethanolamine phospholipase C that promotes nonalcoholic fatty liver disease.

Sphingomyelin synthase (SMS)-related protein (SMSr) is a phosphatidylethanolamine phospholipase C (PE-PLC) that is conserved and ubiquitous in mammals. However, its biological function is still not clear. We previously observed that SMS1 deficiency-mediated glucosylceramide accumulation caused nonalcoholic fatty liver diseases (NAFLD), including nonalcoholic steatohepatitis (NASH) and liver fibrosis. Here, first, we evaluated high-fat diet/fructose-induced NAFLD in Smsr KO and WT mice. Second, we evaluated whether SMSr deficiency can reverse SMS1 deficiency-mediated NAFLD, using Sms1/Sms2 double and Sms1/Sms2/Smsr triple KO mice. We found that SMSr/PE-PLC deficiency attenuated high-fat diet/fructose-induced fatty liver and NASH, and attenuated glucosylceramide accumulation-induced NASH, fibrosis, and tumor formation. Further, we found that SMSr/PE-PLC deficiency reduced the expression of many inflammatory cytokines and fibrosis-related factors, and PE supplementation in vitro or in vivo mimicked the condition of SMSr/PE-PLC deficiency. Furthermore, we demonstrated that SMSr/PE-PLC deficiency or PE supplementation effectively prevented membrane-bound ß-catenin transfer to the nucleus, thereby preventing tumor-related gene expression. Finally, we observed that patients with NASH had higher SMSr protein levels in the liver, lower plasma PE levels, and lower plasma PE/phosphatidylcholine ratios, and that human plasma PE levels are negatively associated with tumor necrosis factor-α and transforming growth factor ß1 levels. In conclusion, SMSr/PE-PLC deficiency causes PE accumulation, which can attenuate fatty liver, NASH, and fibrosis. These results suggest that SMSr/PE-PLC inhibition therapy may mitigate NAFLD.

The formation of migrasomes is initiated by the assembly of sphingomyelin synthase 2 foci at the leading edge of migrating cells.

The migrasome is an organelle of migrating cells with diverse physiological functions. How migrasome formation is initiated is unknown. We found that sphingomyelin is enriched in migrasomes and identified sphingomyelin synthase 2 (SMS2) as an essential protein for migrasome biogenesis. SMS2 assembles into immobile foci that adhere on the basal membrane at the leading edge. When cells migrate away, the SMS2 foci 'move' out of cells and into retraction fibres, where they become migrasome formation sites and eventually grow into migrasomes. Mechanistically, SMS2 foci seed migrasomes by converting ceramide to sphingomyelin, which is essential for migrasome formation. Furthermore, CerS5, which is required for the synthesis of long-chain ceramide, and CERT, which transports ceramide from the endoplasmic reticulum to Golgi, are both required for migrasome formation. Our data reveal the essential role of ceramide and sphingomyelin in migrasome formation and suggest that SMS2 forms basal membrane-surface-connecting structures that pre-determine where migrasomes will grow.