Sensory axons induce epithelial lipid microdomain remodeling and determine the distribution of junctions in the epidermis

Epithelial cell properties are determined by the polarized distribution of membrane lipids, the cytoskeleton, and adhesive junctions. Epithelia are often profusely innervated, but little work has addressed how contact with neurites affects the polarized organization of epithelial components. In previous work, we found that basal keratinocytes in the larval zebrafish epidermis wrap around axons to enclose them in ensheathment channels sealed by autotypic cell junctions. In this study, we used live imaging to characterize how sensory axons remodel cell membranes, the actin cytoskeleton, and adhesive junctions in basal keratinocytes. At the apical surface of basal keratinocytes, axons promoted the formation of lipid microdomains quantitatively enriched in reporters for PI(4,5)P2 and liquid-ordered (Lo) membranes. Lipid microdomains supported the formation of cadherin-enriched F-actin protrusions, which wrapped around axons, likely initiating the formation of ensheathment channels. Lo reporters, but not reporters of liquid-disordered (Ld) membranes, became progressively enriched at axon-associated membrane domains as autotypic junctions matured at ensheathment channels. In the absence of axons, cadherin-enriched lipid microdomains still formed on basal cell membranes, but were not organized into the contiguous domains normally associated with axons. Instead, these isolated domains formed ectopic heterotypic junctions with overlying periderm cells, a distinct epithelial cell type in the epidermis. Thus, axons inhibit the formation of epithelial heterotypic junctions by recruiting cadherin-rich lipid microdomains to form autotypic junctions at ensheathment channels. These findings demonstrate that sensory nerve endings dramatically remodel polarized epithelial components and regulate the adhesive properties of the epidermis.

The bacterial membrane fission protein FisB requires homo-oligomerization and lipid-binding to catalyze membrane scission

ABSTRACTLittle is known about mechanisms of membrane fission in bacteria despite their requirement for cytokinesis. The only known dedicated membrane fission machinery in bacteria, FisB, is expressed during sporulation in Bacillus subtilis and is required to release the developing spore into the mother cell cytoplasm. Here we characterized the requirements for FisB-mediated membrane fission. FisB forms mobile clusters of ~12 molecules that give way to an immobile cluster at the engulfment pole containing ~40 proteins at the time of membrane fission. Function mutants revealed that binding to acidic lipids and homo-oligomerization are both critical for targeting FisB to the engulfment pole and membrane fission. Our results suggest that FisB is a robust and unusual membrane fission protein that relies on homo-oligomerization, lipid-binding and likely the unique membrane topology generated during engulfment for localization and membrane scission, but surprisingly, not on lipid microdomains or negative-curvature lipids.

Curcumin Analogue C1 Promotes Hex and Gal Recruitment to the Plasma Membrane via mTORC1-Independent TFEB Activation

The monocarbonyl analogue of curcumin (1E,4E)-1,5-Bis(2-methoxyphenyl)penta-1,4-dien-3-one (C1) has been used as a specific activator of the master gene transcription factor EB (TFEB) to correlate the activation of this nuclear factor with the increased activity of lysosomal glycohydrolases and their recruitment to the cell surface. The presence of active lysosomal glycohydrolases associated with the lipid microdomains has been extensively demonstrated, and their role in glycosphingolipid (GSL) remodeling in both physiological and pathological conditions, such as neurodegenerative disorders, has been suggested. Here, we demonstrate that Jurkat cell stimulation elicits TFEB nuclear translocation and an increase of both the expression of hexosaminidase subunit beta (HEXB), hexosaminidase subunit alpha (HEXA), and galactosidase beta 1 (GLB1) genes, and the recruitment of β-hexosaminidase (Hex, EC 3.2.1.52) and β-galactosidase (Gal, EC 3.2.1.23) on lipid microdomains. Treatment of Jurkat cells with the curcumin analogue C1 also resulted in an increase of both lysosomal glycohydrolase activity and their targeting to the cell surface. Similar effects of C1 on lysosomal glycohydrolase expression and their recruitment to lipid microdomains was observed by treating the SH-SY5Y neuroblastoma cell line; the effects of C1 treatment were abolished by TFEB silencing. Together, these results clearly demonstrate the existence of a direct link between TFEB nuclear translocation and the transport of Hex and Gal from lysosomes to the plasma membrane.

Syntaxin 17 Recruits ACSL3 to Lipid Microdomains in Lipid Droplet Biogenesis

During lipid droplet (LD) formation, several key enzymes for neutral lipid biosynthesis, such as acyl-CoA synthetase 3 (ACSL3), translocate from the bilayer of the endoplasmic reticulum membrane or mitochondria-associated membrane to the monolayer surface of LDs. It has been recently shown that syntaxin 17 (Stx17) in cooperation with synaptosomal-associated protein of 23 kDa (SNAP23) facilitates the translocation of ACSL3 from the endoplasmic reticulum/mitochondria-associated membrane to LDs. In this study, we investigated whether lipid microdomains enriched in cholesterol and sphingolipids are important for the formation of LDs and the interaction of Stx17 with ACSL3 and SNAP23. Cholesterol depletion and blockage of ceramide synthesis by chemicals inhibited oleic acid (OA)-induced LD biogenesis and decreased the interaction of Stx17 with ACSL3 and SNAP23, whereas blockage of ganglioside GD3 synthesis by sialyltransferase knockdown interfered with LD biogenesis by affecting the interaction of Stx17 with SNAP23 but not ACSL3. Consistent with the requirement of GD3 in LD biogenesis, Stx17 was found to associate with GD3-containing membranes upon OA loading. SNAP23 and a minor fraction of Stx17 were found to reside in detergent-resistant membranes (DRMs), whereas OA treatment caused redistribution of ACSL3 and Stx17 to DRMs. Importantly, the redistribution of ACSL3 to DRMs was abrogated upon depletion of Stx17 or SNAP23. Taken together, our results highlight the importance of lipid microdomains enriched in cholesterol and sphingolipids as a platform for the interaction of Stx17 with ACSL3 and SNAP23 in LD biogenesis.

Nuclear Lipid Microdomains Regulate Daunorubicin Resistance in Hepatoma Cells

Daunorubicin is an anticancer drug, and cholesterol is involved in cancer progression, but their relationship has not been defined. In this study, we developed a novel experimental model that utilizes daunorubicin, cholesterol, and daunorubicin plus cholesterol in the same cells (H35) to search for the role of nuclear lipid microdomains, rich in cholesterol and sphingomyelin, in drug resistance. We find that the daunorubicin induces perturbation of nuclear lipid microdomains, localized in the inner nuclear membrane, where active chromatin is anchored. As changes of sphingomyelin species in nuclear lipid microdomains depend on neutral sphingomyelinase activity, we extended our studies to investigate whether the enzyme is modulated by daunorubicin. Indeed the drug stimulated the sphingomyelinase activity that induced reduction of saturated long chain fatty acid sphingomyelin species in nuclear lipid microdomains. Incubation of untreated-drug cells with high levels of cholesterol resulted in the inhibition of sphingomyelinase activity with increased saturated fatty acid sphingomyelin species. In daunodubicin-treated cells, incubation with cholesterol reversed the action of the drug by acting via neutral sphingomyelinase. In conclusion, we suggest that cholesterol and sphingomyelin-forming nuclear lipid microdomains are involved in the drug resistance.