Ca2+-activated sphingomyelin scrambling and turnover mediate ESCRT-independent lysosomal repair

Abstract

Lysosomes are vital organelles vulnerable to injuries from diverse materials. Failure to repair or sequester damaged lysosomes poses a threat to cell viability. Here we report that cells exploit a sphingomyelin-based lysosomal repair pathway that operates independently of ESCRT to reverse potentially lethal membrane damage. Various conditions perturbing organelle integrity trigger a rapid calcium-activated scrambling and cytosolic exposure of sphingomyelin. Subsequent metabolic conversion of sphingomyelin by neutral sphingomyelinases on the cytosolic surface of injured lysosomes promotes their repair, also when ESCRT function is compromised. Conversely, blocking turnover of cytosolic sphingomyelin renders cells more sensitive to lysosome-damaging drugs. Our data indicate that calcium-activated scramblases, sphingomyelin, and neutral sphingomyelinases are core components of a previously unrecognized membrane restoration pathway by which cells preserve the functional integrity of lysosomes.

Pathogenic variants of sphingomyelin synthase SMS2 disrupt lipid landscapes in the secretory pathway

“…SM adopts an asymmetric distribution across the bilayers of late secretory and endolysosomal organelles, with the bulk of SM residing in the luminal/exoplasmic leaflet. However, using GFP-tagged EqtSM as cytosolic SM reporter (EqtSM cyto ), we found that perturbation of lysosome or PM integrity by pore-forming chemicals or toxins disrupts transbilayer SM asymmetry by triggering a rapid transbilayer movement of SM catalyzed by Ca 2+ -activated scramblases (Niekamp et al, 2022). To perform its central task in membrane biogenesis, the ER harbors constitutively active scramblases that enable a rapid equilibration of newly synthesized phospholipids across its bilayer (Pomorski and Menon, 2016).…”

“…How nSMase-2 gains access to SM, which is normally concentrated in the exoplasmic leaflet, is unclear. We recently showed that minor lesions in PM integrity initiates a rapid SM scrambling mediated by the Ca 2+ -activated scramblase TMEM16F (Niekamp et al, 2022). Intriguingly, loss of TMEM16F leads to decreased mineral deposition in skeletal tissues (Ehlen et al, 2013), suggesting that this process may require a TMEM16F-mediated supply of exoplasmic SM to nSMase-2 for SM hydrolysis in the cytosolic leaflet.…”

“…The constructs encoding FLAG-tagged EqtSM (pET28a-EQ-SM-3xFLAG), GFP-tagged EqtSM SS (pN1-EqtSM SS -oxGFP) and GFP-tagged EqtSol SS (pN1-EqtSol SS -oxGFP) were described in (Deng et al, 2016). The constructs encoding GFP-tagged EqtSM cyto (pN1-EqtSMcyto-oxGFP), GFP-tagged EqtSol cyto (pN1-EqtSol cyto -oxGFP) and mKate-tagged EqtSMcyto (pN1-EqtSMcyto-mkate) were described in (Niekamp et al ., 2022). The construct encoding mCherry-tagged VAPA was described in (Jain et al, 2017).…”

“…Human fibroblasts, human cervical carcinoma HeLa cells (ATCC CCL-2), human osteosarcoma epithelial U2OS cells (ATCC HTB-96), and human embryonic kidney 293 cells transformed with Simian Virus 40 large T antigen (HEK293T, ATCC CRL-3216) were cultured in high glucose Dulbecco's modified Eagle's medium (DMEM) containing 2 mM L-glutamine and 10% FBS, unless indicated otherwise. A HeLa cell-line lacking SMS1 and SMS2 (ΔSMS1/2) was described previously (Niekamp et al, 2022). DNA transfections were carried out using Lipofectamine 3000 (Thermo Fisher Scientific, L3000001) according to the manufacturer's instructions.…”

The ESCRT Machinery: Remodeling, Repairing, and Sealing Membranes

“…An additional factor could be the Parkinson s-disease-related leucine-rich repeat kinase 2 (LRRK2), which upon lysosome damage triggers the recruitment of the small GTPase Rab8A and subsequently the ESCRT-III protein CHMP4B to damaged organelles in macrophages [129]. Interestingly, Ca 2+ efflux can also trigger additional ESCRT-independent lysosomal repair mechanisms mediated by annexins A1 and A2 [130] and sphingomyelin scrambling and turnover [131].…”