MERIT, a cellular system coordinating lysosomal repair, removal and replacement

Membrane integrity is essential for cellular survival and function. The spectrum of mechanisms protecting cellular and intracellular membranes is not fully known. Our recent work has uncovered a cellular system termed MERIT for lysosomal membrane repair, removal and replacement. Specifically, lysosomal membrane damage induces, in succession, ESCRT-dependent membrane repair, macroautophagy/autophagy-dominant removal of damaged lysosomes, and initiation of lysosomal biogenesis via transcriptional programs. The MERIT system is governed by galectins, a family of cytosolically synthesized lectins recognizing β-galactoside glycans. We found in this study that LGALS3 (galectin 3) detects membrane damage by detecting exposed lumenal glycosyl groups, recruits and organizes ESCRT components PDCD6IP/ALIX, CHMP4A, and CHMPB at damaged sites on the lysosomes, and facilitates ESCRT-driven repair of lysosomal membrane. At later stages, LGALS3 cooperates with TRIM16, an autophagy receptor-regulator, to engage autophagy machinery in removal of excessively damaged lysosomes. In the absence of LGALS3, repair and autophagy are less efficient, whereas TFEB nuclear translocation increases to compensate lysosomal deficiency via de novo lysosomal biogenesis. The MERIT system protects endomembrane integrity against a broad spectrum of agents damaging the endolysosomal network including lysosomotropic drugs, Mycobacterium tuberculosis, or neurotoxic MAPT/tau. Abbreviations: AMPK: AMP-activated protein kinase; APEX2: engineered ascorbate peroxidase 2; ATG13: autophagy related 13; ATG16L1: autophagy related 16 like 1; BMMs: bone marrow-derived macrophages; ESCRT: endosomal sorting complexes required for transport; GPN: glycyl-L-phenylalanine 2-naphthylamide; LLOMe: L-leucyl-L-leucine methyl ester; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MERIT: membrane repair, removal and replacement; MTOR: mechanistic target of rapamycin kinase; TFEB: transcription factor EB; TFRC: transferrin receptor; TRIM16: tripartite motif-containing 16

Galectin-3 Coordinates a Cellular System for Lysosomal Repair and Removal

Jia et al. show that Galectin-3 recruits ESCRT components to damaged lysosomes for repair and restoration of their function. During sustained lysosomal injury, galectins induce autophagy and lysosomal biogenesis for a staged repair, removal, and replacement program. This response is deployed during damage with neurotoxic tau or Mycobacterium tuberculosis infection

Dysferlin Forms a Dimer Mediated by the C2 Domains and the Transmembrane Domain In Vitro and in Living Cells

Dysferlin was previously identified as a key player in muscle membrane repair and its deficiency leads to the development of muscular dystrophy and cardiomyopathy. However, little is known about the oligomerization of this protein in the plasma membrane. Here we report for the first time that dysferlin forms a dimer in vitro and in living adult skeletal muscle fibers isolated from mice. Endogenous dysferlin from rabbit skeletal muscle exists primarily as a ∼460 kDa species in detergent-solubilized muscle homogenate, as shown by sucrose gradient fractionation, gel filtration and cross-linking assays. Fluorescent protein (YFP) labeled human dysferlin forms a dimer in vitro, as demonstrated by fluorescence correlation spectroscopy (FCS) and photon counting histogram (PCH) analyses. Dysferlin also dimerizes in living cells, as probed by fluorescence resonance energy transfer (FRET). Domain mapping FRET experiments showed that dysferlin dimerization is mediated by its transmembrane domain and by multiple C2 domains. However, C2A did not significantly contribute to dimerization; notably, this is the only C2 domain in dysferlin known to engage in a Ca-dependent interaction with cell membranes. Taken together, the data suggest that Ca-insensitive C2 domains mediate high affinity self-association of dysferlin in a parallel homodimer, leaving the Ca-sensitive C2A domain free to interact with membranes

Phosphorylated Phospholamban Stabilizes a Compact Conformation of the Cardiac Calcium-ATPase

The sarcoendoplasmic reticulum calcium ATPase (SERCA) plays a key role in cardiac calcium handling and is considered a high-value target for the treatment of heart failure. SERCA undergoes conformational changes as it harnesses the chemical energy of ATP for active transport. X-ray crystallography has provided insight into SERCA structural substates, but it is not known how well these static snapshots describe in vivo conformational dynamics. The goals of this work were to quantify the direction and magnitude of SERCA motions as the pump performs work in live cardiac myocytes, and to identify structural determinants of SERCA regulation by phospholamban. We measured intramolecular fluorescence resonance energy transfer (FRET) between fluorescent proteins fused to SERCA cytoplasmic domains. We detected four discrete structural substates for SERCA expressed in cardiac muscle cells. The relative populations of these discrete states oscillated with electrical pacing. Low FRET states were most populated in low Ca (diastole), and were indicative of an open, disordered structure for SERCA in the E2 (Ca-free) enzymatic substate. High FRET states increased with Ca (systole), suggesting rigidly closed conformations for the E1 (Ca-bound) enzymatic substates. Notably, a special compact E1 state was observed after treatment with β -adrenergic agonist or with coexpression of phosphomimetic mutants of phospholamban. The data suggest that SERCA calcium binding induces the pump to undergo a transition from an open, dynamic conformation to a closed, ordered structure. Phosphorylated phospholamban stabilizes a unique conformation of SERCA that is characterized by a compact architecture