Connexin43 promotes exocytosis of damaged lysosomes through actin remodelling.

A robust and efficient cellular response to lysosomal membrane damage prevents leakage from the lysosome lumen into the cytoplasm. This response is understood to happen through either lysosomal membrane repair or lysophagy. Here we report exocytosis as a third response mechanism to lysosomal damage, which is further potentiated when membrane repair or lysosomal degradation mechanisms are impaired. We show that Connexin43 (Cx43), a protein canonically associated with gap junctions, is recruited from the plasma membrane to damaged lysosomes, promoting their secretion and accelerating cell recovery. The effects of Cx43 on lysosome exocytosis are mediated by a reorganization of the actin cytoskeleton that increases plasma membrane fluidity and decreases cell stiffness. Furthermore, we demonstrate that Cx43 interacts with the actin nucleator Arp2, the activity of which was shown to be necessary for Cx43-mediated actin rearrangement and lysosomal exocytosis following damage. These results define a novel mechanism of lysosomal quality control whereby Cx43-mediated actin remodelling potentiates the secretion of damaged lysosomes.

Upregulation of cholesterol synthesis by lysosomal defects requires a functional mitochondrial respiratory chain.

Mitochondria and lysosomes are two organelles that carry out both signaling and metabolic roles in cells. Recent evidence has shown that mitochondria and lysosomes are dependent on one another, as primary defects in one cause secondary defects in the other. Although there are functional impairments in both cases, the signaling consequences of primary mitochondrial dysfunction and lysosomal defects are dissimilar. Here, we used RNA sequencing to obtain transcriptomes from cells with primary mitochondrial or lysosomal defects to identify the global cellular consequences associated with mitochondrial or lysosomal dysfunction. We used these data to determine the pathways affected by defects in both organelles, which revealed a prominent role for the cholesterol synthesis pathway. We observed a transcriptional upregulation of this pathway in cellular and murine models of lysosomal defects, while it is transcriptionally downregulated in cellular and murine models of mitochondrial defects. We identified a role for the posttranscriptional regulation of transcription factor SREBF1, a master regulator of cholesterol and lipid biosynthesis, in models of mitochondrial respiratory chain deficiency. Furthermore, we found that retention of Ca2+ in lysosomes of cells with mitochondrial respiratory chain defects contributes to the differential regulation of the cholesterol synthesis pathway in the mitochondrial and lysosomal defects tested. Finally, we verified in vivo, using a model of mitochondria-associated disease in Caenorhabditis elegans that normalization of lysosomal Ca2+ levels results in partial rescue of the developmental delay induced by the respiratory chain deficiency.

Role of lipids in interorganelle communication.

Cell homeostasis and function rely on well-orchestrated communication between different organelles. This communication is ensured by signaling pathways and membrane contact sites between organelles. Many players involved in organelle crosstalk have been identified, predominantly proteins and ions. The role of lipids in interorganelle communication remains poorly understood. With the development and broader availability of methods to quantify lipids, as well as improved spatiotemporal resolution in detecting different lipid species, the contribution of lipids to organelle interactions starts to be evident. However, the specific roles of various lipid molecules in intracellular communication remain to be studied systematically. We summarize new insights in the interorganelle communication field from the perspective of organelles and discuss the roles played by lipids in these complex processes.

Up-regulation of cholesterol synthesis by lysosomal defects requires a functional mitochondrial respiratory chain.

Mitochondria and lysosomes are two organelles that carry out both signaling and metabolic roles in the cells. Recent evidence has shown that mitochondria and lysosomes are dependent on one another, as primary defects in one cause secondary defects in the other. Nevertheless, the signaling consequences of primary mitochondrial malfunction and of primary lysosomal defects are not similar, despite in both cases there are impairments of mitochondria and of lysosomes. Here, we used RNA sequencing to obtain transcriptomes from cells with primary mitochondrial or lysosomal defects, to identify what are the global cellular consequences that are associated with malfunction of mitochondria or lysosomes. We used these data to determine what are the pathways that are affected by defects in both organelles, which revealed a prominent role for the cholesterol synthesis pathway. This pathway is transcriptionally up-regulated in cellular and mouse models of lysosomal defects and is transcriptionally down-regulated in cellular and mouse models of mitochondrial defects. We identified a role for post-transcriptional regulation of the transcription factor SREBF1, a master regulator of cholesterol and lipid biosynthesis, in models of mitochondrial respiratory chain deficiency. Furthermore, the retention of Ca 2+ in the lysosomes of cells with mitochondrial respiratory chain defects contributes to the differential regulation of the cholesterol synthesis pathway in the mitochondrial and lysosomal defects tested. Finally, we verified in vivo , using models of mitochondria-associated diseases in C. elegans , that normalization of lysosomal Ca 2+ levels results in partial rescue of the developmental arrest induced by the respiratory chain deficiency.

Loss of the lysosomal protein CLN3 modifies the lipid content of the nuclear envelope leading to DNA damage and activation of YAP1 pro-apoptotic signaling.

Batten disease is characterized by early-onset blindness, juvenile dementia and death during the second decade of life. The most common genetic causes are mutations in the CLN3 gene encoding a lysosomal protein. There are currently no therapies targeting the progression of the disease, mostly due to the lack of knowledge about the disease mechanisms. To gain insight into the impact of CLN3 loss on cellular signaling and organelle function, we generated CLN3 knock-out cells in a human cell line (CLN3-KO), and performed RNA sequencing to obtain the cellular transcriptome. Following a multi-dimensional transcriptome analysis, we identified the transcriptional regulator YAP1 as a major driver of the transcriptional changes observed in CLN3-KO cells. We further observed that YAP1 pro-apoptotic signaling is hyperactive as a consequence of CLN3 functional loss in retinal pigment epithelia cells, and in the hippocampus and thalamus of CLN3exΔ7/8 mice, an established model of Batten disease. Loss of CLN3 activates YAP1 by a cascade of events that starts with the inability of releasing glycerophosphodiesthers from CLN3-KO lysosomes, which leads to perturbations in the lipid content of the nuclear envelope and nuclear dysmorphism. This results in increased number of DNA lesions, activating the kinase c-Abl, which phosphorylates YAP1, stimulating its pro-apoptotic signaling. Altogether, our results highlight a novel organelle crosstalk paradigm in which lysosomal metabolites regulate nuclear envelope content, nuclear shape and DNA homeostasis. This novel molecular mechanism underlying the loss of CLN3 in mammalian cells and tissues may open new c-Abl-centric therapeutic strategies to target Batten disease.