Mechanism of polarized lysosome exocytosis in epithelial cells

Abstract

There was an error published in J. Cell Sci. 125, 5937-5943.In Fig. 4B the same image was inadvertently presented as the xy section (top panels) of syntaxin 4 staining in MDCK wild-type and MDCK m1A-KD cells. In the correct version (shown below), the appropriate xy image for m1A-KD (top panel, middle) matches the corresponding xz panel (bottom panel) that was part of the original figure.The mistake in the figure did not affect the conclusions of the paper. Fig. 4B is shown below. The correctThe authors apologise for this mistake. Summary Fusion of lysosomes with the plasma membrane is a calcium-dependent process that is crucial for membrane repair, limiting pathogen entry and clearing cellular debris. In non-polarized cells, lysosome exocytosis facilitates rapid resealing of torn membranes. Here, we investigate the mechanism of lysosome exocytosis in polarized epithelia, the main barrier between the organism and the external environment and the first line of defense against pathogens. We find that in polarized Madin-Darby canine kidney (MDCK) cells, calcium ionophores or pore-forming toxins cause lysosomes to fuse predominantly with the basolateral membrane. This polarized exocytosis is regulated by the actin cytoskeleton, membrane cholesterol and the clathrin adaptor AP-1. Depolymerization of actin, but not microtubules, causes apical lysosome fusion, supporting the hypothesis that cortical actin is a barrier to exocytosis. Overloading lysosomes with cholesterol inhibits exocytosis, suggesting that excess cholesterol paralyzes lysosomal traffic. The clathrin adaptor AP-1 is responsible for accurately targeting syntaxin 4 to the basolateral domain. In cells lacking either the ubiquitous AP-1A or the epithelialspecific AP-1B, syntaxin 4 is non-polar. This causes lysosomes to fuse with both the apical and basolateral membranes. Consistent with these findings, RNAi-mediated depletion of syntaxin 4 inhibits basolateral exocytosis in wild-type MDCK, and both apical and basolateral exocytosis in cells lacking AP-1A or AP-1B. Our results provide fundamental insight into the molecular machinery involved in membrane repair in polarized epithelia and suggest that AP-1 is a crucial regulator of this process.

Protective responses to sublytic complement in the retinal pigment epithelium

“…Lysosome exocytosis causes transient appearance of lysosomal membrane proteins (e.g., LAMP2) on the plasma membrane and release of lysosomal hydrolases such as β-hexosaminidase (β-hex) into the extracellular medium (25). We detected LAMP2 on the RPE cell surface after a 10-min exposure to either 10% NHS (Fig.…”

“…We have shown that excess cholesterol in RPE with vitamin A dimers activates ASMase, which interferes with organelle traffic by acetylating microtubules (MT) (20). To investigate how excess cholesterol and MT acetylation impacts CD59 trafficking, we treated RPE monolayers with either U18666A to increase cholesterol (25) and, thereby, acetylated MT (20), or with the HDAC6 inhibitor tubacin, which prevents MT deacetylation (26). Similar to A2E, both drugs decreased cell surface CD59 and increased MAC deposition after complement exposure (Fig.…”

“…One of the earliest detectable events after complement exposure is an increase in intracellular calcium ([Ca 2+ ] i ) via influx through MAC pores (24) and/or activation of endogenous ion channels (27). In many cell types, elevated [Ca 2+ ] i has been shown to trigger fusion of lysosomes with the plasma membrane (25,28). Lysosome exocytosis, mediated by the lysosomal calcium sensor synaptotagmin 7 (Syt7), is vital for preserving membrane integrity after exposure to pore-forming toxins (29).…”

“…For surface immunostaining, live cells were incubated with primary antibodies (30 min, 4°C), fixed, permeabilized, and processed as above for ZO-1 labeling. Antibodies used were as follows: CD59 (1:100; ThermoFisher), LAMP2 (1:500; AbDSerotec), acetylated tubulin (1:1000; Sigma), C5b-9 (1:100; Novus), and ZO-1 (1:3000) (25). Other reagents were as follows: rhodaminephalloidin (1:200, PHDR1; Cytoskeleton), Alexa-conjugated secondary antibodies (1:500), and DAPI (1:200), both from ThermoFisher.…”

Lysosomal exocytosis and lipid storage disorders

“…In polarized epithelial cells, lysosomal exocytosis is directed toward the basolateral membranes, but not the apical membranes ( 74 ). The directionality of lysosomal exocytosis in these cells could be caused by the localization of the plasma membrane SNARE, syntaxin4, exclusively at the basolateral membrane, resulting in a "polarized" formation of the SNARE complex ( 74 ).…”