Rab11 is required for lysosome exocytosis through the interaction with Rab3a, Sec15 and GRAB

Abstract

Lysosomes are dynamic organelles, capable of undergoing exocytosis. This process is crucial for several cellular functions, namely plasma membrane repair. Nevertheless, the molecular machinery involved in this process is poorly understood. Here, we identify Rab11a and Rab11b as regulators of calcium-induced lysosome exocytosis. Interestingly, Rab11-positive vesicles transiently interact with lysosomes at the cell periphery, indicating that this interaction is required for the last steps of lysosome exocytosis. Additionally, we found that the silencing of the exocyst subunit Sec15, a Rab11 effector, impairs lysosome exocytosis, suggesting that Sec15 acts together with Rab11 in the regulation of lysosome exocytosis. Furthermore, we show that Rab11 binds the guanine nucleotide exchange factor for Rab3a (GRAB) and also Rab3a, which we described previously as a regulator of the positioning and exocytosis of lysosomes. Thus, our study identifies new players required for lysosome exocytosis and suggest the existence of a Rab11-Rab3a cascade involved in this process.

Current methods to analyze lysosome morphology, positioning, motility and function

“…Moreover, this is a Ca 2+ ‐regulated process that occurs in almost all cell types. Therefore, Ca 2+ ionophores like ionomycin (Sigma, I9657) 201,278 or agonists of the Ca 2+ channel present in the lysosome membrane —TRPML1— such as ML‐SA1 (Sigma, SML0627) and MK6‐83 (AbMole, M10315), are commonly used to induce lysosome exocytosis 279 . Furthermore, the extracellular acidic environment found in tumours 280,281 and altered expression of genes that regulate lysosome positioning and motility 282 can also lead to an increase in lysosome exocytosis.…”

“…The unprocessed and processed (mature) forms of lysosomal enzymes can be detected in the extracellular media, by WB, using specific antibodies that are commercially available ( e.g ., anti‐cathepsin D from Abcam, ab6313; anti‐cathepsin B from R&D Systems, AF953; and anti‐cathepsin L from R&D Systems, AF952) 286,287 . The activity of secreted lysosomal enzymes, such as β‐hexosaminidase (β‐hex), acid‐phosphatase, β‐N‐acerylglucosaminidase, β‐galactosidase, acid sphingomyelinase and cathepsins can be measured, using appropriate fluorometric or colorimetric substrates 201,278,288–290 . For example, the activity of secreted β‐hex can be measured by incubating cell supernatants and cell lysates with the substrate 4‐MU‐β‐d‐glucosaminide (4‐MU‐β‐D‐GlcNAc, Glycosynth, 44007).…”

“…Thus, detection of LAMP1 with a monoclonal antibody for a luminal epitope of the protein (e.g., anti-LAMP1 H4A3) provides a good readout of lysosome exocytosis. 279,283 Cell surface LAMP1 can be measured by flow cytometry 201,278 or fluorescence microscopy. 279,284 When live cells are used, cell should be kept at 4 C to avoid antibody endocytosis.…”

“…286,287 The activity of secreted lysosomal enzymes, such as β-hexosaminidase (β-hex), acidphosphatase, β-N-acerylglucosaminidase, β-galactosidase, acid sphingomyelinase and cathepsins can be measured, using appropriate fluorometric or colorimetric substrates. 201,278,[288][289][290] For example, the activity of secreted β-hex can be measured by incubating cell supernatants and cell lysates with the substrate 4-MU-β-d-glucosaminide (4-MU-β-D-GlcNAc, Glycosynth, 44007). The detection of LAMP1 at the plasma membrane can be easily performed and does not require much expertise.…”

The Dark Side of Melanin Secretion in Cutaneous Melanoma Aggressiveness

“…Indeed, in CM cells, lysosome exocytosis enhances plasma membrane repair after UVr exposure, as well as the release of the lysosomal proteases Cathepsin B and K, which in turn degrade the ECM, thereby promoting CM invasion and metastasis in vitro and in vivo ( 181 – 184 ). Rab11 was also shown by us to cooperate with Rab3a in plasma membrane repair in HeLa cells ( 185 ). Curiously, both Rab11a and Rab11b regulate melanin release from melanocytes and stimulate EMT in CM cells by switching the expression of E-cadherin to N-cadherin at the cell surface ( 168 , 170 , 186 , 187 ).…”