Plasma Membrane Repair Is Regulated Extracellularly by Proteases Released from Lysosomes

Abstract

Eukaryotic cells rapidly repair wounds on their plasma membrane. Resealing is Ca2+-dependent, and involves exocytosis of lysosomes followed by massive endocytosis. Extracellular activity of the lysosomal enzyme acid sphingomyelinase was previously shown to promote endocytosis and wound removal. However, whether lysosomal proteases released during cell injury participate in resealing is unknown. Here we show that lysosomal proteases regulate plasma membrane repair. Extracellular proteolysis is detected shortly after cell wounding, and inhibition of this process blocks repair. Conversely, surface protein degradation facilitates plasma membrane resealing. The abundant lysosomal cysteine proteases cathepsin B and L, known to proteolytically remodel the extracellular matrix, are rapidly released upon cell injury and are required for efficient plasma membrane repair. In contrast, inhibition of aspartyl proteases or RNAi-mediated silencing of the lysosomal aspartyl protease cathepsin D enhances resealing, an effect associated with the accumulation of active acid sphingomyelinase on the cell surface. Thus, secreted lysosomal cysteine proteases may promote repair by facilitating membrane access of lysosomal acid sphingomyelinase, which promotes wound removal and is subsequently downregulated extracellularly by a process involving cathepsin D.

Lysosome exocytosis is required for mitosis

“…To probe the functional requirement of lysosome exocytosis specifically during the small time window encompassed by mitosis we decided to employ acute treatment of cells with pharmacological inhibitors. Silencing of lysosomal SNARE proteins to inhibit exocytosis was considered but rejected as an experimental approach for two reasons: 1) Lysosomal SNAREs such as VAMP7 are also known to be abundant on late endosomes (Chiaruttini et al, 2016). Therefore, siRNA depletion of this protein would potentially influence the behaviour of endocytic compartments other than lysosomes; 2) siRNA would also require transfection of cells for many days which would increase the chances of observing effects on lysosome physiology unrelated to mitosis.…”

“…In addition lysosomes are important calcium (Ca 2+ ) signalling platforms (Kilpatrick et al, 2013) and can facilitate repair of mechanical damage to the PM in most cell types (Rodriguez et al, 1997). This latter function of lysosomes depends upon their ability to undergo Ca 2+ -dependent exocytosis at the PM and a combination of lysosomal membrane material and soluble lysosomal hydrolases appear important in the process of wound healing (Castro-Gomes et al, 2016). More recently it has been proposed that excessive lysosomal exocytosis may in-part be responsible for the enhanced invasiveness of cancer cells (Liu et al, 2012;Machado et al, 2015).…”

Lysosome-Mediated Plasma Membrane Repair Is Dependent on the Small GTPase Arl8b and Determines Cell Death Type in Mycobacterium tuberculosis Infection

“…During trafficking and maturation, pro-cathepsin-D is cleaved resulting in a heavy chain of 48 kDa and a light chain of 33 kDa, which represents the active cathepsin-D (46). In agreement with our analysis of the LAMP-1 translocation, supernatants from Arl8b shRNA treated cells contained less cleaved cathepsin-D protein as compared to the supernatants of control shRNA treated cells (Fig 2E, compare lane 2 to lane 4).…”

Cellular mechanisms and signals that coordinate plasma membrane repair

“…Ceramide is formed directly as a result of injury-triggered release of acid sphingomyelinase (ASM) due to dysferlin and synaptotagmin-mediated lysosome exocytosis (see discussion above) [32,75]. Locally high concentrations of ASM catalyze local formation of ceramide from plasma membrane sphingomyelin [134]). Ceramide clusters may facilitate repair by inducing removal of the injured membrane by endocytosis or by ectocytic shedding [31,135].…”