(D) BAPTA (2.5 mM) was added to chelate extracellular Ca 2+, and cyclopiazonic acid (CPA, 20 µM) to inhibit SERCAs, before the addition of GPN (200 µM) to fluo 8-loaded HEK cells. from 3–4 ROIs in a single cell (summarised in Fig. 3F,H). (C) Time courses of GPN-evoked changes in fluorescence (F/F 0) of the pH ly indicators. Increased pH causes fluorescence to decrease for LysoTracker Red and increase for the other indicators. (B) HEK cells loaded with LysoTracker Red, or with dextran conjugates of Oregon Green or fluorescein report an increase in pH ly after addition of GPN (200 µM for 200 s). (A) GPN is proposed to disrupt lysosomes because its cleavage by cathepsin C (blue arrow) causes osmotic lysis. GPN changes pH and c without rupturing lysosomes. However, Acridine Orange and LysoTracker, like other lysosomotropic agents, will redistribute across intact lysosomal membranes when pH ly increases. Repnik et al., 2017), but many more papers mistakenly assume that loss of Acridine Orange or LysoTracker signifies rupture of lysosomes (e.g. There is evidence (from release of dextran-conjugated fluorophores) that modified dipeptides can rupture lysosomes (e.g. It is assumed that GPN disrupts lysosomes because it is degraded within them by cathepsin C (hence, its selectivity for lysosomes) then the dipeptide accumulates, generating osmotic stress that ruptures lysosome membranes ( Berg et al., 1994). GPN ( Fig. 1A), another synthetic substrate of cathepsin C, has been used extensively to perturb lysosomes, with ∼100 publications reporting its use (e.g. The likely mechanism involves an initial lysosomotropic accumulation of LLOMe within lysosomes, causing pH ly to increase cathepsin C then catalyses polymerisation of the de-esterified dipeptide and, as the hydrophobic polymer accumulates, it perturbs the lysosomal membrane, rendering it permeable to small molecules (molecular mass<10 kDa) ( Repnik et al., 2017 Thiele and Lipsky, 1990). For example, the esterified dipeptide, l-leucyl- l-leucine methyl ester (LLOMe, also known as Leu-Leu-OMe), disrupts lysosomes and, thereby, triggers apoptosis. These properties of cathepsin C have been exploited to allow selective disruption of lysosomes. At neutral pH, cathepsin C can also polymerise dipeptides ( McGuire et al., 1992 Thiele and Lipsky, 1990). There are, however, few pharmacological opportunities to disable lysosomes: inhibition of the lysosomal V-ATPase (with bafilomycin A 1 or concanamycin A) allows the lysosomal pH gradient to be dissipated ( Drose and Altendorf, 1997), and glycyl- l-phenylalanine 2-naphthylamide ( l-GPN, hereafter referred to as GPN) is widely used, purportedly to disrupt lysosomal membranes ( Fig. 1A).Ĭathepsin C (CTSC, also known as dipeptidyl peptidase 1) is widely supposed to be expressed mainly within lysosomes ( Rao et al., 1997), where it cleaves a pair of N-terminal residues from its peptide substrates until it reaches a proline or basic residue. These proteins allow Ca 2+ to regulate lysosomal behaviour and allow lysosomes to contribute to cytosolic Ca 2+ signalling ( López Sanjurjo et al., 2013). Lysosomes also sequester Ca 2+ and express Ca 2+-permeable channels, notably transient receptor protein mucolipin (TRPML1), two pore channel type 2 (TPC2, encoded by TPCN2) and ATP-regulated P 2X 4 receptor (P2RX4) ( Morgan et al., 2011). They also mediate transfer of cholesterol and other lipids between membranes, and they contribute to membrane repair ( Thelen and Zoncu, 2017). Lysosomes coordinate responses to nutrient deprivation through their ability to sense amino acids, and regulate the biogenesis of lysosomes and autophagy proteins ( Sabatini, 2017). More than fifty degradative enzymes within lysosomes allow them to degrade materials imported by endocytosis, and to recycle intracellular materials by autophagy ( Luzio et al., 2014 Rubinsztein et al., 2012). Lysosomes are dynamic, membrane-bound organelles that maintain a low luminal pH (pH ly ∼4.5) ( Johnson et al., 2016).
0 Comments
Leave a Reply. |