were considered separately. The earlier directly perturb lysosomal ion transportation, while the latter directly perturb lysosomal morphology. 

[0277] As elaborated in the following subsections, the effects of the aforementioned lysosomal stressors on lyso› somal physiology were considered in the context of 1) biologically-determined variations in lysosomal morphol› ogy, 2) drug induced changes in lysosomal volume and surface area, 3) changes in lysosomal volume and surface area as may happen during endocytosis or exocytosis. 

[0278] Modeling the Effects of Individual Alteration of Lysosomal Proton and Chloride Transportation on Lyso› somal Physiology 

[0279] First, the effects of stress inducers that perturb lysosomal ion transporters (V-ATPase, CLC7, and mem› brane proton permeability) and ion content (cytoplasmic chloride) were studied in the context of a spherical lysosome (FIG. 23A). Among the stressors modeled, maximum reduc› tion in the number of V-ATPase molecule per lysosome induced significant physiological perturbation in a spherical lysosome, as evidenced by the following changes: 3.55 pH unit increment in lysosomal pH and 142.2 mM reduction in lysosomal Cl accumulation from their respective baseline values of 4.53 pH unit and 224.6 mM of an unperturbed lysosome (FIG. 23A). A similar physiological perturbation was observed following the modeling of the effect of increasing the lysosomal membrane proton permeability on lysosomal ion homeostasis (FIG. 23B). This indicates that lysosomal stressors that directly affect lysosomal proton level by perturbing either proton influx or efflux have similar effect not only on lysosomal pH, but also on lysosomal chloride homeostasis. To the contrary, CLC7 and cytoplas› mic Cl stressors induced comparatively less perturbation to the overall lysosomal physiology as they only affected lysosomal pH and membrane potential when chloride trans› port or concentration were completely abolished. 

[0280] To understand how the same lysosomal ion stress› ors affect lysosomal ion homeostasis of different lysosomal morphologies, the lysosomal ion stressors were each varied along with lysosomal volume stressors that simultaneously reduced lysosomal radius and volume from an unperturbed lysosomal radius of 340 nm and a corresponding volume of 1.65xl0- 16 L, to 40 nm and 2.88xl0- 17 L, respectively. These dimensional changes resulted in tubular lysosomes, which are known to be mainly prevalent before and during the initiation of material uptake (Knapp et al., 1990, supra). Accordingly, they are generally narrower and more elon› gated than spherical lysosomes (Swanson et al., 1987, supra). In spite of the lysosomal radius and volume differ› ences, tubular lysosomes have the same surface area as spherical lysosomes. This assumption is reasonable because balance in cellular membrane material is maintained as a result of the regulation of membrane trafficking upon tubular lysosome mediated exocytosis of endocytosed material from the subcellular spherical lysosome to the plasma membrane. Thus, based on the results, the lysosomal ion stressors showed to have similar effects on the physiology of tubular lysosome as that of spherical lysosome, reflected by the very similar changes in lysosomal pH, chloride, and membrane potential (FIG. 23). 

[0281] Modeling the Effects of Lysosomal Expansion on Lysosomal Ion Homeostasis 

[0282] Next, the effect of lysosomal swelling on lyso› somal physiology was modeled by simultaneously increas- 

ing lysosomal radius and surface area while maintaining a fixed lysosomal volume of a spherical lysosomal morphol› ogy, which equals 1.65xl0- 16 L. Starting from a non› perturbed spherical lysosome, which is 340 nm in radius with a corresponding surface area of 1.45xl0- 8 cm 2 , the lysosomal radius and the corresponding surface area were expanded up to 10 um and 6.28xl0- 6 cm 2 (433.3 fold change), respectively. Such lysosomal swelling led to a 2. 7 4 pH units increment in lysosomal pH, 125.6 mM reduction in lysosomal Cl accumulation, and 39.9m V increment in lyso› somal membrane potential (FIG. 24A). Thus, lysosomal expansion compromised the maintenance of physiological lysosomal ion homeostasis, similar to that of V-ATPase inhibition and membrane proton permeabilization in tubular and spherical lysosomes (FIG. 23). 

[0283] Identifying the Mechanisms of Lysosomal Stress Tolerance in Response to Lysosomal welling 

[0284] To understand the mechanism by which cells can withstand stress generated from simultaneous lysosomal radius and surface area expansion, key parameters that mediate lysosomal stress tolerance (referred to as "lyso› somal stress tolerance inducers") were identified by varying V-ATPase numbers and membrane proton permeability. For this purpose, V-ATPase number and membrane proton per› meability were individually increased and decreased, respectively, 0 to 20 and Oto 433 .3 fold from their respective baseline input values. This was performed in order to maintain a constant number of proton influx and efflux mediated by V-ATPase and membrane proton permeability, respectively, for a given lysosomal surface area. IT was observed that physiological ion homeostasis was fully restored when either the number ofV-ATPase molecules or the membrane proton permeability was kept proportional to the lysosomal surface area expansion at a fixed lysosomal volume of 1.65xl0-$^{16 }$L (FIG. 24B). 

[0285] Modeling the Effects of Altering Multiple Lyso› somal Ion Transport Pathways on Spherical, Tubular and Disc-Shaped Lysosomal Physiology 

[0286] Next, the mechanism by which various combina› tions of lysosomal ion stressors exert their effects on the physiology of lysosomes with distinct morphology was investigated. Different combinations of various ranges of lysosomal parameters to represent V-ATPase-CLC7 stress› ors, V-ATPase-cytoplasmic chloride stressors, V-ATPase -membrane proton permeability stressors, and CLC7-cyto› plasmic chloride stressors were used. These stressor com› binations were simulated to study their effects on the physi› ology of spherical, tubular, and disc-shaped lysosomes. 

[0287] For all of the lysosomal morphologies, maximum alteration of either CLC7 or cytoplasmic chloride param› eters in combination with alteration in either V-ATPase number or membrane proton permeability generally induced perturbation of lysosomal physiology due to the build-up of membrane potential by up to >250 mV (FIGS. 25B, 26 and 27). 

[0288] Although such increment in membrane potential is associated with the increment in lysosomal pH and reduction in lysosomal chloride accumulation, greater perturbations in both variables (>4 pH unit increment in lysosomal pH and >150 mM reduction in lysosomal chloride accumulation) were observed when at least either the V-ATPase number (FIGS. 25B, 25C, 26B, 27 and 28) or membrane proton permeability (FIGS. 25C, 28, 30 and 31) were maximally altered from their respective baseline values. 

