US 2019/0262453 Al  29  Aug. 29, 2019  [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.  [0282]  Next, the effect of lysosomal swelling on lyso› somal physiology was modeled by simultaneously increas- [0281]  Modeling the Effects of Lysosomal Expansion on  Lysosomal Ion Homeostasis  [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).  [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.  [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).  [0285]  Modeling the Effects of Altering Multiple Lyso› somal Ion Transport Pathways on Spherical, Tubular and  Disc-Shaped Lysosomal Physiology  [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).  [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.  [0283]  Identifying the Mechanisms of Lysosomal Stress  Tolerance in Response to Lysosomal welling  [0278]  Modeling the Effects of Individual Alteration of  Lysosomal Proton and Chloride Transportation on Lyso› somal Physiology  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).  [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.  were considered separately. The earlier directly perturb  lysosomal ion transportation, while the latter directly perturb  lysosomal morphology. 