Lysosomal size matters

Abstract Lysosomes are key cellular catabolic centers that also perform fundamental metabolic, signaling and quality control functions. Lysosomes are not static and they respond dynamically to intra‐ and extracellular stimuli triggering changes in organelle numbers, size and position. Such physical changes have a strong impact on lysosomal activity ultimately influencing cellular homeostasis. In this review, we summarize the current knowledge on lysosomal size regulation, on its physiological role(s) and association to several disease conditions.


| INTRODUCTION
Initially described by Christian de Duve, 1,2 lysosomes are key mediators of protein degradation with a pivotal role in the coordination of cellular metabolism and intracellular signaling. 3 Lysosomes participate in many biological processes, including antigen presentation, plasma membrane repair, exosome release, cellular adhesion and migration, apoptosis, gene regulation, tumor invasion and metastasis (reviewed in References [3][4][5] ).
The complexity of lysosomes is emphasized by data from proteome analyses: ion channels and transporters regulate the luminal ion composition, 6,7 a dedicated H+-ATPase maintains the luminal acidic pH, 8,9 tethering factors and SNARE proteins at the membrane control fission and fusion with communicating cellular compartments. 10,11 In addition, around 200 glycosylated and non-glycosylated integral membranes proteins fulfill several critical functions including, for example, the transport of metabolites and the protection of the organelle membrane from degradation. 5,12 In response to diverse intra-and extracellular stimuli, lysosomes continuously adjust their numbers, size and position. It has been previously demonstrated that the localization and motility of those organelles significantly affect their activity. 13,14 Compromised lysosomal positioning is associated with detrimental effects present in several disease conditions, most prominently cancer. [15][16][17] It is also intuitively clear that changes in lysosome number influence the degree to which a certain function can be performed. Interestingly, among the different parameters, the contribution of lysosomal size remained somewhat obscure. Does size matter and if so, to what extent is it linked to organelle numbers?
Various terms are used to differentiate specific intracellular compartments, including organelles of the endocytic and autophagic routes. For clarity reasons, we would like to define the terms used in this review. Late endosomes fuse with terminal lysosomes forming a hybrid compartment called endolysosome. 18 Endolysosomes and terminal lysosomes coexist in a dynamic equilibrium that is maintained by lysosomal reformation. 19 Unless otherwise stated, we use the term lysosome to identify both endolysosomes and lysosomes, jointly corresponding to very late stages-frequently the very end of the endocytic pathway. Whenever necessary, endolysosomes and terminal lysosomes are discriminated as such. As far as autophagy is concerned, autolysosomes arise from the fusion of autophagosomes with lysosomes. 20 In the following, we recapitulate general aspects of lysosomal geometry and size, based on two-dimensional (2D) electron microscopy (EM) snapshots, morphometric measurements, counts and calculations. As already demonstrated by the pioneers in the field, 21 terminal lysosomes are typically spherical organelles, appearing as round or ovoid/elliptical section profiles (eg, Figure 1A), with occasional, short-lived tubular extensions (eg, during lysosome reformation 22 ). For practical reasons, we exclude the less common, tube-shaped lysosomes 22,23 from the following considerations. EM-based morphometry 24 is unique in providing a solid basis for the precise detection of even moderate qualitative and quantitative alterations of organelle ultrastructure, especially when cryo-fixation methods 25 are used for sample preparation. Cryo-fixation also traps transient and/or labile membrane configurations 26,27 and efficiently prevents uneven artifactual organelle shrinkage, that selectively affects different maturation stages of endocytic compartments. 28 To mention an exemplary case, HT1080 cells (human fibrosarcoma cell line carrying an N-ras mutation 29 ) have globular lysosomes with an average diameter of 410 nm. 30 In contrast, lysosomes of HT1080 cells, where components of the BORC (BLOC-1 related complex) complex have been deleted by CRISPR/Cas9 genome editing, are only 311 nm wide ( Figure 1 and Reference 30). Further discussion of this difference follows later in this article. Although a ≈25%reduction of organelle diameter might appear minor at first glance, simple arithmetic allows the conversion of the 2D-value into 3D-reality. As illustrated in Table 1, a 25% diameter reduction yields about half the surface area or the volume of spherical organelles-a quite relevant physiological difference. Bearing this F I G U R E 1 Electron micrographs of snap-frozen lysosomes displaying general morphology and size variations of the almost spherical organelles in HT1080 human fibrosarcoma cells. The term lysosome is used in a broad sense comprising both terminal lysosomes resulting from cargo endocytosis as well as autolysosomes. Those organelles are characterized by an opaque matrix, containing frequently more or less degraded (membrane) material and/or a completely electron-dense core. A, Under wildtype conditions the lysosomal diameter (white arrow) is on average 400 nm. B, After deletion of BLOC-1 related complex (BORC) the mean diameter decreases to approximately 300 nm; m = mitochondrion; scale bar = 400 nm T A B L E 1 Estimation of lysosomal (LY) size changes as calculated from ultrastructure morphometry measurements of cryo-fixed samples  33 In addition, it has been shown that increased lysosomal size negatively influences exocytosis. 34 Enlarged lysosomes in fibroblasts from Chediak-Higashi patients as well as from beige mice displayed reduced exocytosis that could be reverted by treatment with E-64d, a protease inhibitor, reducing lysosomal size in those cells. 34 Mechanistically, E-64d treatment protects PKC from ceramide-induced, calpain-mediated degradation thereby reducing lysosomal size. 35 The protease inhibitor also increases lysosomal elastase and cathepsin G activity. 35 These results imply that the impaired exocytosis observed could be attributed to the abnormal size of lysosomes.
Importantly, since lysosomal size is altered in several human diseases this might directly or indirectly contribute to the pathophysiology of the different conditions. Below, we summarize the current knowledge of factors contributing to the regulation of lysosomal size and frequency and describe human disorders associated with alterations of endolysosome-, phagolysosome-and/or lysosome-size. In Figure 2 we schematically guide the reader through the following chapters and introduce key components and protein machineries of late endosomes/lysosomes/autophagosomes and their involvement in the regulation of organelle size. Importantly, the rate of acidification is not uniform among terminal endocytic organelles, strongly depending on the organelle's position within a cell 36 and on its role within the reformation cycle. 19 In brief, terminal lysosomes are only moderately acidic, smaller and acidhydrolase inactive, whereas endolysosomes tend to be bigger, acidify and are catabolically active. Although not formally proven, it has been proposed that pH fluctuations might facilitate the function of lysosomal enzymes with neutral or alkaline pH optima. 37 Inhibition of the V-ATPase blocks the transport of endocytic markers from early endosomes to more acidic organelles. In other words, the V-ATPase is required for the formation of intermediate compartments between early and late endosomes, generally designated as multivesicular bodies (MVBs). 38 In addition, it has been proposed since two decades that the V-ATPase regulates both fission and fusion events, thereby contributing to the control of the lysosome/vacuole size (Figure 2A

| PI METABOLISM AND ORGANELLE SIZE
Weak base compounds like Chloroquine were some of the first agents found to trigger swelling of lysosomes, 54 followed by experiments using Wortmannin. 55 In melanoma cells, the latter one, a PI 3-kinase inhibitor, leads to inflation of the organelles in a process dependent on endocytic membrane influx. Importantly, Wortmannin inhibition also triggers a decrease in the number of intraluminal vesicles of MVBs. 55 These observations, highlighted the importance of phosphatidylinositol-3phosphate (PtdIns(3)P) the product of PI3K activity, in maintaining the size and structure of late endocytic organelles. Interestingly, PtdIns(3)P levels also control autophagosomal size. 53,56 Two decades ago, PtdIns(3)P was found to accumulate within MVBs. 57 Among the different PtdIns ( subunit and RAB7-GTP. 62,63 In turn, Rab7 activity is kept in check by TBC1D5, a Rab7 GAP that depends on retromer for its membrane association ( Figure 2C ). 64 Of note, Rab7 hyperactivation, either by overexpression or because of impairment of TBC1D5 function, leads to the formation of enlarged endolysosomes, increased transport of cargo along retromer dependent routes and a decrease of mitophagy. 65,66 Mechanistically, the increase in Rab7 prevents autophagic lysosome reformation (ALR), thereby triggering the accumulation of enlarged organelles. 22 Like HRS, PIKfyve (also known as 1-phosphatidylinositol 3-phosphate

5-kinase) is a PtdIns(3)P effector that contains an FYVE domain critical
for its membrane localization. 67  The ternary complex is known as PAS (PIKfyve-ArPIKfyve-Sac3) complex. 70 ArPIKfyve, the structural mediator, maintains the integrity of the complex via homo and heteromeric interactions with the remaining subunits. 71 Overall, the PAS complex is organized to provide optimal PIKfyve functionality. 72 In addition, PAS contains two enzymes with opposing effects, PIKfyve that catalyzes PtdIns(3,5)P 2 production, and sac3 that converts it into PtdIns(3)P. A peculiarity of PAS is that the presence of the phosphatase sac3 is also required for PIKfyve activation. 73,74 PtdIns(3,5)P 2 is a low abundant lipid, typically accounting for 0.04% to 0.08% of total inositol phospholipids present in human cells. 75 Despite the minute expression levels, PtdIns(3,5)P 2 is an essential lipid species and its depletion in mice, Drosophila melanogaster and Caenorhabditis elegans is embryonic lethal. [76][77][78][79] In mammalian cells, ablation of PtdIns(3,5)P 2 induces an imbalance in endosomal membrane homeostasis resulting in the enlargement of both early and late endosomal/lysosomal compartments. 80 Interestingly, too much PtdIns(3,5)P 2 can also be detrimental to lysosomal homeostasis. In Raptor. [83][84][85] Of note, deletion of Atg18 triggers enlargement of the vacuole that is phenotypically indistinguishable from fab1/PIKfyve ablation. 84 In addition, the presence of PtdIns(3,5)P 2 is recognized by the PX domain containing sorting nexins 1 and 2 (SNX1 and SNX2) 86,87 and the sec14 domain containing clavesin. 88 Interestingly, clavesin knockdown in neurons also enlarges lysosomal-associated membrane protein 1 (LAMP1) positive structures. 88 In plants, PtdIns  27 Under these circumstances, organelles coalesce, thereby decreasing their numbers and increasing their size. 94 Both of these processes, retrograde transport and lysosomal reformation utterly, depend on efficient fission. In contrast, the contribution of an imbalanced endocytic pathway to PIKfyve ablation phenotypes seems still a matter of debate with some authors claiming a block in the degradation of both EGFR and c-Met, 91 whereas others observe no clear defects. 80 Mechanistically, the size of an organelle is to a large extent dependent on the rate of membrane exchange, separated in the relative contributions of fusion and fission processes. In lysosomes, high levels of juxta-organellar calcium, released from the intraluminal pool, were detected in the vicinity of fusion/fission sites. Interestingly, PtdIns(3,5)P 2 interacts with the mucolipin transient receptor potential channels (TRPMLs), activating them and promoting calcium transport across the organelle membrane 95 ( Figure 2B). In a reverse manner, PtdIns(4,5)P 2 was shown to inhibit TRPML1 activity. 96  TPC are lysosomal two-pore channel proteins that were initially identified as mediators of nicotinic acid adenine dinucleotide phosphate (NAADP)-dependent calcium release. 7 Later, TPCs were shown to transport sodium upon activation by PtdIns(3,5)P 2 . 102,103 The ion selectivity properties of TPC remains a controversial topic and might be dependent on the experimental conditions. 104 Importantly, TPCmediated calcium transport was shown to rapidly reduce and reverse the membrane potential, thereby promoting organelle fusion ( Figure 2E ). In accordance to its mode of action, TPC overexpression induced enlarged lysosomes. 102   Surprisingly, recent work revealed that PtdIns(4,5)P 2 negatively regulates autophagosome-lysosome fusion. In brief, its production triggers the dissociation of Rab7 and PLEKHM1 from the organelle membrane. 113 Whether PtdIns(4,5)P 2 activates a Rab7 GAP remains to be formally proven. In any case, PtdIns(4,5)P 2 is emerging as pivotal player in the control of both fusion and reformation events actively shifting the balance towards the latter.
Another critical aspect of ALR is the retention mechanism that prevents diffusion of cargo and/or regulatory proteins from the parental autolysosome to the forming proto-lysosome. In PI4KIIIβ depleted cells, lysosomal luminal components were aberrantly found in reformation tubules indicating that PI4KIIIβ actively contributes to retention. 114 In addition, PI4KIIIβ ablation leads to hypertubulation that could only be rescued by expression of the catalytically active form of PI4KIIIβ. These data imply that PI4KIIIβ's control of lysosome tubulation and cargo retention is mediated by PtdIns(4)P. 114 Disturbances in ALR are currently linked to hereditary spastic paraplegia and Parkinson disease. In brief, mutations in spastizin and spatacsin trigger the two most common forms of autosomal recessive hereditary spastic paraplegia. Their ablation leads to the accumulation of enlarged autolysosomes at the expense of lysosomes, a phenotype typical of ALR. 115 In addition, spatacsin knockout mice display loss of cortical neurons and Purkinje cells consistent with a spastic paraplegia-like phenotype. 116 Although not formally proven, it has been proposed that ALR defects may result in a reduction of functionally active lysosomes that in turn would reduce autophagic clearance.  30 and is additionally dependent on LAMTOR/ Ragulator complex ( Figure 2B). Interestingly, both LAMTOR and BORC knockout cells showed reduced lysosomal size, reaching only about 75% of those in WT cells. 26,30 Of note, the reduction of lysosomal diameter was associated with increased lysosomal frequency 26,30 probably reflecting some kind of physiological compensation.
Finally, it is worth mentioning that we observed a further, additive effect on the size reduction of lysosomes in LAMTOR plus BORC double knockouts. 30 These data imply the existence of two independent mechanisms contributing to the observed size regulation. Nowadays, it is clear that the spectrum of LSD must also include other genetic alterations that disturb the synthesis and/or transport of lysosomal proteins and cargo and that are causal for the characteristically enlarged structures. Independently of its origin, the accumulation of nondegradable material within lysosomes has a profound impact on the organelle's physiology, size, trafficking and overall degradative capacity. [145][146][147][148] As an example, sphingomyelin accumulation in Niemann-Pick disease cells, blocks TRPML1 and calcium-dependent lysosomal functions. 149 In general, defects on soluble, luminal proteins in lysosomes are more frequent than those triggered by depletion of lysosomal membrane proteins. 150 The lysosomal-associated membrane protein The presence of inclusion bodies in several immunological cell types, including macrophages, T-lymphocytes, neutrophils and leucocytes serves as diagnostic marker for CHS. 179

| Treatment options for LSD and related diseases
There is light at the end of the tunnel for those suffering from LSD.
We have seen a rapid increase in our understanding of the processes regulating lysosomal homeostasis and function, as well as on the molecular basis of LSD and their associated pathophysiology. In paral- F I G U R E 3 Model summarizing the working hypotheses underlying LSD pathophysiology. Under healthy conditions (left part of the scheme), correct acidification, fusion and fission events maintain the organelle's function and morphology. In contrast, LSD display alterations of the regulatory mechanisms controlling the organelle's size (right part of the scheme), either as an impairment of autophagosome-lysosome fusion (highlighted in green, I) and/or as a reduction of lysosomal reformation (highlighted in green, II) that subsequently lead to the accumulation of autophagic compartments and enlarged endolysosomes. These alterations are accompanied by acidification (blue color gradient) defects that promote the accumulation of undegradable cargo in catabolically inactive organelles. Finally, these events are amplified in a feedforward loop that culminates in the progressive phenotype characteristic of LSD 7 | CONCLUDING REMARKS: PERSPECTIVES 7.1 | Does lysosomal size really matter?
Size, in our point of view, is a morphological readout of organelle homeostasis. As such, size is tightly maintained by the coordinated action of a large number of factors directly or indirectly regulating lysosomal fusion, fission and frequency. These include, but are not restricted to, the V-ATPase, phosphoinositides, mTORC1 signaling, coating and tethering factors, SNAREs, calcium transporters, actin and small GTPases. This impressive list of regulators of lysosomal size is far from being complete. As an example, recent work on organelle contact sites underscored their involvement in the regulation of size: ER contact sites define the position and timing of fission of early and late endosomes, whereas mitochondria-lysosome contacts were shown to promote Rab7 hydrolysis thereby regulating lysosomal size. 203,204 7.2 | What is the contribution of lysosomal size to disease?
A decade ago, it was proposed that LSD pathophysiology might root on reduced fusion efficiency between autophagosomes and lysosomes. 146 This block in autophagy would activate a compensatory feedback mechanism increasing autophagosome formation (Figure 3 right side, annotated in green as I). The variation in the severity of the symptoms expressed in the different LSD would depend on the strength of the fusion block and on the extent of the expansion of autophagosome/ lysosomal compartments generated as a consequence. 150 A second hypothesis raised to explain LSD progression, is based on impaired lysosomal reformation (Figure 3 right side, annotated in green as II). In brief, fibroblasts from patients with different LSD (Scheie syndrome, Fabry disease and Aspartylglucosaminuria) show compromised mTOR reactivation and ALR defects. 22 The tight interplay between lysosomal catabolism and ALR would explain why minor defects in either of two processes are amplified in a positive feedback cycle, eventually triggering the progressive pathology of LSD. 205 These hypotheses have two aspects in common. First, independently of the initiating mechanism, the secondary progressive accumulation of nondegradable cargo (from damaged organelles to ubiquitinated proteins) seems to play a pivotal role in LSD pathology.
Second, both of the mechanisms proposed control the organelle's size.
In Figure 3 we present a model that tries to summarize the working hypotheses underlying LSD pathophysiology, including fusion, fission and acidification defects, cargo accumulation and increased organelle size.
The emerging mechanistic and phenotypical similarities between LSD and other diseases, in particular those with increased lysosomal size, highlight the need to reconsider disease boundaries. 191 In all of these disorders, independently of the initiating factor, the intertwined dynamic balance between fission, fusion, and number of lysosomes is impaired and actively contributes to disease progression. To cut it short, lysosomal size does indeed matter.

ACKNOWLEDGMENTS
This work was supported by the Austrian Science Fund (FWF DK W11 to GL and stand-alone P 32608 to LAH) and the Molecular Cell Biology and Oncology PhD program (MCBO) at the Medical University of Innsbruck.