Biogenesis of lysosome‐related organelles complex‐1 (BORC) regulates late endosomal/lysosomal size through PIKfyve‐dependent phosphatidylinositol‐3,5‐bisphosphate

Abstract Mechanisms that control lysosomal function are essential for cellular homeostasis. Lysosomes adapt in size and number to cellular needs but little is known about the underlying molecular mechanism. We demonstrate that the late endosomal/lysosomal multimeric BLOC‐1‐related complex (BORC) regulates the size of these organelles via PIKfyve‐dependent phosphatidylinositol‐3,5‐bisphosphate [PI(3,5)P2] production. Deletion of the core BORC component Diaskedin led to increased levels of PI(3,5)P2, suggesting activation of PIKfyve, and resulted in enhanced lysosomal reformation and subsequent reduction in lysosomal size. This process required AMP‐activated protein kinase (AMPK), a known PIKfyve activator, and was additionally dependent on the late endosomal/lysosomal adaptor, mitogen‐activated protein kinases and mechanistic target of rapamycin activator (LAMTOR/Ragulator) complex. Consistently, in response to glucose limitation, AMPK activated PIKfyve, which induced lysosomal reformation with increased baseline autophagy and was coupled to a decrease in lysosomal size. These adaptations of the late endosomal/lysosomal system reversed under glucose replete growth conditions. In summary, our results demonstrate that BORC regulates lysosomal reformation and size in response to glucose availability.

the endosomal system is the lysosome, it participates in numerous vital cellular processes, such as microbial killing and antigen presentation, detoxification, recycling of metabolic building blocks (amino acids, glucose and cholesterol), apoptosis, cell migration, cancer invasion and metastasis. 2,3 To adapt to cellular needs and environmental conditions, lysosomes must reform and continuously adapt in size and number. Many lysosomal functions depend on the position of the organelle in the cell. Lysosomes move readily back and forth between the perinuclear region and the cell periphery, 4 with a majority of them showing a steady state distribution close to the Microtubule Organizing Center (MTOC). The bidirectional movement of lysosomes along microtubules is important for distributing the degradative activity of lysosomes to all regions of the cell, as well as for many other lysosomal functions including antigen presentation, microbial killing, autophagy, metabolic signaling, cell adhesion and migration and tumor invasion and metastasis. 2,3,[5][6][7] To control lysosomal function and signaling capacity, many scaffolding complexes populate the limiting membrane of lysosomes, where they recruit and orchestrate multiple signal transduction cascades.
Two such multimeric endosomal scaffold complexes are the biogenesis of lysosome-related organelles complex 1 (BLOC-1) and the late endosomal/lysosomal BLOC-1-related complex (BORC). BLOC-1 comprises the subunits BLOS1, BLOS2, BLOS3, Snapin, Pallidin, Muted, Cappuccino and Dysbindin. [8][9][10][11] Recent observations suggest that BLOC-1 may participate in the biogenesis of recycling endosomes by coordinating endosomal tubule generation. 12 BORC consists only of a subset of BLOC proteins, BLOS1, BLOS2 and Snapin, and additionally employs the specific subunits of this complex KXD1, MEF2BNB, Myrlysin, Lyspersin and Diaskedin. 2 BORC associates with the cytoplasmic leaflet of the limiting late endosomal/lysosomal membrane, at least partially through an N-terminal myristoyl group on Myrlysin. 2 The main function attributed to BORC is the recruitment of the small Arf-like GTPase Arl8b from the cytoplasm onto the lysosomal membrane, which promotes the kinesin-dependent movement of lysosomes toward the cell periphery along microtubule tracks. 2,13 Consistently, BORC deficient cells accumulate lysosomes in the perinuclear region and exhibit reduced spreading and motility. Recently, we demonstrated that the C-terminus of the BORC subunit Lyspersin is essential and sufficient for BORC-dependent recruitment of Arl8b to lysosomes. 14 In addition, we established Lyspersin as the linker between BORC and LAMTOR/Ragulator complexes and identified LAMTOR/Ragulator as a negative regulator of BORC-and Arl8b-dependent lysosomal transport to the cell periphery. Importantly, growth factor stimulation as well as amino acid availability control lysosomal positioning through a LAM-TOR/Ragulator dependent, but mammalian target of rapamycin complex 1 (mTORC1)-independent pathway. 14,15 The BORC complex together with Arl8b is required to allow the movement of a subset of lysosomes to the periphery of the cell. 2,16 Here, we present evidence that BORC is also involved in the control of late endosomal/lysosomal size, which is mediated via regulation of phosphatidylinositol-3,5-bisphosphate [PI(3,5)P 2 ] levels. Cells lacking the core BORC components, Diaskedin or Myrlysin had smaller late endosomes/lysosomes compared to their wild type (WT) counterparts.
In contrast, deletion of a third core BORC component Lyspersin did not affect the size of these organelles, pointing toward a specific function of Diaskedin and Myrlysin in this process. This size difference was retained even under conditions, where fundamental lysosomal properties such as pH were compromised. The morphological changes of the late endosomes/lysosomes in BORC deficient cells were caused by enhanced lysosomal reformation-a process, directly linked to PIKfyve function. Recently, it was reported that inhibiting PIKfyve impairs generation of terminal lysosomes, since PIKfyve activity regulates extensive membrane remodeling that initiates reformation of lysosomes from acidic and hydrolase-active, enlarged endolysosomes. 17,18 Upstream from PIKfyve, we have identified AMPK as key activator.
Endosomal size regulation was correlated with cellular energy status and AMPK activation. Under glucose starvation, a well-established activator of AMPK signaling, 19,20 a significant reduction of late endosomal size in WT cells was achieved, which was morphologically undistinguishable from the Diaskedin deletion mutant, bearing intrinsically activated AMPK signaling. Organelle size could then be reverted to the steady state dimensions upon glucose restimulation, pointing toward an energy sensing involvement of the BORC complex. In addition, the LAM-TOR/Ragulator complex was found as a modulator of this process.

LAMTOR/Ragulator deficient cells phenocopy BORC deletion mutants
in terms of endosomal/lysosomal size and abundance. Overall, our data suggest that BORC controls the size of late endosomes and lysosomes in addition to its established role in controlling lysosomal transport. Finally, baseline autophagy in Diaskedin and Myrlysin deletion mutants was also significantly upregulated. We could link this phenotype to the decrease of endosomal/lysosomal size, as both processes are ultimately regulated by PI(3,5)P 2 production.

| Deletion of the BORC component Diaskedin reduces the size of lysosomes
Recently, we and others 14,15 have demonstrated that the BORC subunit Lyspersin is specifically required for establishing the interaction between BORC and LAMTOR/Ragulator on the surface of late endosomes/lysosomes. Therefore, we hypothesized that other BORC subunits might as well fulfill dedicated functions. To test this assumption, we first selected Diaskedin for further analysis since it was shown to interact in equimolar ratios with Myrlysin, the proposed membrane anchor of the complex. 21 Upon CRISPR/Cas9 mediated knock-out (KO) of Diaskedin in HeLa cells, we noticed that the protein levels of the BORC subunit, Lyspersin, were downregulated, whereas the protein levels of the late endosomal marker lysosome-associated membrane protein 1 (LAMP1) were upregulated ( Figure 1A). Consistently, the morphology of the late endosomal/lysosomal system was severely affected in Diaskedin deficient (KO) cells. The total number of LAMP1 positive late endosomes/lysosomes was significantly increased compared with WT cells (Figure 1B,C) and those organelles typically clustered in the perinuclear region ( Figure 1B,F), which resembled the phenotype of Lyspersin depleted cells. 14 The number  Figure 1D,E). Additionally, morphometry from electron microscopy (EM) images ( Figure 1F) of high-pressure frozen HeLa cells further confirmed an increased frequency of lysosomes, their perinuclear accumulation, as well as their smaller diameter upon Diaskedin deletion ( Figure 1F,G).

| Reduction of endosomal size upon deletion of different BORC components in HT1080 cells
To corroborate these observations in another cell line, we generated a  Figure 2B and Movie S1). Thus, we concluded that those oversized endocytic compartments were catabolically active late endosomes/ lysosomes and we refer to them as "enlarged late endosomes" hereafter. Their exaggerated diameter greatly facilitated light microscopic monitoring of organelle alterations. Importantly, upon Diaskedin deletion, there was a lower frequency of cells with enlarged endosomes ( Figure 2C). In the Diaskedin HT1080 rescue cell line, re-expressing HA-tagged WT Diaskedin, the frequency of enlarged late endosomes was similar to WT HT1080 cells ( lysosomes ( Figure 2I). Overall, these results suggested that Diaskedin localizes to late endosomes/lysosomes and controls their size and abundance.

| Deletions of Diaskedin and Myrlysin but not Lyspersin abolish the formation of characteristic enlarged endosomes in HT1080
We next asked if other BORC components were also required to control the size of late endosomes/lysosomes similar to Diaskedin ( Figure 2C and Figure 3A,B). Therefore, we also deleted Myrlysin, the   17,18,23 The lipid kinase PIKfyve phosphorylates PI(3)P to generate PI(3,5)P 2 on late endosomes/lysosomes. PI(3,5)P 2 plays an important role in late endosomal biogenesis. The reduction of PI(3,5)P 2 levels leads to the enlargement of late endosomes/lysosomes and contributes to a variety of diseases. 18,[23][24][25] To address the link between BORC and PI(3,5)P 2 levels on endosomal size regulation, we used two established inhibitors, which block PI(3,5)P 2 production; either by directly inhibiting PIKfyve with YM201636, or by blocking the upstream production of PI ( Based on these results we speculated that loss of Diasekdin resulted in hyperactive PIKfyve, which generated higher PI(3,5)P 2 levels leading to the reduction of late endosomal/lysosomal size.
To directly test this idea, we determined the changes in phosphoinositides (PtdInsP) species in Diaskedin deficient HT1080 cells compared to their WT counterparts. Therefore, HT1080 cells were incubated with 3 H-myo-inositol to label PtdInsPs, followed by lipid extraction and deacylation, and the amount of PtdInsP species was

| Contribution of the LAMTOR/Ragulator complex
The LAMTOR/Ragulator complex is a major player in the regulation of late endosomal/lysosomal signaling as it recruits and facilitates MAPK, AMPK and mTORC1 activation on late endosomes/lysosomes. 30,34 Since BORC and the LAMTOR/Ragulator complex interact on late endosomes/lysosomes, 14,15 we addressed how their interaction contributed to endosomal size regulation. Therefore, we generated

| Enhanced lysosomal reformation correlates with autophagy induction in BORC deficient cells
Having established that lysosomal biogenesis was severely altered upon deletion of core BORC components, we next investigated whether this would also affect autophagy, since lysosomal reformation and autophagy are tightly interconnected and depend on each other. 40 The lysosomal calcium channel Mucolipin-1 (ML-1), also known as TRPML1 (transient receptor potential cation channel, Mucolipin subfamily, member 1), has been shown to be required for proper TFEB dephosphorylation and thus TFEB translocation into the nucleus, which consequently is a prerequisite for autophagy initiation. Interestingly, ML-1 activation is dependent on PI(3,5)P 2 levels on lysosomes. 41,42 This led us to the hypothesis that  Figure 3C). 14 In particular, we found that members of the BORC complex negatively regulate PIKfyve activity and thereby affect PI(3,5)P 2 levels and consequently late endosomal/lysosomal size. The direct quantification of PtdInsP species revealed that PI(3,5)P 2 was highly enriched in Diaskedin KO cells ( Figure 4C and Supplementary Figure S3D). This  Figure S5A,B), it is tempting to speculate that PI(3,5)P 2 levels could regulate BORC stability and/or localization in a feedback mechanism and thereby ultimately control lysosomal reformation and late endosomal/lysosomal size reduction.

| Glucose levels regulate late endosomal/lysosomal size
When subjected to energy stress, cells react by reprogramming anabolic and catabolic processes, favoring the latter to maintain cellular homeostasis and to survive stress conditions. The endolysosomal system is a main regulator of those processes with a central role for the lysosomal residential LAMTOR/Ragulator complex in sensing intracellular AA and glucose levels and subsequent lysosomal mTORC1 activation. 28  distribution of late endosomes, it is clear that BORC is upstream of LAMTOR and that both complexes work together to position late endosome/lysosomes. Deletion of LAMTOR/Ragulator is associated with translocation of late endosomes/lysosomes toward the cell periphery. 27 Notably, this distinct mislocalization phenotype was dependent on BORC ( Figure 7B).
In our current work, we demonstrated that energy stress, triggered by glucose deprivation, is accompanied by significant morphological changes of the endolysosomal system, in particular size reduction of late endosomes/lysosomes. This is most likely achieved by inducing lysosomal reformation via PIKfyve activation and subsequent PI(3,5)P 2 production and could have several beneficial effects for the cell. For instance, autophagy, a process which is also boosted during glucose starvation, requires newly formed lysosomes to efficiently degrade cargo molecules and thereby recycle metabolic building blocks on which cells rely during nutrient shortage. Surprisingly, the acute morphological changes in the endolysosomal system following glucose limitation were reminiscent of Diaskedin deletion ( Figure 8A,B). Accordingly, late endosomes/lysosomes of Diaskedin KO cells appeared to be constantly undergoing lysosomal reformation, even when not under starvation stress. Therefore, we propose that the BORC might participate in an energy sensing machinery, which ultimately affects endosomal size and morphology.
Recently Zong and colleagues suggested that glucose sensing by the lysosomal AMPK activation pathway serves as a surveillance system monitoring the availability of glucose and switching off anabolic pathways. 38 Our data might provide a link for the lysosomal noncanonical glucose-sensing role of AMPK 30 toward the executing BORC machinery for late endosomal/lysosomal adaption. We propose a model (Supplementary Figure S5C) in which low energy levels, caused for instance by glucose starvation, activate lysosomal reformation via the "BORC-AMPK-PIKfyve-ML-1 axis." BORC is embedded in regulatory circuitry involving AMPK, PIKfyve, ML-1, TFEB and autophagy.
Activating one of those players could potentially set in motion an entire feed-forward feedback mechanism. Upon glucose restimulation, BORC is then required to limit lysosomal reformation by negatively regulating AMPK-dependent PIKfyve activation. In Diaskedin deletion mutants, this process is no longer restricted and might lead to a constant energy stress-like status, which is manifested by activated AMPK signaling and subsequently increased PIKfyve activity and lysosomal reformation.
Furthermore, constant energy stress-like status also leads to increased baseline autophagy, which we observed in Diaskedin KO and Myrlysin KO cell lines. While TFEB dephosphorylation and its subsequent nuclear translocation can promote autophagy, it also promotes lysosomal biogenesis including regulation of LAMP1 levels, which might explain why some BORC deletion mutants display higher LAMP1 levels ( Figure 1A, 2A and Supplementary Figure S1D). This is manifested by increased autophagosome generation and we believe that it is functionally linked to tubule formation and the resulting reduction of endosomal size (Supplementary Figure  S5C).

| Plasmid generation
The template for generating a rescue HA-Diaskedin construct was

| Subcellular fractionation
Cells grown on 15 cm petri dishes were cooled on metal plates on ice and washed with ice cold phosphate buffer saline (PBS). PBS, containing 0.5x protease inhibitors was added and cells were scraped, transferred to Falcon tubes and centrifuged in a precooled centrifuge at 160g for 5 minutes. The supernatant was removed and the pellet wash washed (without disturbing its integrity) with Homogenization

| EM, tomography, immunogold labeling and ultrastructural morphometry
Specimen preparation, immunoelectron microscopy and electron tomography were performed as previously described in detail. 36,56 Briefly, for morphology, the cells were cultured on sapphire discs suitable for high-pressure freezing, 57

| Quantification and statistical tests
Endosomal count in HeLa cells was based on 10 cells per phenotype, where endosomes were automatically counted using Imaris software.
Endosomal diameter in HeLa cells was based on measurements of at least 100 endosomes from at least six cells per genotype imaged. The percentage of cells, displaying enlarged late endosomes in HT1080 cells was determined, based on acquired immunofluorescent images, using LAMP1 antibody, where at least 300 cells from a minimum of three biological replicates were assigned to either "normal" or "enlarged" group based on general size and abundancy of late endosomes. These were quantified by applying an unpaired Student's t test (*P ≤ .05; **P ≤ .01; ***P ≤ .001).  Lukas A. Huber https://orcid.org/0000-0003-1116-2120