Arl8 and SKIP act together to link lysosomes to kinesin-1

Lysosomes move bidirectionally on microtubules, and this motility can be stimulated by overexpression of the small GTPase Arl8. By using affinity chromatography, we find that Arl8-GTP binds to the soluble protein SKIP (SifA and kinesin-interacting protein, aka PLEKHM2). SKIP was originally identified as a target of the Salmonella effector protein SifA and found to bind the light chain of kinesin-1 to activate the motor on the bacteria's replicative vacuole. We show that in uninfected cells both Arl8 and SKIP are required for lysosomes to distribute away from the microtubule-organizing center. We identify two kinesin light chain binding motifs in SKIP that are required for lysosomes to accumulate kinesin-1 and redistribute to the cell periphery. Thus, Arl8 binding to SKIP provides a link from lysosomal membranes to plus-end-directed motility. A splice variant of SKIP that lacks a light chain binding motif does not stimulate movement, suggesting fine-tuning by alternative splicing.     

Interconversion of two GDP-bound conformations and their selection in an Arf-family small G protein

ADP-ribosylation factor (Arf) and other Arf-family small G proteins participate in many cellular functions via their characteristic GTP/GDP conformational cycles, during which a nucleotide(?)Mg(2+)-binding site communicates with a remote N-terminal helix. However, the conformational interplay between the nucleotides, the helix, the protein core, and Mg(2+) has not been fully delineated. Herein, we report a study of the dynamics of an Arf-family protein, Arl8, under various conditions by means of NMR relaxation spectroscopy. The data indicated that, when GDP is bound, the protein core, which does not include the N-terminal helix, reversibly transition between an Arf-family GDP form and another conformation that resembles the Arf-family GTP form. Additionally, we found that the N-terminal helix and Mg(2+), respectively, stabilize the aforementioned former and latter conformations in a population-shift manner. Given the dynamics of the conformational changes, we can describe the Arl8 GTP/GDP cycle in terms of an energy diagram.     

mTOR controls lysosome tubulation and antigen presentation in macrophages and dendritic cells

Macrophages and dendritic cells exposed to lipopolysaccharide (LPS) convert their lysosomes from small, punctate organelles into a network of tubules. Tubular lysosomes have been implicated in phagosome maturation, retention of fluid phase, and antigen presentation. There is a growing appreciation that lysosomes act as sensors of stress and the metabolic state of the cell through the kinase mTOR. Here we show that LPS stimulates mTOR and that mTOR is required for LPS-induced lysosome tubulation and secretion of major histocompatibility complex II in macrophages and dendritic cells. Specifically, we show that the canonical phosphatidylinositol 3-kinase–Akt–mTOR signaling pathway regulates LPS-induced lysosome tubulation independently of IRAK1/4 and TBK. Of note, we find that LPS treatment augmented the levels of membrane-associated Arl8b, a lysosomal GTPase required for tubulation that promotes kinesin-dependent lysosome movement to the cell periphery, in an mTOR-dependent manner. This suggests that mTOR may interface with the Arl8b-kinesin machinery. To further support this notion, we show that mTOR antagonists can block outward movement of lysosomes in cells treated with acetate but have no effect in retrograde movement upon acetate removal. Overall our work provides tantalizing evidence that mTOR plays a role in controlling lysosome morphology and trafficking by modulating microtubule-based motor activity in leukocytes.     

Sorting Nexin 11 Regulates Lysosomal Degradation of Plasma Membrane TRPV3

The trafficking of ion channels to/from the plasma membrane is considered an important mechanism for cellular activity and an interesting approach for disease therapies. The transient receptor potential vanilloid 3 (TRPV3) ion channel is widely expressed in skin keratinocytes, and its trafficking mechanism to/from the plasma membrane is unknown. Here, we report that the vesicular trafficking protein sorting nexin 11 (SNX11) downregulates the level of the TRPV3 plasma membrane protein. Overexpression of SNX11 causes a decrease in the level of TRPV3 current and TRPV3 plasma membrane protein in TRPV3‐transfected HEK293T cells. Subcellular localizations and western blots indicate that SNX11 interacts with TRPV3 and targets it to lysosomes for degradation, which is blocked by the lysosomal inhibitors chloroquine and leupeptin. Both TRPV3 and SNX11 are highly expressed in HaCaT cells. We show that TRPV3 agonists‐activated Ca²⁺ influxes and the level of native TRPV3 total protein in HaCaT cells are decreased by overexpression of SNX11 and increased by knockdown of SNX11. Our findings reveal that SNX11 promotes the trafficking of TRPV3 from the plasma membrane to lysosomes for degradation via protein‐protein interactions, which demonstrates a previously unknown function of SNX11 as a regulator of TRPV3 trafficking from the plasma membrane to lysosomes.     

The lysosomal trafficking of sphingolipid activator proteins (SAPs) is mediated by sortilin

Most soluble lysosomal proteins bind the mannose 6-phosphate receptor (M6P-R) to be sorted to the lysosomes. However, the lysosomes of I-cell disease (ICD) patients, a condition resulting from a mutation in the phosphotransferase that adds mannose 6-phosphate to hydrolases, have near normal levels of several lysosomal proteins, including the sphingolipid activator proteins (SAPs), GM2AP and prosaposin. We tested the hypothesis that SAPs are targeted to the lysosomal compartment via the sortilin receptor. To test this hypothesis, a dominant-negative construct of sortilin and a sortilin small interfering RNA (siRNA) were introduced into COS-7 cells. Our results showed that both the truncated sortilin and the sortilin siRNA block the traffic of GM2AP and prosaposin to the lysosomal compartment. This observation was confirmed by a co-immunoprecipitation, which demonstrated that GM2AP and prosaposin are interactive partners of sortilin. Furthermore, a dominant-negative mutant GGA prevented the trafficking of prosaposin and GM2AP to lysosomes. In conclusion, our results show that the trafficking of SAPs is dependent on sortilin, demonstrating a novel lysosomal trafficking.     

Two di-leucine motifs regulate trafficking of mucolipin-1 to lysosomes

Mutations in the mucolipin-1 gene have been linked to mucolipidosis type IV, a lysosomal storage disorder characterized by severe neurological and ophthalmologic abnormalities. Mucolipin-1 is a membrane protein containing six putative transmembrane domains with both its N- and C-termini localized facing the cytosol. To gain information on the sorting motifs that mediate the trafficking of this protein to lysosomes, we have generated chimeras in which the N- and C- terminal tail portions of mucolipin-1 were fused to a reporter gene. In this article, we report the identification of two separate di-leucine-type motifs that co-operate to regulate the transport of mucolipin-1 to lysosomes. One di-leucine motif is positioned at the N-terminal cytosolic tail and mediates direct transport to lysosomes, whereas the other di-leucine motif is found at the C-terminal tail and functions as an adaptor protein 2-dependent internalization motif. We have also found that the C-terminal tail of mucolipin-1 is palmitoylated and that this modification might regulate the efficiency of endocytosis. Finally, the mutagenesis of both di-leucine motifs abrogated lysosomal accumulation and resulted in cell-surface redistribution of mucolipin-1. Taken together, these results reveal novel information regarding the motifs that regulate mucolipin-1 trafficking and suggest a role for palmitoylation in protein sorting.     

A novel antimicrobial peptide M1-8 targets the lysosomal pathway to inhibit autolysosome formation and promote apoptosis in liver cancer cells

Lysosomes, a central regulator of autophagy, play a critical role in tumour growth. Lysosomal protease cathepsin D can initiate apoptosis when released from lysosomes into the cytosol. In this study, we observed that Musca domestica cecropin (Mdc) 1–8 (M1-8), a small anti-tumour peptide derived from Mdc, inhibits hepatoma cell growth by blocking autophagy–lysosome fusion. This effect is likely achieved by targeting lysosomes to activate lysosomal protease D. Additionally, we examined whether lysosomal content and cathepsin D release were involved in M1-8-induced apoptosis. After exposure to M1-8, human hepatoma HepG2 cells rapidly co-localized with lysosomes, disrupted lysosomal integrity, caused leakage of lysosomal protease cathepsin D, caspase activation and mitochondrial membrane potential changes; and promoted cell apoptosis. Interestingly, in M1-8-treated HepG2 cells, autophagic protein content increased and the lysosome–autophagosome fusion was inhibited, suggesting that M1-8 can cause apoptosis through autophagy and lysosomes. This result indicates that a small accumulation of autophagy and autolysosome inhibition in cells can cause cell death. Taken together, these data suggest a novel insight into the regulatory mechanisms of M1-8 in autophagy and lysosomes, which may facilitate the development of M1-8 as a potential cancer therapeutic agent.     

The needs of a synapse—How local organelles serve synaptic proteostasis

Synaptic function crucially relies on the constant supply and removal of neuronal membranes. The morphological complexity of neurons poses a significant challenge for neuronal protein transport since the machineries for protein synthesis and degradation are mainly localized in the cell soma. In response to this unique challenge, local micro‐secretory systems have evolved that are adapted to the requirements of neuronal membrane protein proteostasis. However, our knowledge of how neuronal proteins are synthesized, trafficked to membranes, and eventually replaced and degraded remains scarce. Here, we review recent insights into membrane trafficking at synaptic sites and into the contribution of local organelles and micro‐secretory pathways to synaptic function. We describe the role of endoplasmic reticulum specializations in neurons, Golgi‐related organelles, and protein complexes like retromer in the synthesis and trafficking of synaptic transmembrane proteins. We discuss the contribution of autophagy and of proteasome‐mediated and endo‐lysosomal degradation to presynaptic proteostasis and synaptic function, as well as nondegradative roles of autophagosomes and lysosomes in signaling and synapse remodeling. We conclude that the complexity of neuronal cyto‐architecture necessitates long‐distance protein transport that combines degradation with signaling functions.     

Arf-like GTPase Arl8b regulates lytic granule polarization and natural killer cell–mediated cytotoxicity

Natural killer (NK) lymphocytes contain lysosome-related organelles (LROs), known as lytic granules, which upon formation of immune synapse with the target cell, polarize toward the immune synapse to deliver their contents to the target cell membrane. Here, we identify a small GTP-binding protein, ADP-ribosylation factor-like 8b (Arl8b), as a critical factor required for NK cell–mediated cytotoxicity. Our findings indicate that Arl8b drives the polarization of lytic granules and microtubule-organizing centers (MTOCs) toward the immune synapse between effector NK lymphocytes and target cells. Using a glutathione S-transferase pull-down approach, we identify kinesin family member 5B (KIF5B; the heavy chain of kinesin-1) as an interaction partner of Arl8b from NK cell lysates. Previous studies showed that interaction between kinesin-1 and Arl8b is mediated by SifA and kinesin-interacting protein (SKIP) and the tripartite complex drives the anterograde movement of lysosomes. Silencing of both KIF5B and SKIP in NK cells, similar to Arl8b, led to failure of MTOC-lytic granule polarization to the immune synapse, suggesting that Arl8b and kinesin-1 together control this critical step in NK cell cytotoxicity.     

The position of lysosomes within the cell determines their luminal pH

We examined the luminal pH of individual lysosomes using quantitative ratiometric fluorescence microscopy and report an unappreciated heterogeneity: peripheral lysosomes are less acidic than juxtanuclear ones despite their comparable buffering capacity. An increased passive (leak) permeability to protons, together with reduced vacuolar H⁺–adenosine triphosphatase (V-ATPase) activity, accounts for the reduced acidifying ability of peripheral lysosomes. The altered composition of peripheral lysosomes is due, at least in part, to more limited access to material exported by the biosynthetic pathway. The balance between Rab7 and Arl8b determines the subcellular localization of lysosomes; more peripheral lysosomes have reduced Rab7 density. This in turn results in decreased recruitment of Rab-interacting lysosomal protein (RILP), an effector that regulates the recruitment and stability of the V1G1 component of the lysosomal V-ATPase. Deliberate margination of lysosomes is associated with reduced acidification and impaired proteolytic activity. The heterogeneity in lysosomal pH may be an indication of a broader functional versatility.     

The FTLD risk factor TMEM106B and MAP6 control dendritic trafficking of lysosomes

TMEM106B is a major risk factor for frontotemporal lobar degeneration with TDP‐43 pathology. TMEM106B localizes to lysosomes, but its function remains unclear. We show that TMEM106B knockdown in primary neurons affects lysosomal trafficking and blunts dendritic arborization. We identify microtubule‐associated protein 6 (MAP6) as novel interacting protein for TMEM106B. MAP6 over‐expression inhibits dendritic branching similar to TMEM106B knockdown. MAP6 knockdown fully rescues the dendritic phenotype of TMEM106B knockdown, supporting a functional interaction between TMEM106B and MAP6. Live imaging reveals that TMEM106B knockdown and MAP6 overexpression strongly increase retrograde transport of lysosomes in dendrites. Downregulation of MAP6 in TMEM106B knockdown neurons restores the balance of anterograde and retrograde lysosomal transport and thereby prevents loss of dendrites. To strengthen the link, we enhanced anterograde lysosomal transport by expressing dominant‐negative Rab7‐interacting lysosomal protein (RILP), which also rescues the dendrite loss in TMEM106B knockdown neurons. Thus, TMEM106B/MAP6 interaction is crucial for controlling dendritic trafficking of lysosomes, presumably by acting as a molecular brake for retrograde transport. Lysosomal misrouting may promote neurodegeneration in patients with TMEM106B risk variants.     

Drosophila Arl8 is a general positive regulator of lysosomal fusion events

The small GTPase Arl8 is known to be involved in the periphery-directed motility of lysosomes. However, the overall importance of moving these vesicles is still poorly understood. Here we show that Drosophila Arl8 is required not only for the proper distribution of lysosomes, but also for autophagosome-lysosome fusion in starved fat cells, endosome-lysosome fusion in garland nephrocytes, and developmentally programmed secretory granule degradation (crinophagy) in salivary gland cells. Moreover, proper Arl8 localization to lysosomes depends on the shared subunits of the BLOC-1 and BORC complexes, which also promote autophagy and crinophagy. In conclusion, we demonstrate that Arl8 is responsible not only for positioning lysosomes but also acts as a general lysosomal fusion factor.     

Autophagy in lysosomal storage disorders

Lysosomes are ubiquitous intracellular organelles that have an acidic internal pH, and play crucial roles in cellular clearance. Numerous functions depend on normal lysosomes, including the turnover of cellular constituents, cholesterol homeostasis, downregulation of surface receptors, inactivation of pathogenic organisms, repair of the plasma membrane and bone remodeling. Lysosomal storage disorders (LSDs) are characterized by progressive accumulation of undigested macromolecules within the cell due to lysosomal dysfunction. As a consequence, many tissues and organ systems are affected, including brain, viscera, bone and cartilage. The progressive nature of phenotype development is one of the hallmarks of LSDs. In recent years biochemical and cell biology studies of LSDs have revealed an ample spectrum of abnormalities in a variety of cellular functions. These include defects in signaling pathways, calcium homeostasis, lipid biosynthesis and degradation and intracellular trafficking. Lysosomes also play a fundamental role in the autophagic pathway by fusing with autophagosomes and digesting their content. Considering the highly integrated function of lysosomes and autophagosomes it was reasonable to expect that lysosomal storage in LSDs would have an impact upon autophagy. The goal of this review is to provide readers with an overview of recent findings that have been obtained through analysis of the autophagic pathway in several types of LSDs, supporting the idea that LSDs could be seen primarily as "autophagy disorders."     

Autophagy in lysosomal storage disorders

Lysosomes are ubiquitous intracellular organelles that have an acidic internal pH, and play crucial roles in cellular clearance. Numerous functions depend on normal lysosomes, including the turnover of cellular constituents, cholesterol homeostasis, downregulation of surface receptors, inactivation of pathogenic organisms, repair of the plasma membrane and bone remodeling. Lysosomal storage disorders (LSDs) are characterized by progressive accumulation of undigested macromolecules within the cell due to lysosomal dysfunction. As a consequence, many tissues and organ systems are affected, including brain, viscera, bone and cartilage. The progressive nature of phenotype development is one of the hallmarks of LSDs. In recent years biochemical and cell biology studies of LSDs have revealed an ample spectrum of abnormalities in a variety of cellular functions. These include defects in signaling pathways, calcium homeostasis, lipid biosynthesis and degradation and intracellular trafficking. Lysosomes also play a fundamental role in the autophagic pathway by fusing with autophagosomes and digesting their content. Considering the highly integrated function of lysosomes and autophagosomes it was reasonable to expect that lysosomal storage in LSDs would have an impact upon autophagy. The goal of this review is to provide readers with an overview of recent findings that have been obtained through analysis of the autophagic pathway in several types of LSDs, supporting the idea that LSDs could be seen primarily as “autophagy disorders.”     

Inhibition of lipid kinase PIKfyve reveals a role for phosphatase Inpp4b in the regulation of PI(3)P-mediated lysosome dynamics through VPS34 activity

Lysosome membranes contain diverse phosphoinositide (PtdIns) lipids that coordinate lysosome function and dynamics. The PtdIns repertoire on lysosomes is tightly regulated by the actions of diverse PtdIns kinases and phosphatases; however, specific roles for PtdIns in lysosomal functions and dynamics are currently unclear and require further investigation. It was previously shown that PIKfyve, a lipid kinase that synthesizes PtdIns(3,5)P₂ from PtdIns(3)P, controls lysosome “fusion-fission” cycle dynamics, autophagosome turnover, and endocytic cargo delivery. Furthermore, INPP4B, a PtdIns 4-phosphatase that hydrolyzes PtdIns(3,4)P₂ to form PtdIns(3)P, is emerging as a cancer-associated protein with roles in lysosomal biogenesis and other lysosomal functions. Here, we investigated the consequences of disrupting PIKfyve function in Inpp4b-deficient mouse embryonic fibroblasts. Through confocal fluorescence imaging, we observed the formation of massively enlarged lysosomes, accompanied by exacerbated reduction of endocytic trafficking, disrupted lysosome fusion-fission dynamics, and inhibition of autophagy. Finally, HPLC scintillation quantification of ³H-myo-inositol labeled PtdIns and PtdIns immunofluorescence staining, we observed that lysosomal PtdIns(3)P levels were significantly elevated in Inpp4b-deficient cells due to the hyperactivation of phosphatidylinositol 3-kinase catalytic subunit VPS34 enzymatic activity. In conclusion, our study identifies a novel signaling axis that maintains normal lysosomal homeostasis and dynamics, which includes the catalytic functions of Inpp4b, PIKfyve, and VPS34.     

Syntaxin 11 binds Vti1b and regulates late endosome to lysosome fusion in macrophages

Syntaxin 11 (Stx11) is a SNARE protein enriched in cells of the immune system. Loss or mutation of Stx11 results in familial hemophagocytic lymphohistiocytosis type-4 (FHL-4), an autosomal recessive disorder of immune dysregulation characterized by high levels of inflammatory cytokines along with defects in T-cell and natural killer cell function. We show here Stx11 is located on endosomal membranes including late endosomes and lysosomes in macrophages. While Stx11 did not form a typical trans-SNARE complex, it did bind to the Q-SNARE Vti1b and was able to regulate the availability of Vti1b to form the Q-SNARE complexes Stx6/Stx7/Vtib and Stx7/Stx8/Vti1b. The mutant form of Stx11 sequestered Vti1b from forming the Q-SNARE complex that mediates late endosome to lysosome fusion. Depletion of Stx11 in activated macrophages leads to an accumulation of enlarged late endocytic compartments, increased trafficking to the cell surface and inhibition of late endosome to lysosome fusion. These phenotypes are rescued by the expression of an siRNA-resistant Stx11 construct in Stx11-depleted cells. Our results suggest that by regulating the availability of Vti1b, Stx11 regulates trafficking steps between late endosomes, lysosomes and the cell surface in macrophages.     

Differential Regulation of Amyloid-β Endocytic Trafficking and Lysosomal Degradation by Apolipoprotein E Isoforms

Aggregation of amyloid-β (Aβ) peptides leads to synaptic disruption and neurodegeneration in Alzheimer disease (AD). A major Aβ clearance pathway in the brain is cellular uptake and degradation. However, how Aβ traffics through the endocytic pathway and how AD risk factors regulate this event is unclear. Here we show that the majority of endocytosed Aβ in neurons traffics through early and late endosomes to the lysosomes for degradation. Overexpression of Rab5 or Rab7, small GTPases that function in vesicle fusion for early and late endosomes, respectively, significantly accelerates Aβ endocytic trafficking to the lysosomes. We also found that a portion of endocytosed Aβ traffics through Rab11-positive recycling vesicles. A blockage of this Aβ recycling pathway with a constitutively active Rab11 mutant significantly accelerates cellular Aβ accumulation. Inhibition of lysosomal enzymes results in Aβ accumulation and aggregation. Importantly, apolipoprotein E (apoE) accelerates neuronal Aβ uptake, lysosomal trafficking, and degradation in an isoform-dependent manner with apoE3 more efficiently facilitating Aβ trafficking and degradation than apoE4, a risk factor for AD. Taken together, our results demonstrate that Aβ endocytic trafficking to lysosomes for degradation is a major Aβ clearance pathway that is differentially regulated by apoE isoforms. A disturbance of this pathway can lead to accumulation and aggregation of cellular Aβ capable of causing neurotoxicity and seeding amyloid.     

LYST Affects Lysosome Size and Quantity,but not Trafficking or Degradation Through Autophagy or Endocytosis

Mutations in the large BEACH domain‐containing protein LYST causes Chediak–Higashi syndrome. The diagnostic hallmark is enlarged lysosomes and lysosome‐related organelles in various cell types. Dysfunctional secretion of enlarged lysosome‐related organelles has been observed in cells with mutations in LYST, but the capacity of the enlarged lysosomes to degrade endogenous proteins has not been studied. Here, we show for the first time that small interfering RNA‐depletion of LYST in human cell lines recapitulates the LYST mutant phenotype of enlarged lysosomes. We found no evidence for an effect of LYST depletion on autophagy or endocytic degradation. Autophagosomes are formed in normal size and quantities and are able to fuse to the enlarged lysosomes, leading to normal rates of degradation. Degradation of the epidermal growth factor receptor (EGFR) was similarly not affected, indicating that the enlarged lysosomes are fully functional in degrading endogenous proteins. Retrograde trafficking of toxins as well as the localization of transporters of lysosomal proteins, adaptor protein‐3 (AP‐3) and cation‐independent mannose‐6‐phosphate receptor (CI‐MPR), were all found to be unaffected by LYST. Quantitative analysis of the enlarged lysosomes shows that LYST depletion causes a reduction in vesicle quantity per cell, while the total enzymatic content and vesicular pH are unaffected, supporting a role for LYST in lysosomal fission and/or fusion events.      

Aminopeptidase-resistant peptides are targeted to lysosomes and subsequently degraded

Most cytoplasmic and nuclear proteins are degraded via the ubiquitin-proteasome system into peptides, which are subsequently hydrolyzed by downstream aminopeptidases. Inefficient degradation can lead to accumulation of protein fragments, and subsequent aggregation and toxicity. Whereas the role of the proteasome and the effect of its impairment on aggregation have been intensively studied, little is known about how cells deal with peptides that show resistance to degradation by aminopeptidases. Here, we introduced peptidase-resistant peptides into living cells and show that these peptides rapidly and irreversibly accumulate into puncta in the perinuclear region of the cell. Accumulation appears to be independent of peptide sequence but is less efficient for longer peptides. The puncta colocalize with autophagosomal and lysosomal markers, suggesting that these peptides end up within lysosomes via macroautophagy. Surprisingly, the peptides still accumulate within lysosomes when macroautophagy is impaired, suggesting a trafficking route independent of macroautophagy. Upon lysosomal uptake, peptides are degraded, suggesting that cells can clear peptidase-resistant proteasomal products by an alternative pathway, which targets them to lysosomes.