Role of the Cytosolic Domain of the a3 Subunit of V-ATPase in the Interaction with Rab7 and Secretory Lysosome Trafficking in Osteoclasts.

We previously reported that the a3 subunit of proton-pumping vacuolar-type ATPase (V-ATPase) interacts with Rab7 and its guanine nucleotide exchange factor, Mon1a-Ccz1, and recruits them to secretory lysosomes in osteoclasts, which is essential for anterograde trafficking of secretory lysosomes. The a3 subunit interacts with Mon1a-Ccz1 through its cytosolic N-terminal domain. Here, we examined the roles of this domain in the interaction with Rab7 and trafficking of secretory lysosomes. Immunoprecipitation experiments showed that a3 interacted with Rab7 through its cytosolic domain, similar to the interaction with Mon1a-Ccz1. We connected this domain with a lysosome localization signal and expressed it in a3-knockout (a3KO) osteoclasts. Although the signal connected to the cytosolic domain was mainly detected in lysosomes, impaired lysosome trafficking in a3KO osteoclasts was not rescued. These results indicate that the cytosolic domain of a3 can interact with trafficking regulators, but is insufficient to induce secretory lysosome trafficking. The C-terminal domain of a3 and other subunits of V-ATPase are likely required to form a fully functional complex for secretory lysosome trafficking.

Rab7 localized on zymogen granules is involved in maturation but not in autophagy or regulated exocytosis in pancreatic acinar cells.

Rab7 is known to function in the autophagy and endocytosis pathways in eukaryocytes and is related to various diseases. We recently reported that Rab7 plays a protective role against acute pancreatitis. However, its physiological function in exocytic cells remains unclear. Therefore, we investigated the role of Rab7 in pancreas-specific Rab7 knockout mice (Rab7Δpan). Immunofluorescence microscopy revealed that Rab7 colocalized with amylase in pancreatic acinar cells of wild-type mice, but not in Rab7Δpan mice. Western blotting confirmed Rab7 localization in the zymogen granule (ZG) membranes of wild-type mice. Cholecystokinin (CCK)-stimulated amylase secretion examined using isolated pancreatic acini was similar in Rab7Δpan and wild-type mice. In contrast, electron microscopy revealed that the diameters of ZGs were shorter and the number of ZGs was larger in the pancreatic acinar cells of Rab7Δpan mice than in those of wild-type mice. However, the number of ZGs decreased in both Rab7Δpan and wild-type mice after 24 h of starvation. In addition, the amount of amylase in the pancreas was decreased in both Rab7Δpan and wild-type mice. These data indicate that Rab7 localized on ZGs plays a crucial role in the maturation of ZGs but not in their autophagy or regulated exocytosis in pancreatic acinar cells.

Rab32 and Rab38 maintain bone homeostasis by regulating intracellular traffic in osteoclasts.

Osteoclasts play a crucial role in bone homeostasis by forming resorption pits on bone surfaces, resulting in bone resorption. The osteoclast expression of Rab38 protein is highly induced during differentiation from macrophages. Here we generated mice with double knockout (DKO) of Rab38 and its paralogue, Rab32, to investigate the roles of these proteins in osteoclasts. Bone marrow-derived macrophages from Rab32/38 DKO mice differentiated normally into osteoclasts in vitro. However, DKO osteoclasts showed reduced bone resorption activity. These osteoclasts also demonstrated defective secretion of tartrate-resistant acid phosphatase and cathepsin K into culture medium. Furthermore, the plasma membrane localization of a3, an osteoclast-specific a subunit of V-ATPase, was abrogated in DKO mice, substantiating the reduced resorption activity. In vivo, Rab32- and Rab38-positive cells were attached to the bone surface. Eight-week-old DKO mice showed significantly thickened trabecular bones in micro-CT and histomorphometry analysis, as well as reduced serum levels of cross-linked C-telopeptide of type I collagen, indicating diminished bone resorption in vivo. In DKO male mice over 10 weeks of age, hyperostosis appeared at the talofibular syndesmosis, the distal junction of the tibia and fibula. Furthermore, middle-aged mice (10 to 12 months of age) exhibited kyphosis, which is not usually observed in wild-type male mice until around 24 months of age. These results indicate that Rab32 and Rab38 contribute to osteoclast function by supporting intracellular traffic, thereby maintaining normal bone homeostasis.Key words: Rab32, Rab38, osteoclast, lysosome-related organelle, secretory lysosome.

Characterization of Rab32- and Rab38-positive lysosome-related organelles in osteoclasts and macrophages.

Both the biogenesis and functions of osteoclasts and macrophages involves dynamic membrane traffic. We screened transcript levels for Rab family small GTPases related to osteoclasts and identified Rab38. Rab38 expression is upregulated during osteoclast differentiation and maturation. In osteoclasts, both Rab38 and its paralog, Rab32, colocalize to lysosome-related organelles (LROs). In macrophages, Rab32 is also found in LROs. LROs are part of the endocytic pathway but are distinct from lysosomes. After receptor activator of NF-κB ligand stimulation, LROs contain cathepsin K and tartrate-resistant acid phosphatase inside and help both proteins to accumulate around bone resorption pits. After osteoclast maturation, these enzymes are hardly found within LROs. In macrophages derived from Rab32 and Rab38 double knockout mice, both acidification and V-ATPase a3 localization were severely compromised. Both the double knockout macrophage and bafilomycin-treated wildtype macrophage show an increase in Lamp1-positive organelles, implying that biogenesis of lysosomes and LROs are related. These results indicate that Rab32 and Rab38 both play a crucial role in LRO biogenesis in macrophages and in osteoclasts.

Exploring the Link between Vacuolar-Type Proton ATPase and Epithelial Cell Polarity.

Vacuolar-type H+-ATPase (V-ATPase) was first identified as an electrogenic proton pump that acidifies the lumen of intracellular organelles. Subsequently, it was observed that the proton pump also participates in the acidification of extracellular compartments. V-ATPase plays important roles in a wide range of cell biological processes and physiological functions by generating an acidic pH; therefore, it has attracted much attention not only in basic research but also in pathological and clinical aspects. Emerging evidence indicates that the luminal acidic endocytic organelles and their trafficking may function as important hubs that connect and coordinate various signaling pathways. Various pharmacological analyses have suggested that acidic endocytic organelles are important for the maintenance of cell polarity. Recently, several studies using genetic approaches have revealed the involvement of V-ATPase in the establishment and maintenance of apico-basal polarity. This review provides a brief overview of the relationship between the polarity of epithelial cells and V-ATPase as well as V-ATPase driven luminal acidification.

The a subunit isoforms of vacuolar-type proton ATPase exhibit differential distribution in mouse perigastrulation embryos.

Vacuolar-type H+-ATPases (V-ATPases) are large multi-subunit complexes that play critical roles in the acidification of a variety of intracellular or extracellular compartments. Mammalian cells contain four isoforms of the membrane integral subunit a (a1-a4); these isoforms contain the information necessary to target the enzyme to different cellular destinations. They are also involved in regulating the efficiency of ATP hydrolysis and proton transport. Previously, we showed that early embryogenesis requires V-ATPase function, and the luminal acidic endocytic and lysosomal compartments in the visceral endoderm of mouse embryos at the pre-gastrulation stage (E6.5) are essential for both nutrition and signal transduction during early embryogenesis. In this study, we examined the expression and distribution of a subunit isoforms in mouse embryos at E6.5. We found that all four isoforms expressed and exhibited differential distribution in the E6.5 embryo. At this developmental stage, the embryos establish highly elaborate endocytic compartments called apical vacuoles, on which the a3 isoform specifically accumulated.

The lysosomal V-ATPase a3 subunit is involved in localization of Mon1-Ccz1, the GEF for Rab7, to secretory lysosomes in osteoclasts.

We have shown previously that the lysosomal a3 isoform of the a subunit of vacuolar-type ATPase (V-ATPase) interacts with inactive (GDP-bound form) Rab7, a small GTPase that regulates late endosome/lysosome trafficking, and that a3 recruits Rab7 to secretory lysosomes in mouse osteoclasts. This is essential for outward trafficking of secretory lysosomes and thus for bone resorption. However, the molecular mechanism underlying the recruitment of Rab7 by a3 remains to be fully elucidated. Here, we showed that a3 interacts with the Mon1A-Ccz1 complex, a guanine nucleotide exchange factor (GEF) for Rab7, using HEK293T cells. The interaction was mediated by the amino-terminal half domain of a3 and the longin motifs of Mon1A and Ccz1. Exogenous expression of the GEF promoted the interaction between a3 and Rab7. Mon1A mutants that interact inefficiently with Rab7 interacted with a3 at a similar level to wild-type Mon1A. Lysosomal localization of endogenous Ccz1 was abolished in osteoclasts lacking a3. These results suggest that the lysosomal a3 isoform of V-ATPase interacts with Mon1A-Ccz1, and that a3 is important for Mon1A-Ccz1 localization to secretory lysosomes, which mediates Rab7 recruitment to the organelle.

Vacuolar-type proton ATPase is required for maintenance of apicobasal polarity of embryonic visceral endoderm.

The endocytic compartments keep their interior acidic through the inward flow of protons and anions from the cytosol. Acidification is mediated by a proton pump known as vacuolar-type ATPase (V-ATPase) and transporters conferring anion conductance to the organellar membrane. In this study, we analysed the phenotype of mouse embryos lacking the V-ATPase c-subunit. The mutant embryos differentiated embryonic epithelial tissues, primitive endoderm, epiblast, and extraembryonic ectoderm; however, the organisation of these epithelia was severely affected. The apical-basal polarity in the visceral endoderm layer was not properly established in the mutant embryos, resulting in abnormal epithelial morphology. Thus, the function of V-ATPase is imperative for the establishment and/or maintenance of epithelial cell polarity, which is required for early embryogenesis.

Functional complementation of V-ATPase a subunit isoforms in osteoclasts.

In osteoclasts, the a3 isoform of the proton-pumping V-ATPase plays essential roles in anterograde trafficking of secretory lysosomes and extracellular acidification required for bone resorption. This study examined functional complementation of the a isoforms by exogenously expressing the a1, a2 and a3 isoforms in a3-knockout (KO) osteoclasts. The expression levels of a1 and a2 in a3KO osteoclasts were similar, but lower than that of a3. a1 significantly localized to lysosomes, whereas a2 slightly did. On the other hand, a2 interacted with Rab7, a regulator of secretory lysosome trafficking in osteoclasts, more efficiently than a1. a1 partly complemented the functions of a3 in secretory lysosome trafficking and calcium phosphate resorption, while a2 partly complemented the former but not the latter function.

Rab7-Mediated Endocytosis Establishes Patterning of Wnt Activity through Inactivation of Dkk Antagonism.

Endocytosis has been proposed to modulate cell signaling activities. However, the role of endocytosis in embryogenesis, which requires coordination of multiple signaling inputs, has remained less understood. We previously showed that mouse embryos lacking a small guanosine triphosphate (GTP)-binding protein Rab7 implicated in endocytic flow are defective in gastrulation. Here, we investigate how subcellular defects associated with Rab7 deficiency are related to the observed developmental defects. Rab7-deficient embryos fail to organize mesodermal tissues due to defects in Wnt-ß-catenin signaling. Visceral endoderm (VE)-specific ablation of Rab7 results in patterning defects similar to systemic Rab7 deletion. Rab7 mutants accumulate the Wnt antagonist Dkk1 in the extracellular space and in intracellular compartments throughout the VE epithelium. These data indicate that Rab7-dependent endocytosis regulates the concentration and availability of extracellular Dkk1, thereby relieving the epiblast of antagonism. This intercellular mechanism therefore organizes distinct spatiotemporal patterns of canonical Wnt activity during the peri-gastrulation stages of embryonic development.

V-ATPase a3 isoform mutations identified in osteopetrosis patients abolish its expression and disrupt osteoclast function.

The a3 isoform of vacuolar-type proton-pumping ATPase (V-ATPase) is essential for bone resorption by osteoclasts. Although more than 90 mutations of the human a3 gene have been identified in patients with infantile malignant osteopetrosis, it is unclear whether they lead to osteoclast dysfunction. We have established an in vitro assay to induce osteoclasts from spleen macrophages derived from a3-knockout mice. Here, we examined the effects of these mutations in a3-knockout osteoclasts. We were interested in four mutations, two short deletions and two missense mutations, previously identified in the a3 cytosolic domain. a3 harboring either of the two short deletions was hardly expressed in osteoclasts and calcium phosphate resorption was impaired. On the other hand, osteoclasts expressing a3 with either of the two missense mutations exhibited no defects. Specifically, expression levels of the mutant proteins, V-ATPase assembly, and calcium phosphate resorption activity were similar to those of the wild type. Moreover, these missense mutants interacted with Rab7, a small GTPase that regulates lysosomal trafficking. These results suggest that the short deletions impair a3 expression and thus disrupt V-ATPase subunit assembly essential for bone resorption, while the missense mutations do not cause osteoclast dysfunction without an additional mutation(s) or impair resorption of bone, but not of calcium phosphate.

Observations From a Mouse Model of Forebrain Voa1 Knockout: Focus on Hippocampal Structure and Function.

Voa protein is a subunit of V-ATPase proton pump which is essential to acidify intracellular organelles including synaptic vesicles. Voa1 is one of the four isoforms of Voa family with strong expression in neurons. Our present study was aimed to examine the role of Voa1 protein in mammalian brain neurons. To circumvent embryonic lethality, we generated conditional Voa1 knockout mice in which Voa1 was selectively deleted from forebrain pyramidal neurons. We performed experiments in the Voa1 knockout mice of ages 5-6 months to assess the persistent effects of Voa1 deletion. We found that the Voa1 knockout mice exhibited poor performance in the Morris water maze test compared to control mice. In addition, synaptic field potentials of the hippocampal CA1 region were greatly diminished in the Voa1 knockout mice when examined in brain slices in vitro. Furthermore, brain histological experiments showed severe degeneration of dorsal hippocampal CA1 neurons while CA3 neurons were largely preserved. The CA1 neurodegeneration was associated with general brain atrophy as overall hemispheric areas were reduced in the Voa1 cKO mice. Despite the CA1 degeneration and dysfunction, electroencephalographic recordings from the hippocampal CA3 area revealed aberrant spikes and non-convulsive discharges in the Voa1 knockout mice but not in control mice. These hippocampal spikes were suppressed by single intra-peritoneal injection of diazepam which is a benzodiazepine GABAA receptor enhancer. Together these results suggest that Voa1 related activities are essential for the survival of the targeted neurons in the dorsal hippocampal CA1 as well as other forebrain areas. We postulate that the Voa1 knockout mice may serve as a valuable model for further investigation of V-ATPase dysfunction related neuronal degeneration and functional abnormalities in forebrain areas particularly the hippocampus.

Isoform-specific gene disruptions reveal a role for the V-ATPase subunit a4 isoform in the invasiveness of 4T1-12B breast cancer cells.

The vacuolar H+-ATPase (V-ATPase) is an ATP-driven proton pump present in various intracellular membranes and at the plasma membrane of specialized cell types. Previous work has reported that plasma membrane V-ATPases are key players in breast cancer cell invasiveness. The two subunit a-isoforms known to target the V-ATPase to the plasma membrane are a3 and a4, and expression of a3 has been shown to correlate with plasma membrane localization of the V-ATPase in various invasive human breast cancer cell lines. Here we analyzed the role of subunit a-isoforms in the invasive mouse breast cancer cell line, 4T1-12B. Quantitation of mRNA levels for each isoform by quantitative RT-PCR revealed that a4 is the dominant isoform expressed in these cells. Using a CRISPR/Cas9-based approach to disrupt the genes encoding each of the four V-ATPase subunit a-isoforms, we found that ablation of only the a4-encoding gene significantly inhibits invasion and migration of 4T1-12B cells. Additionally, cells with disrupted a4 exhibited reduced V-ATPase expression at the leading edge, suggesting that the a4 isoform is primarily responsible for targeting the V-ATPase to the plasma membrane in 4T1-12B cells. These findings suggest that different subunit a-isoforms may direct V-ATPases to the plasma membrane of different invasive breast cancer cell lines. They further suggest that expression of V-ATPases at the cell surface is the primary factor that promotes an invasive cancer cell phenotype.

Vacuolar-type ATPase: A proton pump to lysosomal trafficking.

Vacuolar-type ATPase (V-ATPase), initially identified in yeast and plant vacuoles, pumps protons into the lumen of organelles coupled with ATP hydrolysis. The mammalian counterpart is found ubiquitously in endomembrane organelles and the plasma membrane of specialized cells such as osteoclasts. V-ATPase is also present in unique organelles such as insulin secretory granules, neural synaptic vesicles, and acrosomes of spermatozoa. Consistent with its diverse physiological roles and unique localization, the seven subunits of V-ATPase have 2-4 isoforms that are organelle- or cell-specific. Subunits of the enzyme function in trafficking organelles and vesicles by interacting with small molecule GTPases. During osteoclast differentiation, one of the four isoforms of subunit a, a3, is indispensable for secretory lysosome trafficking to the plasma membrane. Diseases such as osteopetrosis, renal acidosis, and hearing loss are related to V-ATPase isoforms. In addition to its role as an enzyme, V-ATPase has versatile physiological roles in eukaryotic cells.

Combating herpesvirus encephalitis by potentiating a TLR3-mTORC2 axis.

TLR3 is a sensor of double-stranded RNA that is indispensable for defense against infection with herpes simplex virus type 1 (HSV-1) in the brain. We found here that TLR3 was required for innate immune responses to HSV-1 in neurons and astrocytes. During infection with HSV-1, TLR3 recruited the metabolic checkpoint kinase complex mTORC2, which led to the induction of chemokines and trafficking of TLR3 to the cell periphery. Such trafficking enabled the activation of molecules (including mTORC1) required for the induction of type I interferons. Intracranial infection of mice with HSV-1 was exacerbated by impairment of TLR3 responses with an inhibitor of mTOR and was significantly 'rescued' by potentiation of TLR3 responses with an agonistic antibody to TLR3. These results suggest that the TLR3-mTORC2 axis might be a therapeutic target through which to combat herpes simplex encephalitis.

Essential Role of the a3 Isoform of V-ATPase in Secretory Lysosome Trafficking via Rab7 Recruitment.

Secretory lysosomes are required for the specialised functions of various types of differentiated cells. In osteoclasts, the lysosomal proton pump V-ATPase (vacuolar-type ATPase) is targeted to the plasma membrane via secretory lysosomes and subsequently acidifies the extracellular compartment, providing optimal conditions for bone resorption. However, little is known about the mechanism underlying this trafficking of secretory lysosomes. Here, we demonstrate that the lysosome-specific a3 isoform of the V-ATPase a subunit plays an indispensable role in secretory lysosome trafficking, together with Rab7, a small GTPase involved in organelle trafficking. In osteoclasts lacking a3, lysosomes were not transported to the cell periphery, and Rab7 was not localised to lysosomes but diffused throughout the cytoplasm. Expression of dominant-negative (GDP-bound form) Rab7 inhibited lysosome trafficking in wild-type cells. Furthermore, a3 directly interacted with the GDP-bound forms of Rab7 and Rab27A. These findings reveal a novel role for the proton pump V-ATPase in secretory lysosome trafficking and an unexpected mechanistic link with Rab GTPases.

Disruption of Small GTPase Rab7 Exacerbates the Severity of Acute Pancreatitis in Experimental Mouse Models.

Although aberrations of intracellular vesicle transport systems towards lysosomes including autophagy and endocytosis are involved in the onset and progression of acute pancreatitis, the molecular mechanisms underlying such aberrations remain unclear. The pathways of autophagy and endocytosis are closely related, and Rab7 plays crucial roles in both. In this study, we analyzed the function of Rab7 in acute pancreatitis using pancreas-specific Rab7 knockout (Rab7Δpan) mice. In Rab7Δpan pancreatic acinar cells, the maturation steps of both endosomes and autophagosomes were deteriorated, and the lysosomal functions were affected. In experimental models of acute pancreatitis, the histopathological severity, serum amylase concentration and intra-pancreatic trypsin activity were significantly higher in Rab7Δpan mice than in wild-type mice. Furthermore, the autophagy process was blocked in Rab7Δpan pancreas compared with wild-type mice. In addition, larger autophagic vacuoles that colocalize with early endosome antigen 1 (EEA1) but not with lysosomal-associated membrane protein (LAMP)-1 were much more frequently formed in Rab7Δpan pancreatic acinar cells. Accordingly, Rab7 deficiency exacerbates the severity of acute pancreatitis by impairing the autophagic and endocytic pathways toward lysosomes.

The a3 isoform of subunit a of the vacuolar ATPase localizes to the plasma membrane of invasive breast tumor cells and is overexpressed in human breast cancer.

The vacuolar (H+)-ATPases (V-ATPases) are a family of ATP-driven proton pumps that acidify intracellular compartments and transport protons across the plasma membrane. Previous work has demonstrated that plasma membrane V-ATPases are important for breast cancer invasion in vitro and that the V-ATPase subunit a isoform a3 is upregulated in and critical for MDA-MB231 and MCF10CA1a breast cancer cell invasion. It has been proposed that subunit a3 is present on the plasma membrane of invasive breast cancer cells and is overexpressed in human breast cancer. To test this, we used an a3-specific antibody to assess localization in breast cancer cells. Subunit a3 localizes to the leading edge of migrating breast cancer cells, but not the plasma membrane of normal breast epithelial cells. Furthermore, invasive breast cancer cells express a3 throughout all intracellular compartments tested, including endosomes, the Golgi, and lysosomes. Moreover, subunit a3 knockdown in MB231 breast cancer cells reduces in vitro migration. This reduction is not enhanced upon addition of a V-ATPase inhibitor, suggesting that a3-containing V-ATPases are critical for breast cancer migration. Finally, we have tested a3 expression in human breast cancer tissue and mRNA prepared from normal and cancerous breast tissue. a3 mRNA was upregulated 2.5-47 fold in all breast tumor cDNA samples tested relative to normal tissue, with expression generally correlated to cancer stage. Furthermore, a3 protein expression was increased in invasive breast cancer tissue relative to noninvasive cancer and normal breast tissue. These studies suggest that subunit a3 plays an important role in invasive human breast cancer.

Membrane dynamics in mammalian embryogenesis: Implication in signal regulation.

Eukaryotes have evolved an array of membrane compartments constituting secretory and endocytic pathways that allow the flow of materials. Both pathways perform important regulatory roles. The secretory pathway is essential for the production of extracellular, secreted signal molecules, but its function is not restricted to a mere route connecting intra- and extracellular compartments. Post-translational modifications also play an integral function in the secretory pathway and are implicated in developmental regulation. The endocytic pathway serves as a platform for relaying signals from the extracellular stimuli to intracellular mediators, and then ultimately inducing signal termination. Here, we discuss recent studies showing that dysfunction in membrane dynamics causes patterning defects in embryogenesis and tissue morphogenesis in mammals.

Loss of G2 subunit of vacuolar-type proton transporting ATPase leads to G1 subunit upregulation in the brain.

Vacuolar-type ATPase (V-ATPase) is a primary proton pump with versatile functions in various tissues. In nerve cells, V-ATPase is required for accumulation of neurotransmitters into secretory vesicles and subsequent release at the synapse. Neurons express a specific isoform (G2) of the G subunit of V-ATPase constituting the catalytic sector of the enzyme complex. Using gene targeting, we generated a mouse lacking functional G2 (G2 null), which showed no apparent disorders in architecture and behavior. In the G2-null mouse brain, a G1 subunit isoform, which is ubiquitously expressed in neuronal and non-neuronal tissues, accumulated more abundantly than in wild-type animals. This G1 upregulation was not accompanied by an increase in mRNA. These results indicate that loss of function of neuron-specific G2 isoform was compensated by an increase in levels of the G1 isoform without apparent upregulation of the G1 mRNA.