The nascent phagosome progressively establishes an acidic milieu by acquiring a proton pump, the vacuolar-type ATPase (V-ATPase). However, the origin of phagosomal V-ATPase remains poorly understood. ...We found that phagosomes were enriched with the V-ATPase a3 subunit, which also accumulated in late endosomes and lysosomes. We modified the mouse Tcirg1 locus encoding subunit a3, to express an a3-GFP fusion protein. Live-cell imaging and immunofluorescence microscopy revealed that nascent phagosomes received the a3-GFP from tubular structures extending from lysosomes located in the perinuclear region. Macrophages from a3-deficient mice exhibited impaired acidification of phagosomes and delayed digestion of bacteria. These results show that lysosomal V-ATPase is recruited directly to the phagosomes via tubular lysosomes to establish the acidic environment hostile to pathogens.
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.
The metabolism of membrane phosphoinositides is critical for a variety of cellular processes. Phosphatidylinositol-3,5-bisphosphate PtdIns(3,5)P ₂ controls multiple steps of the intracellular ...membrane trafficking system in both yeast and mammalian cells. However, other than in neuronal tissues, little is known about the physiological functions of PtdIns(3,5)P ₂ in mammals. Here, we provide genetic evidence that type III phosphatidylinositol phosphate kinase (PIPKIII), which produces PtdIns(3,5)P ₂, is essential for the functions of polarized epithelial cells. PIPKIII-null mouse embryos die by embryonic day 8.5 because of a failure of the visceral endoderm to supply the epiblast with maternal nutrients. Similarly, although intestine-specific PIPKIII-deficient mice are born, they fail to thrive and eventually die of malnutrition. At the mechanistic level, we show that PIPKIII regulates the trafficking of proteins to a cell’s apical membrane domain. Importantly, mice with intestine-specific deletion of PIPKIII exhibit diarrhea and bloody stool, and their gut epithelial layers show inflammation and fibrosis, making our mutants an improved model for inflammatory bowel diseases. In summary, our data demonstrate that PIPKIII is required for the structural and functional integrity of two different types of polarized epithelial cells and suggest that PtdIns(3,5)P ₂ metabolism is an unexpected and critical link between membrane trafficking in intestinal epithelial cells and the pathogenesis of inflammatory bowel disease.
During early embryogenesis, before the conceptus forms the placenta, maternal nutrients as well as signaling molecules must reach the embryo proper through a tightly sealed epithelial tissue, the ...visceral endoderm (VE). The VE serves as a signaling center for embryogenesis, where exocytic and endocytic processes integrate signal production, perception and termination. However, the endocytic process in this important tissue has not been well characterized. We show that endocytic delivery to the lysosomes occurs via RAB7-dependent microautophagy. This process is essential for early mammalian development.
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.
We investigated the cell penetration of Sp1 zinc finger proteins (Sp1 ZF) and the mechanism
via
which the total cationic charge and distribution of cationic residues on the protein surface affect ...intracellular trafficking. Sp1 ZFs showed intrinsic cell membrane permeability. The intracellular transfer of Sp1 ZFs other than 1F3 was dependent on the total cationic charge. Investigation of the effect of cationic residue distribution on intracellular membrane permeability revealed that the cellular uptake of unfolded Zn
2+
-non-coordinating Ala mutants was lower than that of the wild type. Therefore, the total cationic charge and distribution of cationic residues on the protein played crucial roles in intracellular translocation. Mutational studies revealed that the two-dimensional cation cluster on the protein surface significantly improved their cellular uptake. This study will contribute to the design of artificial cargoes that can efficiently transport target substances into cells.
Two-dimensional cation clusters formed on the surface of proteins play an important role in their intracellular translocation.
Vacuolar-type proton transporting ATPase (V-ATPase) is involved in the proper development of visual function. Mutations in the Tcirg1 (also known as Atp6V0a3) locus, which encodes the a3 subunit of ...V-ATPase, cause severe autosomal recessive osteopetrosis (ARO) in humans. ARO is often associated with impaired vision most likely because of nerve compression at the optic canal. We examined the ocular phenotype of mice deficient in Tcirg1 function.
X-ray microtomography showed narrowed foramina in the skull, suggesting that optic nerve compression occurred in the a3-deficient (Tcirg1-/-) mice. The retina of the mutant mice had normal architecture, but the number of apoptotic cells was increased at 2-3 wks after birth. In the ocular system, the a3 subunit accumulated in the choriocapillary meshwork in uveal tissues. Two other subunit isoforms a1 and a2 accumulated in the retinal photoreceptor layer. We found that the a4 subunit, whose expression has previously been shown to be restricted to several transporting epithelia, was enriched in pigmented epithelial cells of the retina and ciliary bodies. The expression of a4 in the uveal tissue was below the level of detection in wild-type mice, but it was increased in the mutant choriocapillary meshwork, suggesting that compensation may have occurred among the a subunit isoforms in the mutant tissues.
Our findings suggest that a similar etiology of visual impairment is involved in both humans and mice; thus, a3-deficient mice may provide a suitable model for clinical and diagnostic purposes in cases of ARO.
Vacuolar-type proton ATPase (V-ATPase) is a multi-subunit enzyme that couples ATP hydrolysis to the translocation of protons across membranes. Mammalian cells express four isoforms of the a subunit ...of V-ATPase. Previously, we have shown that V-ATPase with the a3 isoform is highly expressed in pancreatic islets and is located in the membranes of insulin-containing granules in the beta cells. The a3 isoform functions in the regulation of hormone secretion. In this study, we have examined the distribution of a subunit isoforms in endocrine tissues, including the adrenal, parathyroid, thyroid, and pituitary glands, with isoform-specific antibodies. We have found that the a3 isoform is strongly expressed in all these endocrine tissues. Our results suggest that functions of the a3 isoform are commonly involved in the process of exocytosis in regulated secretion.
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