A Deficiency Screen for Zygotic Loci Required for Establishment and Patterning of the Epidermis in Caenorhabditis Elegans

To identify genomic regions required for establishment and patterning of the epidermis, we screened 58 deficiencies that collectively delete at least ~67% of the Caenorhabditis elegans genome. The epidermal pattern of deficiency homozygous embryos was analyzed by examining expression of a marker specific for one of the three major epidermal cell types, the seam cells. The organization of the epidermis and internal organs was also analyzed using a monoclonal antibody specific for epithelial adherens junctions. While seven deficiencies had no apparent effect on seam cell production, 21 were found to result in subnormal, and five in excess numbers of these cells. An additional 23 deficiencies blocked expression of the seam cell marker, in some cases without preventing cell proliferation. Two deficiencies result in multinucleate seam cells. Deficiencies were also identified that result in subnormal numbers of epidermal cells, hyperfusion of epidermal cells into a large syncytium, or aberrant epidermal differentiation. Finally, analysis of internal epithelia revealed deficiencies that cause defects in formation of internal organs, including circularization of the intestine and bifurcation of the pharynx lumen. This study reveals that many regions of the C. elegans genome are required zygotically for patterning of the epidermis and other epithelia.     

ceh-16/engrailed patterns the embryonic epidermis of Caenorhabditis elegans

engrailed is a homeobox gene essential for developmental functions such as differentiation of cell populations and the onset of compartment boundaries in arthropods and vertebrates. We present the first functional study on engrailed in an unsegmented animal: the nematode Caenorhabditis elegans. In the developing worm embryo, ceh-16/engrailed is predominantly expressed in one bilateral row of epidermal cells (the seam cells). We show that ceh-16/engrailed primes a specification cascade through three mechanisms: (1) it suppresses fusion between seam cells and other epidermal cells by repressing eff-1/fusogen expression; (2) it triggers the differentiation of the seam cells through different factors, including the GATA factor elt-5; and (3) it segregates the seam cells into a distinct lateral cellular compartment, repressing cell migration toward dorsal and ventral compartments.     

Mitochondrial Safeguard: a stress response that offsets extreme fusion and protects respiratory function via flickering‐induced Oma1 activation

The connectivity of mitochondria is regulated by a balance between fusion and division. Many human diseases are associated with excessive mitochondrial connectivity due to impaired Drp1, a dynamin‐related GTPase that mediates division. Here, we report a mitochondrial stress response, named mitochondrial safeguard, that adjusts the balance of fusion and division in response to increased mitochondrial connectivity. In cells lacking Drp1, mitochondria undergo hyperfusion. However, hyperfusion does not completely connect mitochondria because Opa1 and mitofusin 1, two other dynamin‐related GTPases that mediate fusion, become proteolytically inactivated. Pharmacological and genetic experiments show that the activity of Oma1, a metalloprotease that cleaves Opa1, is regulated by short pulses of the membrane depolarization without affecting the overall membrane potential in Drp1‐knockout cells. Re‐activation of Opa1 and Mitofusin 1 in Drp1‐knockout cells further connects mitochondria beyond hyperfusion, termed extreme fusion, leading to bioenergetic deficits. These findings reveal an unforeseen safeguard mechanism that prevents extreme fusion of mitochondria, thereby maintaining mitochondrial function when the balance is shifted to excessive connectivity.     

Role of the V-ATPase in regulation of the vacuolar fission-fusion equilibrium

Like numerous other eukaryotic organelles, the vacuole of the yeast Saccharomyces cerevisiae undergoes coordinated cycles of membrane fission and fusion in the course of the cell cycle and in adaptation to environmental conditions. Organelle fission and fusion processes must be balanced to ensure organelle integrity. Coordination of vacuole fission and fusion depends on the interactions of vacuolar SNARE proteins and the dynamin-like GTPase Vps1p. Here, we identify a novel factor that impinges on the fusion-fission equilibrium: the vacuolar H(+)-ATPase (V-ATPase) performs two distinct roles in vacuole fission and fusion. Fusion requires the physical presence of the membrane sector of the vacuolar H(+)-ATPase sector, but not its pump activity. Vacuole fission, in contrast, depends on proton translocation by the V-ATPase. Eliminating proton pumping by the V-ATPase either pharmacologically or by conditional or constitutive V-ATPase mutations blocked salt-induced vacuole fragmentation in vivo. In living cells, fission defects are epistatic to fusion defects. Therefore, mutants lacking the V-ATPase display large single vacuoles instead of multiple smaller vacuoles, the phenotype that is generally seen in mutants having defects only in vacuolar fusion. Its dual involvement in vacuole fission and fusion suggests the V-ATPase as a potential regulator of vacuolar morphology and membrane dynamics.     

fus-1, a pH Shift Mutant of Semliki Forest Virus, Acts by Altering Spike Subunit Interactions via a Mutation in the E2 Subunit

Semliki Forest virus (SFV), an enveloped alphavirus, is a well-characterized paradigm for viruses that infect cells via endocytic uptake and low-pH-triggered fusion. The SFV spike protein is composed of a dimer of E1 and E2 transmembrane subunits, which dissociate upon exposure to low pH, liberating E2 and the fusogenic E1 subunit to undergo independent conformational changes. SFV fusion and infection are blocked by agents such as ammonium chloride, which act by raising the pH in the endosome and inhibiting the low-pH-induced conformational changes in the SFV spike protein. We have previously isolated an SFV mutant, fus-1, that requires more acidic pH to trigger its fusion activity and is therefore more sensitive to inhibition by ammonium chloride. The acid shift in the fusion activity of fus-1 was here shown to be due to a more acidic pH threshold for the initial dissociation of the fus-1 spike dimer, thereby resulting in a more acidic pH requirement for the subsequent conformational changes in both fus-1 E1 and fus-1 E2. Sequence analysis demonstrated that the fus-1 phenotype was due to a mutation in the E2 spike subunit, threonine 12 to isoleucine. fus-1 revertants that have regained the parental fusion phenotype and ammonium chloride sensitivity were shown to have also regained E2 threonine 12. Our results identify a region of the SFV E2 spike protein subunit that regulates the pH dependence of E1-catalyzed fusion by controlling the dissociation of the E1/E2 dimer.     

Kinetics of endosome acidification detected by mutant and wild-type Semliki Forest virus.

The fusogenic properties of Semliki Forest virus (SFV) and its mutants were used to follow the kinetics of acidification during the endocytic uptake of virus by BHK-21 cells. It has previously been shown that the low pH of endocytic vacuoles triggers a conformational change in the SFV spike glycoprotein, activating membrane fusion and initiating virus infection. This conformational alteration was here shown to occur in endosomes and to follow the same time course as the intracellular fusion reaction, demonstrating that fusion occurs rapidly after virus exposure to endosome acidity. The kinetics of endosome acidification were monitored using wild type (wt) SFV and fus-1, an SFV mutant with a lower fusion pH threshold. The results presented here demonstrated that wt and mutant virus were internalized with a t1/2 of 10 min, and that endosomes were acidified to the wt threshold of pH 6.2 with a t1/2 of 15 min. In contrast, endosome pH reached the fus-1 threshold of 5.3 with a much longer t1/2 of 45 min. The subsequent degradation of SFV in lysosomes had a t1/2 of 90 min. It was found that after the initial uptake of virus from the plasma membrane, its transit through the endocytic pathway, exposure to endosome acidity and eventual delivery to lysosomes were markedly asynchronous.     

Vacuolar-type proton ATPase as regulator of membrane dynamics in multicellular organisms

Acidification inside membrane compartments is a common feature of all eukaryotic cells. The acidic milieu is involved in many physiological processes including secretion, protein processing, and others. However, its cellular relevance has not been well established beyond the results of in vitro studies involving cultured cell systems. In the last decade, human and mouse genetics have revealed that the acidification machinery is implicated in multiple pathophysiological disorders, and thus our understanding of physiological consequences of the defective acidification in multicellular organisms has improved. In invertebrates including Drosophila and nematodes, mutations of V-ATPase were found to lead the development of rather unexpected phenotypes. Studies have suggested that V-ATPase may be involved in membrane fusion and vesicle formation, important processes for membrane trafficking, and have further implied its involvement in cell–cell fusion. This rather novel idea arose from the phenotypes associated with genetic disorders involving V-ATPase genes in various genetic model systems. In this article, we focus and overview the non-classical, beyond proton-pumping function of the vacuolar-type ATPase in exo/endocytic systems.     

Intestinal expression of H+ V-ATPase in the mosquito Aedes albopictus is tightly associated with gregarine infection

Vacuolar ATPase (V-ATPase) is a family of ATP-dependent proton pumps expressed on the plasma membrane and endomembranes of eukaryotic cells. Acidification of intracellular compartments, such as lysosomes, endosomes, and parasitophorous vacuoles, mediated by V-ATPase is essential for the entry by many enveloped viruses and invasion into or escape from host cells by intracellular parasites. In mosquito larvae, V-ATPase plays a role in regulating alkalization of the anterior midgut. We extracted RNA from larval tissues of Aedes albopictus, cloned the full-length sequence of mRNA of V-ATPase subunit A, which contains a poly-A tail and 2,971 nucleotides, and expressed the protein. The fusion protein was then used to produce rabbit polyclonal antibodies, which were used as a tool to detect V-ATPase in the midgut and Malpighian tubules of mosquito larvae. A parasitophorous vacuole was formed in the midgut in response to invasion by Ascogregarina taiwanensis, confining the trophozoite(s). Acidification was demonstrated within the vacuole using acridine orange staining. It is concluded that gregarine sporozoites are released by ingested oocysts in the V-ATPase-energized high-pH environment. The released sporozoites then invade and develop in epithelial cells of the posterior midgut. Acidification of the parasitophorous vacuoles may be mediated by V-ATPase and may facilitate exocytosis of the vacuole confining the trophozoites from the infected epithelial cells for further extracellular development.     

The C. elegans developmental fusogen EFF-1 mediates homotypic fusion in heterologous cells and in vivo

During cell-cell fusion, two cells' plasma membranes merge, allowing the cytoplasms to mix and form a syncytium. Little is known about the mechanisms of cell fusion. Here, we asked whether eff-1, shown previously to be essential for fusion in Caenorhabditis elegans, acts directly in the fusion machinery. We show that expression of EFF-1 transmembrane protein drives fusion of heterologous cells into multinucleate syncytia. We obtained evidence that EFF-1-mediated fusion involves a hemifusion intermediate characterized by membrane mixing without cytoplasm mixing. Furthermore, syncytiogenesis requires EFF-1 in both fusing cells. To test whether this mechanism also applies in vivo, we conducted genetic mosaic analysis of C. elegans and found that homotypic epidermal fusion requires EFF-1 in both cells. Thus, although EFF-1-mediated fusion shares characteristics with viral and intracellular fusion, including an apparent hemifusion step, it differs from these reactions in the homotypic organization of the fusion machinery.     

Vacuolar and plasma membrane proton pumps collaborate to achieve cytosolic pH homeostasis in yeast

Vacuolar proton-translocating ATPases (V-ATPases) play a central role in organelle acidification in all eukaryotic cells. To address the role of the yeast V-ATPase in vacuolar and cytosolic pH homeostasis, ratiometric pH-sensitive fluorophores specific for the vacuole or cytosol were introduced into wild-type cells and vma mutants, which lack V-ATPase subunits. Transiently glucose-deprived wild-type cells respond to glucose addition with vacuolar acidification and cytosolic alkalinization, and subsequent addition of K(+) ion increases the pH of both the vacuole and cytosol. In contrast, glucose addition results in an increase in vacuolar pH in both vma mutants and wild-type cells treated with the V-ATPase inhibitor concanamycin A. Cytosolic pH homeostasis is also significantly perturbed in the vma mutants. Even at extracellular pH 5, conditions optimal for their growth, cytosolic pH was much lower, and response to glucose was smaller in the mutants. In plasma membrane fractions from the vma mutants, activity of the plasma membrane proton pump, Pma1p, was 65-75% lower than in fractions from wild-type cells. Immunofluorescence microscopy confirmed decreased levels of plasma membrane Pma1p and increased Pma1p at the vacuole and other compartments in the mutants. Pma1p was not mislocalized in concanamycin-treated cells, but a significant reduction in cytosolic pH under all conditions was still observed. We propose that short-term, V-ATPase activity is essential for both vacuolar acidification in response to glucose metabolism and for efficient cytosolic pH homeostasis, and long-term, V-ATPases are important for stable localization of Pma1p at the plasma membrane.     

Caenorhabditis elegans LET-413 Scribble is essential in the epidermis for growth,viability, and directional outgrowth of epithelial seam cells

The conserved adapter protein Scribble (Scrib) plays essential roles in a variety of cellular processes, including polarity establishment, proliferation, and directed cell migration. While the mechanisms through which Scrib promotes epithelial polarity are beginning to be unraveled, its roles in other cellular processes including cell migration remain enigmatic. In C. elegans, the Scrib ortholog LET-413 is essential for apical–basal polarization and junction formation in embryonic epithelia. However, whether LET-413 is required for postembryonic development or plays a role in migratory events is not known. Here, we use inducible protein degradation to investigate the functioning of LET-413 in larval epithelia. We find that LET-413 is essential in the epidermal epithelium for growth, viability, and junction maintenance. In addition, we identify a novel role for LET-413 in the polarized outgrowth of the epidermal seam cells. These stem cell-like epithelial cells extend anterior and posterior directed apical protrusions in each larval stage to reconnect to their neighbors. We show that the role of LET-413 in seam cell outgrowth is likely mediated largely by the junctional component DLG-1 discs large, which we demonstrate is also essential for directed outgrowth of the seam cells. Our data uncover multiple essential functions for LET-413 in larval development and show that the polarized outgrowth of the epithelial seam cells is controlled by LET-413 Scribble and DLG-1 Discs large.     

Retrieval of the Vacuolar H+-ATPase from Phagosomes Revealed by Live Cell Imaging

Background

The vacuolar H+-ATPase, or V-ATPase, is a highly-conserved multi-subunit enzyme that transports protons across membranes at the expense of ATP. The resulting proton gradient serves many essential functions, among them energizing transport of small molecules such as neurotransmitters, and acidifying organelles such as endosomes. The enzyme is not present in the plasma membrane from which a phagosome is formed, but is rapidly delivered by fusion with endosomes that already bear the V-ATPase in their membranes. Similarly, the enzyme is thought to be retrieved from phagosome membranes prior to exocytosis of indigestible material, although that process has not been directly visualized.

Methodology

To monitor trafficking of the V-ATPase in the phagocytic pathway of Dictyostelium discoideum, we fed the cells yeast, large particles that maintain their shape during trafficking. To track pH changes, we conjugated the yeast with fluorescein isothiocyanate. Cells were labeled with VatM-GFP, a fluorescently-tagged transmembrane subunit of the V-ATPase, in parallel with stage-specific endosomal markers or in combination with mRFP-tagged cytoskeletal proteins.

Principal Findings

We find that the V-ATPase is commonly retrieved from the phagosome membrane by vesiculation shortly before exocytosis. However, if the cells are kept in confined spaces, a bulky phagosome may be exocytosed prematurely. In this event, a large V-ATPase-rich vacuole coated with actin typically separates from the acidic phagosome shortly before exocytosis. This vacuole is propelled by an actin tail and soon acquires the properties of an early endosome, revealing an unexpected mechanism for rapid recycling of the V-ATPase. Any V-ATPase that reaches the plasma membrane is also promptly retrieved.

Conclusions/Signficance

Thus, live cell microscopy has revealed both a usual route and alternative means of recycling the V-ATPase in the endocytic pathway.     

Membrane requirement for folding of the herpes simplex virus 1 gB cytodomain suggests a unique mechanism of fusion regulation

Herpes simplex virus type 1 (HSV-1) enters cells by fusion of its envelope with a host cell membrane, which requires four viral glycoproteins and a cellular receptor. Viral fusion glycoprotein B (gB) mediates membrane fusion through the action of its ectodomain, while its cytoplasmic domain (cytodomain) regulates fusion from the opposite face of the membrane by an unknown mechanism. The gB cytodomain appears to restrict fusion, because point or truncation mutations within it increase the extent of fusion (syn mutations). Previously, we showed that the hyperfusion phenotype correlated with reduced membrane binding in gB syn truncation mutants and proposed that membrane binding was important in regulating fusion. Here, we extended our analysis to three syn point mutants: A855V, R858H, and A874P. These mutations produce local conformational changes, with some affecting membrane interaction, which suggests that while syn mutants may deregulate fusion by somewhat different mechanisms, maintaining the wild-type (WT) conformation is critical for fusion regulation. We further show that the presence of a membrane is necessary for the cytodomain to achieve its fully folded conformation and propose that the membrane-bound form of the cytodomain represents its native conformation. Taken together, our data suggest that the cytodomain of gB regulates fusion by a novel mechanism in which membrane interaction plays a key role.     

Molecular cloning and characterization of a novel V-ATPase associated protein, DVA9.2, from human dendritic cells

The vacuolar proton-ATPase (V-ATPase) is a ubiquitous ATP-driven H(+) transporter that functions in numerous cell processes. Accumulating evidence shows important roles of V-ATPase in tumor metastasis and antigen presentation of dendritic cells (DC). A novel V-ATPase associated protein, designated as DVA9.2 (dendritic cell-derived V-ATPase associated protein of 9.2 kDa), has been identified from a human DC cDNA library by large-scale random sequencing. Full length cDNA of DVA9.2 encodes an 81-residue protein that shares 70-80% homology with human V-ATPase subunit M9.2. Distant relationship is also found with Vma21p, a yeast protein required for V-ATPase assembly. DVA9.2 contains a conserved domain, ATP synthase subunit H (pafm05493), and two membrane-spanning helices. DVA9.2 mRNA is detectable in several human tumor cell lines as well as some human normal cells and tissues. Moreover, the inducible expression of DVA9.2 mRNA in DC during maturation is observed. DVA9.2 displays integration with membrane and main localization in lysosome, endoplasmic reticulum and Golgi-associated organelles, only less at the plasma membrane. In addition, DVA9.2 is co-localized with V(0)-sector subunit a. Silencing of DVA9.2 by small interfering RNA (siRNA) does not affect the V-ATPase activity in cell membrane fractions or attenuate the migration and invasion in breast cancer MDA-MB-231 cells. These results indicate that DVA9.2, as a novel V-ATPase-associated protein, is not essential for the activity of V-ATPase complex and may be involved in functions of DC.     

Interaction between aldolase and vacuolar H+-ATPase: evidence for direct coupling of glycolysis to the ATP-hydrolyzing proton pump

Vacuolar H(+)-ATPases (V-ATPases) are essential for acidification of intracellular compartments and for proton secretion from the plasma membrane in kidney epithelial cells and osteoclasts. The cellular proteins that regulate V-ATPases remain largely unknown. A screen for proteins that bind the V-ATPase E subunit using the yeast two-hybrid assay identified the cDNA clone coded for aldolase, an enzyme of the glycolytic pathway. The interaction between E subunit and aldolase was confirmed in vitro by precipitation assays using E subunit-glutathione S-transferase chimeric fusion proteins and metabolically labeled aldolase. Aldolase was isolated associated with intact V-ATPase from bovine kidney microsomes and osteoclast-containing mouse marrow cultures in co-immunoprecipitation studies performed using an anti-E subunit monoclonal antibody. The interaction was not affected by incubation with aldolase substrates or products. In immunocytochemical assays, aldolase was found to colocalize with V-ATPase in the renal proximal tubule. In osteoclasts, the aldolase-V-ATPase complex appeared to undergo a subcellular redistribution from perinuclear compartments to the ruffled membranes following activation of resorption. In yeast cells deficient in aldolase, the peripheral V(1) domain of V-ATPase was found to dissociate from the integral membrane V(0) domain, indicating direct coupling of glycolysis to the proton pump. The direct binding interaction between V-ATPase and aldolase may be a new mechanism for the regulation of the V-ATPase and may underlie the proximal tubule acidification defect in hereditary fructose intolerance.     

Neurotransmitter release: the dark side of the vacuolar-H+ATPase

Vacuolar-H⁺ATPase (V-ATPase) is a complex enzyme with numerous subunits organized in two domains. The membrane domain V0 contains a proteolipid hexameric ring that translocates protons when ATP is hydrolysed by the catalytic cytoplasmic sector (V1). In nerve terminals, V-ATPase generates an electrochemical proton gradient that is acid and positive inside synaptic vesicles. It is used by specific neurotransmitter-proton antiporters to accumulate neurotransmitters inside their storage organelles. During synaptic activity, neurotransmitters are released from synaptic vesicles docked at specialized portions of the presynaptic plasma membrane, the active zones. A fusion pore opens that allows the neurotransmitter to be released from the synaptic vesicle lumen into the synaptic cleft. We briefly review experimental data suggesting that the membrane domain of V-ATPase could be such a fusion pore.We also discuss the functional implications for quantal neurotransmitter release of the sequential use of the same V-ATPase membrane domain in two different events, neurotransmitter accumulation in synaptic vesicles first, and then release from these organelles during synaptic activity.     

Role of the vacuolar-ATPase in Sindbis virus infection

Bafilomycin A(1) is a specific inhibitor of the vacuolar-ATPase (V-ATPase), which is responsible for pH homeostasis of the cell and for the acidification of endosomes. Bafilomycin A(1) has been commonly used as a method of inhibition of infection by viruses known or suspected to follow the path of receptor-mediated endocytosis and low-pH-mediated membrane fusion. The exact method of entry for Sindbis virus, the prototype alphavirus, remains undetermined. To further investigate the role of the V-ATPase in Sindbis virus infection, the effects of bafilomycin A(1) on the infection of BHK and insect cells by Sindbis virus were studied. Bafilomycin A(1) was found to block the expression of a virus-encoded reporter gene in both infection and transfection of BHK cells. The inhibitory effects of bafilomycin A(1) were found to be reversible. The results suggest that in BHK cells in the presence of bafilomycin A(1), virus RNA enters the cell and is translated, but replication and proper folding of the product proteins requires the function of the V-ATPase. Bafilomycin A(1) had no significant effect on the outcome of infection in insect cells.     

The cytotoxic action of diphtheria toxin and its degradation in intact Vero cells are inhibited by bafilomycin A1, a specific inhibitor of vacuolar-type H(+)-ATPase

The role of vacuolar-type H(+)-ATPase (V-ATPase) in the cytotoxic action of diphtheria toxin (DT) was studied by using bafilomycin A1, a specific inhibitor of V-ATPase. Studies with acridine orange showed that the acidification of intracellular acidic compartments was inhibited strongly when Vero cells were treated with 500 nM bafilomycin A1, indicating that bafilomycin effectively inhibits V-ATPase when it is added to the culture medium. The toxicity of DT to Vero cells, which was determined by the inhibition of protein synthesis by DT, was inhibited partially by bafilomycin at 10 nM and inhibited completely at 500 nM. Therefore, V-ATPase is involved in the expression of the toxicity of DT. Studies using 125I-labeled DT showed that bafilomycin inhibited the degradation of internalized DT, indicating that V-ATPase is also involved in this step. Subcellular fractionation revealed that 125I-DT accumulated mainly in the endosome fraction, and not in the lysosome fraction, when the cells were incubated with 125I-DT in the presence of bafilomycin. Under the cell fractionation conditions similar to those used for the DT-treated cells, we determined the location of 125I-labeled epidermal growth factor in the degradation pathway. The result suggests that bafilomycin A1 does not inhibit the transport of epidermal growth factor to lysosome.