glo-3, a novel Caenorhabditis elegans gene, is required for lysosome-related organelle biogenesis

Gut granules are specialized lysosome-related organelles that act as sites of fat storage in Caenorhabditis elegans intestinal cells. We identified mutations in a gene, glo-3, that functions in the formation of embryonic gut granules. Some glo-3(−) alleles displayed a complete loss of embryonic gut granules, while other glo-3(−) alleles had reduced numbers of gut granules. A subset of glo-3 alleles led to mislocalization of gut granule contents into the intestinal lumen, consistent with a defect in intracellular trafficking. glo-3(−) embryos lacking gut granules developed into adults containing gut granules, indicating that glo-3(+) function may be differentially required during development. We find that glo-3(+) acts in parallel with or downstream of the AP-3 complex and the PGP-2 ABC transporter in gut granule biogenesis. glo-3 encodes a predicted membrane-associated protein that lacks obvious sequence homologs outside of nematodes. glo-3 expression initiates in embryonic intestinal precursors and persists almost exclusively in intestinal cells through adulthood. GLO-3GFP localizes to the gut granule membrane, suggesting it could play a direct role in the trafficking events at the gut granule. smg-1(−) suppression of glo-3(−) nonsense alleles indicates that the C-terminal half of GLO-3, predicted to be present in the cytoplasm, is not necessary for gut granule formation. Our studies identify GLO-3 as a novel player in the formation of lysosome-related organelles.     

Suppression of the cup-5 mucolipidosis type IV-related lysosomal dysfunction by the inactivation of an ABC transporter in C. elegans

Mutations in MCOLN1, which encodes the protein mucolipin 1, result in the lysosomal storage disease mucolipidosis Type IV. Studies on human mucolipin 1 and on CUP-5, the Caenorhabditis elegans ortholog of mucolipin 1, have shown that these proteins are required for lysosome biogenesis/function. Loss of CUP-5 results in a defect in lysosomal degradation, leading to embryonic lethality. We have identified a mutation in the ABC transporter MRP-4 that rescues the degradation defect and the corresponding lethality, owing to the absence of CUP-5. MRP-4 localizes to endocytic compartments and its levels are elevated in the absence of CUP-5. These results indicate that the lysosomal degradation defect is exacerbated in some cells because of the accumulation of MRP-4 in lysosomes rather than the loss of CUP-5 per se. We also show that under some conditions, loss of MRP-4 rescues the embryonic lethality caused by the loss of the cathepsin L protease, indicating that the accumulation of ABC transporters may be a more general mechanism whereby an initial lysosomal dysfunction is more severely compromised.     

Selenium-induced autometallographic demonstration of endogenous zinc in organs of the rainbow trout,Salmo gairdneri

Summary Autometallographic (AMG) silver enhancement of endogenous zinc was studied in seven organs of the rainbow trout Salmo gairdneri. Groups of trout were injected intraperitoneally with sodium selenite in doses ranging from 0.08 to 25 ppm, administered 1 h before being killed. The concentration of selenium obtained by each organ was determined by gamma-spectrometry, and compared with the autometallographic deposition of silver grains. The relative accumulation of selenium in the organs was: liver > spleen > kidney > intestine > gills > brain > muscle. In the fish labelled with 10 and 25 ppm Se, AMG-deposits were found (1) within lysosomes of liver cells, (2) within the granules and on the nuclear membrane of melanophores in the spleen, (3) on the microvilli and in the apical cytoplasm of renal proximal tubular cells, (4) within the granules and along the plasma membrane of intestinal eosinophilic granule cells, and in the apical portion of the intestinal epithelium, and (5) in the gills, within granule cells and on the surface of the ionocytes. In the trouts injected with 5 ppm Se, silver grains were still observed in the liver, the intestine, and the gills, whereas, no such grains were found in preparations from fish having received 1 ppm Se. The use of selenium for the histochemical demonstration of endogenous zinc versus exogenous metals is discussed. Also, consideration is given to the question of which part of the total tissue zinc that is histochemically reactive.     

Transient <Emphasis Type="Italic">in utero</Emphasis>knockout (TIUKO) of C-MYC affects late lung and intestinal development in the mouse

Background  

Developmentally important genes often result in early lethality in knockout animals. Thus, the direct role of genes in late gestation organogenesis cannot be assessed directly. In utero delivery of transgenes was shown previously to result in high efficiency transfer to pulmonary and intestinal epithelial stem cells. Thus, this technology can be used to evaluate late gestation development.     

Intestinal barrier function in response to abundant or depleted mucosal glutathione in Salmonella-infected rats

Background  

Glutathione, the main antioxidant of intestinal epithelial cells, is suggested to play an important role in gut barrier function and prevention of inflammation-related oxidative damage as induced by acute bacterial infection. Most studies on intestinal glutathione focus on oxidative stress reduction without considering functional disease outcome. Our aim was to determine whether depletion or maintenance of intestinal glutathione changes susceptibility of rats to Salmonella infection and associated inflammation.     

Surface displaced alfa-enolase of Lactobacillus plantarum is a fibronectin binding protein

Background  

Lactic acid bacteria of the genus Lactobacillus and Bifidobacterium are one of the most important health promoting groups of the human intestinal microbiota. Their protective role within the gut consists in out competing invading pathogens for ecological niches and metabolic substrates. Among the features necessary to provide health benefits, commensal microorganisms must have the ability to adhere to human intestinal cells and consequently to colonize the gut. Studies on mechanisms mediating adhesion of lactobacilli to human intestinal cells showed that factors involved in the interaction vary mostly among different species and strains, mainly regarding interaction between bacterial adhesins and extracellular matrix or mucus proteins. We have investigated the adhesive properties of Lactobacillus plantarum, a member of the human microbiota of healthy individuals.     

A transferable heterogeneous two-hybrid system in <Emphasis Type="Italic">Escherichia coli</Emphasis> based on polyhydroxyalkanoates synthesis regulatory protein PhaR

Background  

Polyhydroxyalkanoate (PHA) synthesis regulatory protein PhaR contains a DNA binding domain (DBD) and a PHA granule binding domain (GBD), it anchors to the promoter region of PHA granule-associated protein (PhaP) to repress phaP expression. However, PhaR will bind to PHB granules and be released from phaP promoter region when PHA granules are formed in vivo, initiating expression of phaP gene. Based on this regulatory mechanism, a bacterial two-hybrid system was developed: PhaR was separated into two parts: DBD was used to fuse with the bait, GBD with the prey, and phaP was replaced by a reporter gene lacZ. However, GBD protein expressed in vivo formed inclusion bodies. Thus, PhaP with strong binding ability to PHB granules was employed to replace GBD.     

Basis of lethality in C. elegans lacking CUP-5, the Mucolipidosis Type IV orthologue

Mutations in MCOLN1, which encodes the protein h-mucolipin-1, result in the lysosomal storage disease Mucolipidosis Type IV. Studies on CUP-5, the human orthologue of h-mucolipin-1 in Caenorhabditis elegans, have shown that these proteins are required for lysosome biogenesis. We show here that the lethality in cup-5 mutant worms is due to two defects, starvation of embryonic cells and general developmental defects. Starvation leads to apoptosis through a CED-3-mediated pathway. We also show that providing worms with a lipid-soluble metabolite partially rescues the embryonic lethality but has no effect on the developmental defects, the major cause of the lethality. These results indicate that supplementing the metabolic deficiency of Mucolipidosis Type IV patients mat not be sufficient to alleviate the symptoms due to tissue degeneration.     

CXC chemokine receptor 4 expressed in T cells plays an important role in the development of collagen-induced arthritis

Introduction  

Chemokines and their receptors are potential therapeutic targets in rheumatoid arthritis (RA). Among these, several studies suggested the involvement of CXC chemokine 4 (CXCR4) and its ligand CXC ligand 12 (SDF-1) in RA pathogenesis. However, the role of these molecules in T-cell function is not known completely because of embryonic lethality of Cxcr4- and Cxcl12- deficient mice. In this report, we generated T cell-specific Cxcr4 -deficient mice and showed that the CXCR4 in T cells is important for the development of collagen-induced arthritis (CIA).     

Detection of the pediocin gene <Emphasis Type="Italic">pedA</Emphasis> in strains from human faeces by real-time PCR and characterization of <Emphasis Type="Italic">Pediococcus acidilactici</Emphasis>UVA1

Background  

Bacteriocin-producing lactic acid bacteria are commonly used as natural protective cultures. Among them, strains of the genus Pediococcus are particularly interesting for their ability to produce pediocin, a broad spectrum antimicrobial peptide with a strong antagonistic activity against the food-borne pathogen Listeria monocytogenes. Furthermore, there is increasing interest in isolating new bacteriocin-producing strains of human intestinal origin that could be developed for probiotic effects and inhibition of pathogenic bacteria in the gut. In this work, we typed a new strain, co-isolated from baby faeces together with a Bifidobacterium thermophilum strain, and characterized its proteinaceous compound with strong antilisterial activity.     

Caenorhabditis elegans HOPS and CCZ-1 mediate trafficking to lysosome-related organelles independently of RAB-7 and SAND-1

As early endosomes mature, the SAND-1/CCZ-1 complex acts as a guanine nucleotide exchange factor (GEF) for RAB-7 to promote the activity of its effector, HOPS, which facilitates late endosome–lysosome fusion and the consumption of AP-3–containing vesicles. We show that CCZ-1 and the HOPS complex are essential for the biogenesis of gut granules, cell type–specific, lysosome-related organelles (LROs) that coexist with conventional lysosomes in Caenorhabditis elegans intestinal cells. The HOPS subunit VPS-18 promotes the trafficking of gut granule proteins away from lysosomes and functions downstream of or in parallel to the AP-3 adaptor. CCZ-1 also acts independently of AP-3, and ccz-1 mutants mistraffic gut granule proteins. Our results indicate that SAND-1 does not participate in the formation of gut granules. In the absence of RAB-7 activity, gut granules are generated; however, their size and protein composition are subtly altered. These observations suggest that CCZ-1 acts in partnership with a protein other than SAND-1 as a GEF for an alternate Rab to promote gut granule biogenesis. Point mutations in GLO-1, a Rab32/38-related protein, predicted to increase spontaneous guanine nucleotide exchange, specifically suppress the loss of gut granules by ccz-1 and glo-3 mutants. GLO-3 is known to be required for gut granule formation and has homology to SAND-1/Mon1–related proteins, suggesting that CCZ-1 functions with GLO-3 upstream of the GLO-1 Rab, possibly as a GLO-1 GEF. These results support LRO formation occurring via processes similar to conventional lysosome biogenesis, albeit with key molecular differences.     

Pathophysiologic consequences following inhibition of a CFTR-dependent developmental cascade in the lung

Background  

Examination of late gestation developmental genes in vivo may be limited by early embryonic lethality and compensatory mechanisms. This problem is particularly apparent in evaluating the developmental role of the cystic fibrosis transmembrane conductance regulator (CFTR) gene in the cystic fibrosis (CF) phenotype. A previously described transient in utero knockout (TIUKO) technology was used to address the developmental role of CFTR in the rat lung.     

FlyPNS,a database of the <Emphasis Type="Italic">Drosophila</Emphasis> embryonic and larval peripheral nervous system

Background  

The embryonic and larval peripheral nervous system of Drosophila melanogaster is extensively studied as a very powerful model of developmental biology. One main advantage of this system is the ability to study the origin and development of individual sensory cells. However, there remain several discrepancies regarding the organization of sensory organs in each abdominal segment A1-A7.     

Highlights from this issue

Germline P granules are specialized protein/RNA aggregates that are found exclusively in germ cells in C. elegans. During the early embryonic divisions that generate germ blastomeres, aggregate-prone P granule components PGL-1 and PGL-3 that remain in the cytoplasm destined for somatic daughters are selectively removed by autophagy. Loss-of-function of components of the autophagy pathway, including the VPS-34/BEC-1 complex, causes accumulation of PGL-1 and PGL-3 into aggregates in somatic cells (termed PGL granules). Formation of PGL granules depends on SEPA-1, which is an integral component of these granules. SEPA-1 is preferentially degraded by autophagy and is also required for the autophagic degradation of PGL-1 and PGL-3. SEPA-1 functions as a bridging molecule in mediating degradation of P granule components by directly interacting with PGL-3 and also with the autophagy protein LGG-1/Atg8. The defect in embryonic development in autophagy mutants is suppressed by mutation of sepa-1, suggesting that autophagic degradation of PGL granule components may provide nutrients for embryogenesis and/or also prevent the formation of aggregates that could be toxic for animal development. Our study reveals a specific physiological function of selective autophagic degradation during C. elegans development.     

Transcriptional regulation of subclass 5b fimbriae

Background  

Enterotoxigenic Escherichia coli (ETEC) is a major cause of infant and child mortality in developing countries. This enteric pathogen causes profuse watery diarrhea by elaborating one or more enterotoxins that intoxicate eukaryotic cells and ultimately leads to a loss of water to the intestinal lumen. Virulence is also dependent upon fimbrial adhesins that facilitate colonization of the small intestine.     

Life and Death of Sensory Hair Cells Expressing Constitutively Active TRPML3

The varitint-waddler mutation A419P renders TRPML3 constitutively active, resulting in cationic overload, particularly in sustained influx of Ca²⁺. TRPML3 is expressed by inner ear sensory hair cells, and we were intrigued by the fact that hair cells are able to cope with expressing the TRPML3(A419P) isoform for weeks before they ultimately die. We hypothesized that the survival of varitint-waddler hair cells is linked to their ability to deal with Ca²⁺ loads due to the abundance of plasma membrane calcium ATPases (PMCAs). Here, we show that PMCA2 significantly reduced [Ca²⁺]_i increase and apoptosis in HEK293 cells expressing TRPML3(A419P). The deaf-waddler isoform of PMCA2, operating at 30% efficacy, showed a significantly decreased ability to rescue the Ca²⁺ loading of cells expressing TRPML3(A419P). When we combined mice heterozygous for the varitint-waddler mutant allele with mice heterozygous for the deaf-waddler mutant allele, we found severe hair bundle defects as well as increased hair cell loss compared with mice heterozygous for each mutant allele alone. Furthermore, 3-week-old double mutant mice lacked auditory brainstem responses, which were present in their respective littermates containing single mutant alleles. Likewise, heterozygous double mutant mice exhibited severe circling behavior, which was not observed in mice heterozygous for TRPML3(A419P) or PMCA2(G283S) alone. Our results provide a molecular rationale for the delayed hair cell loss in varitint-waddler mice. They also show that hair cells are able to survive for weeks with sustained Ca²⁺ loading, which implies that Ca²⁺ loading is an unlikely primary cause of hair cell death in ototoxic stress situations.Varitint-waddler (Va) mice express a mutant isoform (A419P) of the transient receptor potential channel TRPML3 (murine gene symbol, Mcoln3) that results in profound hearing loss, vestibular defects (circling behavior, imbalance, head bobbing, waddling), pigmentation deficiencies, sterility, and perinatal lethality in homozygous animals (1). A second Mcoln3 variant (Va^J) that arose in the Va background carries two mutations (I362T and A419P) and shows a phenotype with reduced severity, particularly in heterozygous animals (1). The A419P mutation in Va and Va^J mice is located in transmembrane-spanning domain 5(TM5)³ of TRPML3, where it leads to a constitutively open channel, resulting in highly elevated [Ca²⁺]_i (2-5). In contrast to the effect of the A419P mutation on TRPML3 channel activity, the single I362T mutation does not appear to affect [Ca²⁺]_i (3, 5). When combined with the A419P mutation, as found in Va^J mice, the constitutive activity of this mutant TRPML3 isoform is comparable with that of A419P alone (2-5).Here, we show that HEK293 cells expressing TRPML3-(A419P) or TRPML3(I362T/A419P) undergo rapid apoptosis. This apoptosis is suppressed by coexpression of plasma membrane calcium ATPase type 2 (PMCA2). In varitint-waddler mice, sensory hair cells survive for weeks after birth (6), which raised the question of whether this survival could be the result of the hair cells'' ability to deal with normally transient and localized Ca²⁺ influx, a feature that is centered around the high levels of mobile Ca²⁺ buffers and PMCA isoforms found in sensory hair cells (7-10). We decided to test this hypothesis in vivo by utilizing deaf-waddler mice that carry a mutation (G283S) in the Atp2b2 gene encoding mutant PMCA2. Mice homozygous for PMCA2(G283S) (Atp2b2^dfw/dfw) are deaf and have poor balance (11). Compared with Atp2b2 knock-out mice, deaf-waddler mice display a milder phenotype because PMCA2(G283S) retains 30% of its biological activity compared with the wild-type isoform (12). We found that sensory hair cell loss, hearing loss, and vestibular dysfunction were aggravated in mice carrying varitint-waddler and deaf-waddler alleles compared with animals carrying the single mutant alleles. Our results reveal that the Ca²⁺-buffering and Ca²⁺ extrusion abilities of hair cells are powerful enough to prevent cell death for weeks, even in the presence of constitutively active TRPML3(A419P), which is able to induce rapid apoptosis in other cells.     

Chromogranin B: intra‐ and extra‐cellular mechanisms to regulate catecholamine storage and release,in catecholaminergic cells and organisms

Chromogranin B (CHGB) is the major matrix protein in human catecholamine storage vesicles. CHGB genetic variation alters catecholamine secretion and blood pressure. Here, effective Chgb protein under‐expression was achieved by siRNA in PC12 cells, resulting in ~ 48% fewer secretory granules on electron microscopy, diminished capacity for catecholamine uptake (by ~ 79%), and a ~ 73% decline in stores available for nicotinic cholinergic‐stimulated secretion. In vivo, loss of Chgb in knockout mice resulted in a ~ 35% decline in chromaffin granule abundance and ~ 44% decline in granule diameter, accompanied by unregulated catecholamine release into plasma. Over‐expression of CHGB was achieved by transduction of a CHGB‐expressing lentivirus, resulting in ~ 127% elevation in CHGB protein, with ~ 122% greater abundance of secretory granules, but only ~ 14% increased uptake of catecholamines, and no effect on nicotinic‐triggered secretion. Human CHGB protein and its proteolytic fragments inhibited nicotinic‐stimulated catecholamine release by ~ 72%. One conserved‐region CHGB peptide inhibited nicotinic‐triggered secretion by up to ~ 41%, with partial blockade of cationic signal transduction. We conclude that bi‐directional quantitative derangements in CHGB abundance result in profound changes in vesicular storage and release of catecholamines. When processed and released extra‐cellularly, CHGB proteolytic fragments exert a feedback effect to inhibit catecholamine secretion, especially during nicotinic cholinergic stimulation.

     

Analysis of gene expression profiles in HeLa cells in response to overexpression or siRNA-mediated depletion of NASP

Background  

NASP (Nuclear Autoantigenic Sperm Protein) is a linker histone chaperone required for normal cell division. Changes in NASP expression significantly affect cell growth and development; loss of gene function results in embryonic lethality. However, the mechanism by which NASP exerts its effects in the cell cycle is not understood. To understand the pathways and networks that may involve NASP function, we evaluated gene expression in HeLa cells in which NASP was either overexpressed or depleted by siRNA.