UNC-80 and the NCA ion channels contribute to endocytosis defects in synaptojanin mutants

Synaptojanin is a lipid phosphatase required to degrade phosphatidylinositol 4,5 bisphosphate (PIP(2)) at cell membranes during synaptic vesicle recycling. Synaptojanin mutants in C. elegans are severely uncoordinated and are depleted of synaptic vesicles, possibly because of accumulation of PIP(2). To identify proteins that act downstream of PIP(2) during endocytosis, we screened for suppressors of synaptojanin mutants in the nematode C. elegans. A class of uncoordinated mutants called "fainters" partially suppress the locomotory, vesicle depletion, and electrophysiological defects in synaptojanin mutants. These suppressor loci include the genes for the NCA ion channels, which are homologs of the vertebrate cation leak channel NALCN, and a novel gene called unc-80. We demonstrate that unc-80 encodes a novel, but highly conserved, neuronal protein required for the proper localization of the NCA-1 and NCA-2 ion channel subunits. These data suggest that activation of the NCA ion channel in synaptojanin mutants leads to defects in recycling of synaptic vesicles.     

The Francisella tularensis pathogenicity island protein IglC and its regulator MglA are essential for modulating phagosome biogenesis and subsequent bacterial escape into the cytoplasm

The Francisella tularensis subsp. novicida-containing phagosome (FCP) matures into a late endosome-like stage that acquires the late endosomal marker LAMP-2 but does not fuse to lysosomes, for the first few hours after bacterial entry. This modulation in phagosome biogenesis is followed by disruption of the phagosome and bacterial escape into the cytoplasm where they replicate. Here we examined the role of the Francisella pathogenicity island (FPI) protein IglC and its regulator MglA in the intracellular fate of F. tularensis subsp. novicida within human macrophages. We show that F. tularensis mglA and iglC mutant strains are defective for survival and replication within U937 macrophages and human monocyte-derived macrophages (hMDMs). The defect in intracellular replication of both mutants is associated with a defect in disruption of the phagosome and failure to escape into the cytoplasm. Approximately, 80-90% of the mglA and iglC mutants containing phagosomes acquire the late endosomal/lysosomal marker LAMP-2 similar to the wild-type (WT) strain. Phagosomes harbouring the mglA or iglC mutants acquire the lysosomal enzyme Cathepsin D, which is excluded from the phagosomes harbouring the WT strain. In hMDMs in which the lysosomes are preloaded with BSA-gold or Texas Red Ovalbumin, phagosomes harbouring the mglA or the iglC mutants acquire both lysosomal tracers. We conclude that the FPI protein IglC and its regulator MglA are essential for modulating phagosome biogenesis and subsequent bacterial escape into the cytoplasm. Therefore, acquisition of the FPI, within which iglC is contained, is essential for the pathogenic evolution of F. tularensis to evade lysosomal fusion within human macrophages and cause tularemia. This is the first example of specific virulence factors of F. tularensis that are essential for evasion of fusion of the FCP to lysosomes.     

Mitogen-activated protein kinase activity during goat oocyte maturation and the acquisition of meiotic competence

Changes in MPF and MAPK activities during meiotic maturation of goat oocytes were investigated. Detection of MPF activity occurred concomitantly with GVBD, increased at MI, decreased during anaphase-telophase I transition, and increased thereafter in MII oocytes. The appearance of MAPK activity was delayed compared to MPF activity. MAPK activity increased after GVBD and persisted during the MI-MII transition. Whether MAPK was implicated in goat oocyte meiotic competence was also investigated by using oocytes from different follicle size categories that arrest at specific stages of the maturation process (GV, GVBD, MI, and MII). Results indicate that the ability of goat oocytes to resume meiosis is not directly related to the presence of Erk2. The ability to phosphorylate MAPK is acquired by the oocyte during follicular growth after the ability to resume meiosis. GVBD-arrested oocytes exhibited a high level of MPF activity after 27 hr of culture. However, 28% of oocytes from this group contained inactive MAPK, and 72% exhibited high MAPK activity. In addition, 29% of GVBD-arrested oocytes contained a residual interphasic network without recruitment of microtubules around the condensed chromosomes; 71% of GVBD-arrested oocytes displayed recruitment of microtubules near the condensed chromosomes and contained asters of microtubules distributed throughout the cytoplasm. These results indicate that oocytes arrested at GVBD were not exactly at the same point in the meiotic cell cycle progression, and suggest that MAPK could be implicated in the regulation of microtubule organization. The data presented here suggest that in goat oocytes, MAPK is not implicated in the early events of meiosis resumption, but rather in post-GVBD events such as spindle formation and MII arrest. © 1996 Wiley-Liss Inc.     

Development of hypoosmoregulatory ability in allis shad Alosa alosa

When different ages of juvenile allis shad Alosa alosa (18–74 days post-fertilization) were exposed for 48 h to elevated salinity (25 or 30), all ages could tolerate moderates salinity (25) whereas only the older stages could tolerate the higher salinity. In 88 days post-fertilization allis shad, acclimatation to full-strength sea water for 2 weeks did not lead to significant changes in total water content and gill Na⁺/K⁺-ATPase activity whereas total Na⁺ content increased within 5 days to values which remained stable thereafter. Together, these data suggest that juvenile allis shad develop a limited euryhalinity, which allows them to fully adapt to hyperosmotic environment immediately after transfer.     

A Francisella tularensis pathogenicity island protein essential for bacterial proliferation within the host cell cytosol

Francisella tularensis is an intracellular bacterial pathogen, and is a category A bioterrorism agent. Within quiescent human macrophages, the F. tularensis pathogenicity island (FPI) is essential for bacterial growth within quiescent macrophages. The F. tularensis-containing phagosome matures to a late endosome-like stage that does not fuse to lysosomes for 1-8 h, followed by gradual bacterial escape into the macrophage cytosol. Here we show that the FPI protein IglD is essential for intracellular replication in primary human monocyte-derived macrophages (hMDMs). While the parental strain replicates robustly in pulmonary, hepatic and splenic tissues of BALB/c mice associated with severe immunopathologies, the isogenic iglD mutant is severely defective. Within hMDMs, the iglD mutant-containing phagosomes mature to either a late endosome-like phagosome, similar to the parental strain, or to a phagolysosome, similar to phagosomes harbouring the iglC mutant control. Despite heterogeneity and alterations in phagosome biogenesis, the iglD mutant bacteria escape into the cytosol faster than the parental strain within hMDMs and pulmonary cells of BALB/c mice. Co-infections of hMDMs with the wild-type strain and the iglD mutant, or super-infection of iglD mutant-infected hMDMs with the wild-type strain show that the mutant strain replicates robustly within the cytosol of hMDMs coinhabited by the wild strain. However, when the wild-type strain-infected hMDMs are super-infected by the iglD mutant, the mutant fails to replicate in the cytosol of communal macrophages. This is the first demonstration of a F. tularensis novel protein essential for proliferation in the macrophage cytosol. Our data indicate that F. tularensis transduces signals to the macrophage cytosol to remodel it into a proliferative niche, and IglD is essential for transduction of these signals.