Phosphoinositides in phagolysosome and autophagosome biogenesis

Interconversions of phosphoinositides play a pivotal role during phagocytosis and at the subsequent stages of phagosomal maturation into the phagolysosome. Several model systems have been used to study the role of phosphoinositides in phagosomal membrane remodelling. These include phagosomes formed by inanimate objects such as latex beads, or pathogenic bacteria, e.g. Mycobacterium tuberculosis. The latter category provides naturally occurring tools to dissect membrane trafficking processes governing phagolysosome biogenesis. M. tuberculosis persists in infected macrophages by blocking Rab conversion and affecting Rab effectors. One of the major Rab effectors involved in this process is the type III phosphatidylinositol 3-kinase hVPS34. The lipid kinase hVPS34 and its enzymatic product PtdIns3P are critical for the default pathway of phagosomal maturation into phagolysosomes. Mycobacteria block PtdIns3P production and thus arrest phagosomal maturation. PtdIns3P is also critical for the process of autophagy, recently recognized as an effector of innate immunity defenses. Induction of autophagy by pharmacological, physiological, or immunological means, overcomes mycobacterial phagosome maturation block in a PtdIns3P generation dependent manner and eliminates intracellular M. tuberculosis. PtdIns3P and PtdIns3P-dependent processes represent an important cellular nexus where fundamental trafficking processes, disease causing host-pathogen interactions, and innate and adaptive immunity defense mechanisms meet.     

Rab7: a key to lysosome biogenesis

The molecular machinery behind lysosome biogenesis and the maintenance of the perinuclear aggregate of late endocytic structures is not well understood. A likely candidate for being part of this machinery is the small GTPase Rab7, but it is unclear whether this protein is associated with lysosomes or plays any role in the regulation of the perinuclear lysosome compartment. Previously, Rab7 has mainly been implicated in transport from early to late endosomes. We have now used a new approach to analyze the role of Rab7: transient expression of Enhanced Green Fluorescent Protein (EGFP)-tagged Rab7 wt and mutant proteins in HeLa cells. EGFP-Rab7 wt was associated with late endocytic structures, mainly lysosomes, which aggregated and fused in the perinuclear region. The size of the individual lysosomes as well as the degree of perinuclear aggregation increased with the expression levels of EGFP-Rab7 wt and, more dramatically, the active EGFP-Rab7Q67L mutant. In contrast, upon expression of the dominant-negative mutants EGFP-Rab7T22N and EGFP-Rab7N125I, which localized mainly to the cytosol, the perinuclear lysosome aggregate disappeared and lysosomes, identified by colocalization of cathepsin D and lysosome-associated membrane protein-1, became dispersed throughout the cytoplasm, they were inaccessible to endocytosed molecules such as low-density lipoprotein, and their acidity was strongly reduced, as determined by decreased accumulation of the acidotropic probe LysoTracker Red. In contrast, early endosomes associated with Rab5 and the transferrin receptor, late endosomes enriched in the cation-independent mannose 6-phosphate receptor, and the trans-Golgi network, identified by its enrichment in TGN-38, were unchanged. These data demonstrate for the first time that Rab7, controlling aggregation and fusion of late endocytic structures/lysosomes, is essential for maintenance of the perinuclear lysosome compartment.     

Mycobacterium tuberculosis inhibition of phagolysosome biogenesis and autophagy as a host defence mechanism

A marquee feature of the powerful human pathogen Mycobacterium tuberculosis is its macrophage parasitism. The intracellular survival of this microorganism rests upon its ability to arrest phagolysosome biogenesis, avoid direct cidal mechanisms in macrophages, and block efficient antigen processing and presentation. Mycobacteria prevent Rab conversion on their phagosomes and elaborate glycolipid and protein trafficking toxins that interfere with Rab effectors and regulation of specific organellar biogenesis in mammalian cells. One of the major Rab effectors affected in this process is the type III phosphatidylinositol 3-kinase hVPS34 and its enzymatic product phosphatidylinositol 3-phosphate (PI3P), a regulatory lipid earmarking organellar membranes for specific trafficking events. PI3P is also critical for the process of autophagy, recently recognized as an effector of innate and adaptive immunity. Induction of autophagy by physiological, pharmacological or immunological signals, including the major antituberculosis Th1 cytokine IFN-gamma and its downstream effector p47 GTPase LRG-47, can overcome mycobacterial phagosome maturation block and inhibit intracellular M. tuberculosis survival. This review summarizes the findings centred around the PI3P-nexus where the mycobacterial phagosome maturation block and execution stages of autophagy intersect.     

Rab2 regulates presynaptic precursor vesicle biogenesis at the trans-Golgi

Reliable delivery of presynaptic material, including active zone and synaptic vesicle proteins from neuronal somata to synaptic terminals, is prerequisite for successful synaptogenesis and neurotransmission. However, molecular mechanisms controlling the somatic assembly of presynaptic precursors remain insufficiently understood. We show here that in mutants of the small GTPase Rab2, both active zone and synaptic vesicle proteins accumulated in the neuronal cell body at the trans-Golgi and were, consequently, depleted at synaptic terminals, provoking neurotransmission deficits. Ectopic presynaptic material accumulations consisted of heterogeneous vesicles and short tubules of 40 × 60 nm, segregating in subfractions either positive for active zone or synaptic vesicle proteins and LAMP1, a lysosomal membrane protein. Genetically, Rab2 acts upstream of Arl8, a lysosomal adaptor controlling axonal export of precursors. Collectively, we identified a Golgi-associated assembly sequence of presynaptic precursor biogenesis dependent on a Rab2-regulated protein export and sorting step at the trans-Golgi.     

A role for Rab5 activity in the biogenesis of endosomal and lysosomal compartments

Rab5 is a small GTPase that plays roles in the homotypic fusion of early endosomes and regulation of intracellular vesicle transport. We show here that expression of GFP-tagged GTPase-deficient form of Rab5b (Rab5bQ79L) in NRK cells results in the sequential formation of three morphologically and functionally distinct types of endosomes. Expression of GFP-Rab5bQ79L initially caused a homotypic fusion of early endosomes accompanying a redistribution of the TGN-resident cargo molecules, and subsequent fusion with late endosomes/lysosomes, leading to the formation of giant hybrid organelles with features of early endosomes and late endosomes/lysosomes. Surprisingly, the giant endosomes gradually fragmented and shrunk, leading to the accumulation of early endosome clusters and concurrent reformation of late endosomes/lysosomes, a process accelerated by treatment with a phosphatidylinositol-3-kinase (PI(3)K) inhibitor, wortmannin. We postulate that such sequential processes reflect the biogenesis and maintenance of late endosomes/lysosomes, presumably via direct fusion with early endosomes and subsequent fission from hybrid organelles. Thus, our findings suggest a regulatory role for Rab5 in not only the early endocytic pathway, but also the late endocytic pathway, of membrane trafficking in coordination with PI(3)K activity.     

Higher order structure in metaphase chromosomes

The morphology of metaphase chromosome-derived chromatin fibers released from cells by non-ionic detergent cell lysis in the presence of divalent cations has been studied by electron microscopy. In these preparations the euchromatic arms appear as a series of loops, 200–300 Å in diameter, which are composed of closely-apposed nucleosome arrays. The higher order fiber in chromosomes derived from detergent-lysed cells appears to be less stable than chromatin fibers obtained by mechanical cell lysis. The fiber breaks down into a series of non-uniform nucleosome aggregates (superbeads) and finally to chromatin in a beads-on-a-string morphology upon incubation at 31° for 20 min. These observations allow us to suggest a relationship between uniform thick fibers, superbead-containing fibers, and beads-on-a-string chromatin within metaphase chromosomes.     

Lysosome biogenesis requires Rab9 function and receptor recycling from endosomes to the trans-Golgi network

Newly synthesized lysosomal enzymes bind to mannose 6-phosphate receptors (MPRs) in the TGN, and are carried to prelysosomes, where they are released. MPRs then return to the TGN for another round of transport. Rab9 is a ras-like GTPase which facilitates MPR recycling to the TGN in vitro. We show here that a dominant negative form of rab9, rab9 S21N, strongly inhibited MPR recycling in living cells. The block was specific in that the rates of biosynthetic protein transport, fluid phase endocytosis and receptor-mediated endocytosis were unchanged. Expression of rab9 S21N was accompanied by a decrease in the efficiency of lysosomal enzyme sorting. Cells compensated for the presence of the mutant protein by inducing the synthesis of both soluble and membrane- associated lysosomal enzymes, and by internalizing lysosomal enzymes that were secreted by default. These data show that MPRs are limiting in the secretory pathway of cells expressing rab9 S21N and document the importance of MPR recycling and the rab9 GTPase for efficient lysosomal enzyme delivery.     

Higher order structure in ribosomal RNA

[This corrects the article on p. 1111 in vol. 5, PMID: 3720727.].     

Rab11A controls the biogenesis of Birbeck granules by regulating Langerin recycling and stability

The extent to which Rab GTPases, Rab-interacting proteins, and cargo molecules cooperate in the dynamic organization of membrane architecture remains to be clarified. Langerin, a recycling protein accumulating in the Rab11-positive compartments of Langerhans cells, induces the formation of Birbeck granules (BGs), which are membrane subdomains of the endosomal recycling network. We investigated the role of Rab11A and two members of the Rab11 family of interacting proteins, Rip11 and RCP, in Langerin traffic and the biogenesis of BGs. The overexpression of a dominant-negative Rab11A mutant or Rab11A depletion strongly influenced Langerin traffic and stability and the formation of BGs, whereas modulation of other Rab proteins involved in dynamic regulation of the endocytic-recycling pathway had no effect. Impairment of Rab11A function led to a missorting of Langerin to lysosomal compartments, but inhibition of Langerin degradation by chloroquine did not restore the formation of BGs. Loss of RCP, but not of Rip11, also had a modest, but reproducible effect on Langerin stability and BG biogenesis, pointing to a role for Rab11A-RCP complexes in these events. Our results show that Rab11A and Langerin are required for BG biogenesis, and they illustrate the role played by a Rab GTPase in the formation of a specialized subcompartment within the endocytic-recycling system.     

Role of phosphatidylinositol 3-kinase and Rab5 effectors in phagosomal biogenesis and mycobacterial phagosome maturation arrest

Phagosomal biogenesis is a fundamental biological process of particular significance for the function of phagocytic and antigen-presenting cells. The precise mechanisms governing maturation of phagosomes into phagolysosomes are not completely understood. Here, we applied the property of pathogenic mycobacteria to cause phagosome maturation arrest in infected macrophages as a tool to dissect critical steps in phagosomal biogenesis. We report the requirement for 3-phosphoinositides and acquisition of Rab5 effector early endosome autoantigen (EEA1) as essential molecular events necessary for phagosomal maturation. Unlike the model phagosomes containing latex beads, which transiently recruited EEA1, mycobacterial phagosomes excluded this regulator of vesicular trafficking that controls membrane tethering and fusion processes within the endosomal pathway and is recruited to endosomal membranes via binding to phosphatidylinositol 3-phosphate (PtdIns[3]P). Inhibitors of phosphatidylinositol 3'(OH)-kinase (PI-3K) activity diminished EEA1 recruitment to newly formed latex bead phagosomes and blocked phagosomal acquisition of late endocytic properties, indicating that generation of PtdIns(3)P plays a role in phagosomal maturation. Microinjection into macrophages of antibodies against EEA1 and the PI-3K hVPS34 reduced acquisition of late endocytic markers by latex bead phagosomes, demonstrating an essential role of these Rab5 effectors in phagosomal biogenesis. The mechanism of EEA1 exclusion from mycobacterial phagosomes was investigated using mycobacterial products. Coating of latex beads with the major mycobacterial cell envelope glycosylated phosphatidylinositol lipoarabinomannan isolated from the virulent Mycobacterium tuberculosis H37Rv, inhibited recruitment of EEA1 to latex bead phagosomes, and diminished their maturation. These findings define the generation of phosphatidylinositol 3-phosphate and EEA1 recruitment as: (a) important regulatory events in phagosomal maturation and (b) critical molecular targets affected by M. tuberculosis. This study also identifies mycobacterial phosphoinositides as products with specialized toxic properties, interfering with discrete trafficking stages in phagosomal maturation.     

Higher order structure in ribosomal RNA.

The only reliable general method currently available for determining precise higher order structure in the large ribosomal RNAs is comparative sequence analysis. The method is here applied to reveal 'tertiary' structure in the 16S-like rRNAs, i.e. structure more complex than simple double-helical, secondary structure. From a list of computer-generated potential higher order interactions within 16S rRNA one such interaction considered likely was selected for experimental test. The putative interaction involves a Watson-Crick one to one correspondence between positions 570 and 866 in the molecule (E. coli numbering). Using existing oligonucleotide catalog information several organisms were selected whose 16S rRNA sequences might test the proposed co-variation. In all of the (phylogenetically independent) cases selected, full sequence evidence confirms the predicted one to one (Watson-Crick) correspondence. An interaction between positions 570 and 866 is, therefore, considered proven phylogenetically.     

Modulation of biogenesis of the Francisella tularensis subsp. novicida-containing phagosome in quiescent human macrophages and its maturation into a phagolysosome upon activation by IFN-gamma

Francisella tularensis is a highly virulent facultative intracellular pathogen that has been categorized as a class A bioterrorism agent, and is classified into four subsp, tularensis, holarctica, mediasiatica and novicida. Although the ability of F. tularensis subsp. novicida to cause tularemia in mice is similar to the virulent subsp. tularensis and holarctica, it is attenuated in humans. It is not known whether attenuation of F. tularensis subsp. novicida in humans is resulting from a different route of trafficking within human macrophages, compared with the tularensis or holarctica subsp. Here we show that in quiescent human monocytes-derived macrophages (hMDMs), the F. 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. This modulation of phagosome biogenesis by F. tularensis is followed by disruption of the phagosome at 4-12 h and subsequent bacterial escape into cytoplasm where the organism replicates. In IFN-gamma-activated hMDMs, intracellular replication of F. tularensis is completely inhibited, and is associated with failure of the organism to escape from the phagosome into the cytoplasm for up to 24 h after infection. In IFN-gamma-activated hMDMs, the FCPs acquire the lysosomal enzymes Cathepsin D, which is excluded in quiescent hMDMs. When the lysosomes of IFN-gamma-activated hMDMs are preload with Texas Red Ovalbumin or BSA-gold, the FCPs acquire both lysosomal tracers. In contrast, both lysosomal tracers are excluded from the FCPs within quiescent hMDMs. We conclude that although F. tularensis subsp. novicida is attenuated in humans, it modulates biogenesis of its phagosome into a late endosome-like compartment followed by bacterial escape into the cytoplasm within quiescent hMDMs, similar to the virulent subsp. tularensis. In IFN-gamma-activated hMDMs, the organism fails to escape into the cytoplasm and its phagosome fuses to lysosomes, similar to inert particles.     

A core complex of BBS proteins cooperates with the GTPase Rab8 to promote ciliary membrane biogenesis

Primary cilium dysfunction underlies the pathogenesis of Bardet-Biedl syndrome (BBS), a genetic disorder whose symptoms include obesity, retinal degeneration, and nephropathy. However, despite the identification of 12 BBS genes, the molecular basis of BBS remains elusive. Here we identify a complex composed of seven highly conserved BBS proteins. This complex, the BBSome, localizes to nonmembranous centriolar satellites in the cytoplasm but also to the membrane of the cilium. Interestingly, the BBSome is required for ciliogenesis but is dispensable for centriolar satellite function. This ciliogenic function is mediated in part by the Rab8 GDP/GTP exchange factor, which localizes to the basal body and contacts the BBSome. Strikingly, Rab8(GTP) enters the primary cilium and promotes extension of the ciliary membrane. Conversely, preventing Rab8(GTP) production blocks ciliation in cells and yields characteristic BBS phenotypes in zebrafish. Our data reveal that BBS may be caused by defects in vesicular transport to the cilium.     

The CORVET tethering complex interacts with the yeast Rab5 homolog Vps21 and is involved in endo-lysosomal biogenesis

The dynamic equilibrium between vesicle fission and fusion at Golgi, endosome, and vacuole/lysosome is critical for the maintenance of organelle identity. It depends, among others, on Rab GTPases and tethering factors, whose function and regulation are still unclear. We now show that transport among Golgi, endosome, and vacuole is controlled by two homologous tethering complexes, the previously identified HOPS complex at the vacuole and a novel endosomal tethering (CORVET) complex, which interacts with the Rab GTPase Vps21. Both complexes share the four class C Vps proteins: Vps11, Vps16, Vps18, and Vps33. The HOPS complex, in addition, contains Vps41/Vam2 and Vam6, whereas the CORVET complex has the Vps41 homolog Vps8 and the (h)Vam6 homolog Vps3. Strikingly, the CORVET and HOPS complexes can interconvert; we identify two additional intermediate complexes, both consisting of the class C core bound to Vam6-Vps8 or Vps3-Vps41. Our data suggest that modular assembled tethering complexes define organelle biogenesis in the endocytic pathway.     

Higher order assemblies of molluscan hemocyanins

1. The hemocyanins of the Fissurellidae, Naticidae and Melongenidae families of marine gastropods as well as some other molluscs including some members of the Opistobranchia and Bivalvia groups have hemocyanins which exist in solution as tri-decameric and mixed, multi-decameric aggregates characterized by sedimentation coefficients close to 100 S, 130 S, 150 S, 170 S and 200 S to 230 S. 2. The particle masses of the molluscan hemocyanins appear to be integral multiples close to 4.4 x 10(6) daltons. Thus, particle mass values of 4.47 x 10(6), 8.67 x 10(6) and 13.40 x 10(6) daltons were obtained for representative decameric, di-decameric, and tri-decameric components of Stenoplax conspicua, Fasciolaria tulipa and Euspira (Lunatia) heros hemocyanins. For Busycon contrarium, a gastropod with a mixed multidecameric hemocyanin, scanning transmission electron microscopic (STEM) measurements gave particle masses ranging from 8.89 x 10(6) and 13.20 x 10(6) for the di- and tri-decameric components to 38.87 x 10(6) and 43.40 x 10(6) daltons for highest nano- and deca-decameric aggregates. 3. The electron microscopic images of both uranyl acetate-stained and unstained specimens of hemocyanin aggregates indicate a non-random mode of assembly of the multi-decameric particles. This is most apparent from the electron micrographs of the moon snail hemocyanins. The tri-decameric and tetra-decameric particles seem to be assembled from a single di-decameric unit of the Mellema and Klug arrangement, with the collar ends facing outward, to which decameric units have been added from one or both ends, in a unidirectional tail-to-head to tail-to-collar manner. Consequently, all the aggregates including the higher, Melongenidae polymers have the appearance of closed cylinders terminating with the collar ends. 4. The radial distribution of the end-on views of the hemocyanin of the moon-snail Calinatioina oldroydii, show that the radial mass drops to zero at the center of the cylindrical particles consisting of one, two, or three decamers. This suggests that no caps are present at the ends of the hemocyanin particles which would inhibit or terminate their linear assembly. 5. The light-scattering behavior of B. contrarium and Marisa cornarietis hemocyanins examined as a function of increasing reagent concentration using the hydrophobic urea and Hofmeister salt series of reagents, show distinct aggregation and increase in molecular weights at low concentrations of reagent. Together with the stabilizing influence of Mg2+ and Ca2+ ions, this suggests polar and ionic stabilization of the inter-decameric contacts between the central di-decamers and the added decameric units of the higher aggregates of molluscan hemocyanins.(ABSTRACT TRUNCATED AT 400 WORDS)     

Living in a phagolysosome; metabolism of Leishmania amastigotes

Leishmania amastigotes primarily proliferate within macrophages in the mammalian host. Genome-based metabolic reconstructions, combined with biochemical, reverse genetic and mRNA or protein profiling studies are providing new insights into the metabolism of this intracellular stage. We propose that the complex nutritional requirements of amastigotes have contributed to the tropism of these parasites for the amino acid-rich phagolysosome of macrophages. Amastigote metabolism in this compartment is robust because many metabolic mutants are capable of either growing normally or persisting long term in susceptible animals. New approaches for measuring amastigote metabolism in vivo are discussed.     

Roles of plant homologs of Rab1p and Rab7p in the biogenesis of the peribacteroid membrane, a subcellular compartment formed de novo during root nodule symbiosis.

The peribacteroid membrane (PBM) in legume root nodules is derived from plasma membrane following endocytosis of Rhizobium by fusion of newly synthesized vesicles. We studied the roles of plant Rab1p and Rab7p homologs, the small GTP-binding proteins involved in vesicular transport, in the biogenesis of the PBM. Three cDNAs encoding legume homologs of mammalian Rab1p and Rab7p were isolated from soybean (sRab1p, sRab7p) and Vigna aconitifolia (vRab7p). sRab1p was confirmed to be a functional counterpart of yeast Ypt1p (Rab1p) by complementation of a yeast ypt1-1 mutant. Both srab1 and vrab7 genes are induced during nodulation with the level of vrab7 mRNA being 12 times higher than that in root meristem and leaves. This induction directly correlates with membrane proliferation in nodules. Antisense constructs of srab1 and vrab7, under a nodule-specific promoter (leghemoglobin, Lbc3), were made in a binary vector and transgenic nodules were developed on soybean hairy roots obtained through Agrobacterium rhizogenes-mediated transformation. Both antisense srab1 and vrab7 nodules were smaller in size and showed lower nitrogenase activity than controls. The antisense srab1 nodules showed lack of expansion of infected cells, fewer bacteroids per cell and their frequent release into vacuoles. In contrast, antisense vrab7 expressing nodules showed accumulation of late endosomal structure and multivesicular bodies in the perinuclear region. These data suggest that both Rab1p and Rab7p are essential for the development of the PBM compartment in effective symbiosis.     

Higher order chromatin structures in maize and Arabidopsis.

We are investigating the nature of plant genome domain organization by using DNase I- and topoisomerase II-mediated cleavage to produce domains reflecting higher order chromatin structures. Limited digestion of nuclei with DNase I results in the conversion of the >800 kb genomic DNA to an accumulation of fragments that represents a collection of individual domains of the genome created by preferential cleavage at super-hypersensitive regions. The median size of these fragments is approximately 45 kb in maize and approximately 25 kb in Arabidopsis. Hybridization analyses with specific gene probes revealed that individual genes occupy discrete domains within the distribution created by DNase I. The maize alcohol dehydrogenase Adh1 gene occupies a domain of 90 kb, and the maize general regulatory factor GRF1 gene occupies a domain of 100 kb in length. Arabidopsis Adh was found within two distinct domains of 8.3 and 6.1 kb, whereas an Arabidopsis GRF gene occupies a single domain of 27 kb. The domains created by topoisomerase II-mediated cleavage are identical in size to those created by DNase I. These results imply that the genome is not packaged by means of a random gathering of the genome into domains of indiscriminate length but rather that the genome is gathered into specific domains and that a gene consistently occupies a discrete physical section of the genome. Our proposed model is that these large organizational domains represent the fundamental structural loop domains created by attachment of chromatin to the nuclear matrix at loop basements. These loop domains may be distinct from the domains created by the matrix attachment regions that typically flank smaller, often functionally distinct sections of the genome.     

Insights into endosomal maturation of human holo‐transferrin in the enteric parasite Entamoeba histolytica: essential roles of Rab7A and Rab5 in biogenesis of giant early endocytic vacuoles

The pathogenic amoeba Entamoeba histolytica is one of the causative agents of health hazards in tropical countries. It causes amoebic dysentery, colitis and liver abscesses in human. Iron is one of the essential nutritional resources for survival and chronic infection caused by the amoeba. The parasite has developed multiple ways to import, sequester and utilize iron from various iron‐binding proteins from its host. In spite of its central role in pathogenesis, the mechanism of iron uptake by the parasite is largely unknown. Here, we carried out a systematic study to understand the role of some of the amoebic homologues of mammalian endocytic Rab GTPases (Rab5 and Rab21, Rab7A and Rab7B) in intracellular transport of human holo‐transferrin by the parasite. Flow cytometry and quantitative microscopic image analysis revealed that Rab5 and Rab7A are required for the biogenesis of amoebic giant endocytic vacuoles (GEVs) and regulate the early phase of intracellular trafficking of transferrin. Rab7B is involved in the late phase, leading to the degradation of transferrin in the amoebic lysosome‐like compartments. Using time‐lapse fluorescence imaging in fixed trophozoites, we determined the kinetics of the vesicular transport of transferrin through Rab5‐, Rab7A‐ and Rab7B‐positive compartments. The involvement of Rab7A in the early phase of endocytosis by the parasite marks a significant divergence from its host in terms of spatiotemporal regulation by the Rab GTPases.