BRYOPSIS MAXIMA (CHLOROPHYTA) GLUTAMATE DEHYDROGENASE: MULTIPLE GENES AND ISOZYMES

Subcellular localization of glutamate dehydrogenase (GDH) was investigated in the green alga Bryopsis maxima. Both intact and pure chloroplasts and mitochondria were isolated by two methods: successive centrifugation and continuous Percoll density gradient centrifugation. The NADP-dependent GDH activities of the chloroplastic, mitochondrial, and cytosolic portions were estimated as 64.3, 9.8, and 25.9%, respectively, and NAD-dependent GDH activity was observed only in the chloroplasts. Three organelle-specific isozymes—chloroplastic NADP-GDH1, cytosolic/mitochondrial NADP-GDH2, and cytosolic/mitochondrial NADP-GDH3—were purified. The molecular masses of these isozymes were estimated to be the same (280 kDa). K_m values of NADP-GDH1, NADP-GDH2, and NADP-GDH3 for NADPH in the amination reaction were 30, 110, and 34 μM, respectively, and those for NADH were 185, 1490, and 974 μM, respectively, showing different cofactor affinities. Several NADP-GDHs and one NAD-GDH were induced in the chloroplasts during incubation of the collected thalli in either continuous light or darkness in aerated seawater for 0 to 5 days, whereas the cytosolic and mitochondrial NADP-GDHs decreased to an almost undetectable level in 5 days. Two distinct DNA fragments (BmF-1 and BmF-2) encoding B. maxima Okamura GDH were identified and sequenced. They showed 90% homology in their deduced amino acid sequences, whereas synonymous nucleotide substitution was observed in the third position of 52% of the codons. Genomic Southern analysis suggested that the two genes are located at two different loci on the B. maxima chromosome. Thus, B. maxima GDH has been confirmed to be multiple in terms of both protein and gene. The localization of other nitrogen-assimilating enzymes was also determined. Glutamine synthetase was located in the chloroplasts and the cytosol, glutamate synthase was located in the chloroplasts, and nitrate reductase was located in the cytosol.     

The Phosphoinositide‐Gated Lysosomal Ca2+ Channel,TRPML1, Is Required for Phagosome Maturation

Macrophages internalize and sequester pathogens into a phagosome. Phagosomes then sequentially fuse with endosomes and lysosomes, converting into degradative phagolysosomes. Phagosome maturation is a complex process that requires regulators of the endosomal pathway including the phosphoinositide lipids. Phosphatidylinositol‐3‐phosphate and phosphatidylinositol‐3,5‐bisphosphate (PtdIns(3,5)P₂), which respectively control early endosomes and late endolysosomes, are both required for phagosome maturation. Inhibition of PIKfyve, which synthesizes PtdIns(3,5)P₂, blocked phagosome–lysosome fusion and abated the degradative capacity of phagosomes. However, it is not known how PIKfyve and PtdIns(3,5)P₂ participate in phagosome maturation. TRPML1 is a PtdIns(3,5)P₂‐gated lysosomal Ca²⁺ channel. Because Ca²⁺ triggers membrane fusion, we postulated that TRPML1 helps mediate phagosome–lysosome fusion. Using Fcγ receptor‐mediated phagocytosis as a model, we describe our research showing that silencing of TRPML1 hindered phagosome acquisition of lysosomal markers and reduced the bactericidal properties of phagosomes. Specifically, phagosomes isolated from TRPML1‐silenced cells were decorated with lysosomes that docked but did not fuse. We could rescue phagosome maturation in TRPML1‐silenced and PIKfyve‐inhibited cells by forcible Ca²⁺ release with ionomycin. We also provide evidence that cytosolic Ca²⁺ concentration increases upon phagocytosis in a manner dependent on TRPML1 and PIKfyve. Overall, we propose a model where PIKfyve and PtdIns(3,5)P₂ activate TRPML1 to induce phagosome–lysosome fusion.      

Biogenesis and secretion of micronemes in Toxoplasma gondii

One of the hallmarks of the parasitic phylum of Apicomplexa is the presence of highly specialised, apical secretory organelles, called the micronemes and rhoptries that play critical roles in ensuring survival and dissemination. Upon exocytosis, the micronemes release adhesin complexes, perforins, and proteases that are crucially implicated in egress from infected cells, gliding motility, migration across biological barriers, and host cell invasion. Recent studies on Toxoplasma gondii and Plasmodium species have shed more light on the signalling events and the machinery that trigger microneme secretion. Intracellular cyclic nucleotides, calcium level, and phosphatidic acid act as key mediators of microneme exocytosis, and several downstream effectors have been identified. Here, we review the key steps of microneme biogenesis and exocytosis, summarising the still fractal knowledge at the molecular level regarding the fusion event with the parasite plasma membrane.     

The BLOS1-interacting protein KXD1 is involved in the biogenesis of lysosome-related organelles

Biogenesis of lysosome‐related organelles (LROs) complex‐1 (BLOC‐1) is an eight‐subunit complex involved in lysosomal trafficking. Interacting proteins of these subunits expand the understanding of its biological functions. With the implementation of the naïve Bayesian analysis, we found that a human uncharacterized 20 kDa coiled‐coil KxDL protein, KXD1, is a BLOS1‐interacting protein. In vitro binding assays confirmed the interaction between BLOS1 and KXD1. The mouse KXD1 homolog was widely expressed and absent in Kxd1 knockout (KO) mice. BLOS1 was apparently reduced in Kxd1‐KO mice. Mild defects in the melanosomes of the retinal pigment epithelia and in the platelet dense granules of the Kxd1‐KO mouse were observed, mimicking a mouse model of mild Hermansky–Pudlak syndrome that affects the biogenesis of LROs.     

Effect of Exogenous Ammonium on Photosynthetic O2 Evolution and Ultrastructural Organization of Cells in Soybean Callus

The effect of exogenous ammonium on O₂ evolution and structural organization of cells in mixotrophic callus of soybean (Glycine max L.). Chlorophyll content increased in the presence of ammonium in nutrient medium. Under these conditions, the rate of photosynthetic O₂ evolution per unit biomass increased; however, the photosynthetic rate decreased when calculated per unit of chlorophyll content. The presence of ammonium in nutrient medium favored the formation of the protein-synthesizing machinery in cells, which manifested itself in an increase of the number of ribosomes, and directly enhanced protein synthesis, as follows from the expansion of chloroplast membrane systems and greater electron density of mitochondrial matrix and cytoplasm of mixotrophic cells. Possible sites of ammonium participation in the formation of the functional structure in plant cells are discussed.     

Spatiotemporal Control of Intracellular Phase Transitions Using Light-Activated optoDroplets

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A link between mitochondrial gene expression and life stage morphologies in Trypanosoma cruzi

The protozoan Trypanosoma cruzi has a complicated dual-host life cycle, and starvation can trigger transition from the replicating insect stage to the mammalian-infectious nonreplicating insect stage (epimastigote to trypomastigote differentiation). Abundance of some mature RNAs derived from its mitochondrial genome increase during culture starvation of T. cruzi for unknown reasons. Here, we examine T. cruzi mitochondrial gene expression in the mammalian intracellular replicating life stage (amastigote), and uncover implications of starvation-induced changes in gene expression. Mitochondrial RNA levels in general were found to be lowest in actively replicating amastigotes. We discovered that mitochondrial respiration decreases during starvation in insect stage cells, despite the previously observed increases in mitochondrial mRNAs encoding electron transport chain (ETC) components. Surprisingly, T. cruzi epimastigotes in replete medium grow at normal rates when we genetically compromised their ability to perform insertion/deletion editing and thereby generate mature forms of some mitochondrial mRNAs. However, these cells, when starved, were impeded in the epimastigote to trypomastigote transition. Further, they experience a short-flagella phenotype that may also be linked to differentiation. We hypothesize a scenario where levels of mature RNA species or editing in the single T. cruzi mitochondrion are linked to differentiation by a yet-unknown signaling mechanism.