Optical Diffraction Tomography and Raman Confocal Microscopy for the Investigation of Vacuoles Associated with Cancer Senescent Engulfing Cells.

Wild-type p53 cancer therapy-induced senescent cells frequently engulf and degrade neighboring ones inside a massive vacuole in their cytoplasm. After clearance of the internalized cell, the vacuole persists, seemingly empty, for several hours. Despite large vacuoles being associated with cell death, this process is known to confer a survival advantage to cancer engulfing cells, leading to therapy resistance and tumor relapse. Previous attempts to resolve the vacuolar structure and visualize their content using dyes were unsatisfying for lack of known targets and ineffective dye penetration and/or retention. Here, we overcame this problem by applying optical diffraction tomography and Raman spectroscopy to MCF7 doxorubicin-induced engulfing cells. We demonstrated a real ability of cell tomography and Raman to phenotype complex microstructures, such as cell-in-cells and vacuoles, and detect chemical species in extremely low concentrations within live cells in a completely label-free fashion. We show that vacuoles had a density indistinguishable to the medium, but were not empty, instead contained diluted cell-derived macromolecules, and we could discern vacuoles from medium and cells using their Raman fingerprint. Our approach is useful for the noninvasive investigation of senescent engulfing (and other peculiar) cells in unperturbed conditions, crucial for a better understanding of complex biological processes.

Dictyostelium lacking the single atlastin homolog Sey1 shows aberrant ER architecture, proteolytic processes and expansion of the Legionella-containing vacuole.

Dictyostelium discoideum Sey1 is the single ortholog of mammalian atlastin 1-3 (ATL1-3), which are large homodimeric GTPases mediating homotypic fusion of endoplasmic reticulum (ER) tubules. In this study, we generated a D. discoideum mutant strain lacking the sey1 gene and found that amoebae deleted for sey1 are enlarged, but grow and develop similarly to the parental strain. The ∆sey1 mutant amoebae showed an altered ER architecture, and the tubular ER network was partially disrupted without any major consequences for other organelles or the architecture of the secretory and endocytic pathways. Macropinocytic and phagocytic functions were preserved; however, the mutant amoebae exhibited cumulative defects in lysosomal enzymes exocytosis, intracellular proteolysis, and cell motility, resulting in impaired growth on bacterial lawns. Moreover, ∆sey1 mutant cells showed a constitutive activation of the unfolded protein response pathway (UPR), but they still readily adapted to moderate levels of ER stress, while unable to cope with prolonged stress. In D. discoideum ∆sey1 the formation of the ER-associated compartment harbouring the bacterial pathogen Legionella pneumophila was also impaired. In the mutant amoebae, the ER was less efficiently recruited to the "Legionella-containing vacuole" (LCV), the expansion of the pathogen vacuole was inhibited at early stages of infection and intracellular bacterial growth was reduced. In summary, our study establishes a role of D. discoideum Sey1 in ER architecture, proteolysis, cell motility and intracellular replication of L. pneumophila.

Predicting the unexpected in stomatal gas exchange: not just an open-and-shut case.

Plant membrane transport, like transport across all eukaryotic membranes, is highly non-linear and leads to interactions with characteristics so complex that they defy intuitive understanding. The physiological behaviour of stomatal guard cells is a case in point in which, for example, mutations expected to influence stomatal closing have profound effects on stomatal opening and manipulating transport across the vacuolar membrane affects the plasma membrane. Quantitative mathematical modelling is an essential tool in these circumstances, both to integrate the knowledge of each transport process and to understand the consequences of their manipulation in vivo. Here, we outline the OnGuard modelling environment and its use as a guide to predicting the emergent properties arising from the interactions between non-linear transport processes. We summarise some of the recent insights arising from OnGuard, demonstrate its utility in interpreting stomatal behaviour, and suggest ways in which the OnGuard environment may facilitate 'reverse-engineering' of stomata to improve water use efficiency and carbon assimilation.

Apple ALMT9 Requires a Conserved C-Terminal Domain for Malate Transport Underlying Fruit Acidity.

Malate accumulation in the vacuole largely determines apple (Malus domestica) fruit acidity, and low fruit acidity is strongly associated with truncation of Ma1, an ortholog of ALUMINUM-ACTIVATED MALATE TRANSPORTER9 (ALMT9) in Arabidopsis (Arabidopsis thaliana). A mutation at base 1,455 in the open reading frame of Ma1 leads to a premature stop codon that truncates the protein by 84 amino acids at its C-terminal end. Here, we report that both the full-length protein, Ma1, and its naturally occurring truncated protein, ma1, localize to the tonoplast; when expressed in Xenopus laevis oocytes and Nicotiana benthamiana cells, Ma1 mediates a malate-dependent inward-rectifying current, whereas the ma1-mediated transmembrane current is much weaker, indicating that ma1 has significantly lower malate transport activity than Ma1. RNA interference suppression of Ma1 expression in 'McIntosh' apple leaves, 'Empire' apple fruit, and 'Orin' apple calli results in a significant decrease in malate level. Genotyping and phenotyping of 186 apple accessions from a diverse genetic background of 17 Malus species combined with the functional analyses described above indicate that Ma1 plays a key role in determining fruit acidity and that the truncation of Ma1 to ma1 is genetically responsible for low fruit acidity in apple. Furthermore, we identified a C-terminal domain conserved in all tonoplast-localized ALMTs essential for Ma1 function; protein truncations into this conserved domain significantly lower Ma1 transport activity. We conclude that the truncation of Ma1 to ma1 reduces its malate transport function by removing a conserved C-terminal domain, leading to low fruit acidity in apple.

A role for the Salmonella Type III Secretion System 1 in bacterial adaptation to the cytosol of epithelial cells.

Salmonella enterica serovar Typhimurium is a facultative intracellular pathogen that invades the intestinal epithelium. Following invasion of epithelial cells, Salmonella survives and replicates within two distinct intracellular niches. While all of the bacteria are initially taken up into a membrane bound vacuole, the Salmonella-containing vacuole or SCV, a significant proportion of them promptly escape into the cytosol. Cytosolic Salmonella replicates more rapidly compared to the vacuolar population, although the reasons for this are not well understood. SipA, a multi-function effector protein, has been shown to affect intracellular replication and is secreted by cytosolic Salmonella via the invasion-associated Type III Secretion System 1 (T3SS1). Here, we have used a multipronged microscopy approach to show that SipA does not affect bacterial replication rates per se, but rather mediates intra-cytosolic survival and/or initiation of replication following bacterial egress from the SCV. Altogether, our findings reveal an important role for SipA in the early survival of cytosolic Salmonella.

Discovery of gas vesicles in Streptomyces sp. CB03234-S and potential effects of gas vesicle gene overexpression on morphological and metabolic changes in streptomycetes.

Gas vesicles are usually found in aquatic microbes to help against adverse growth conditions and also present applied potentials in various biotechnological areas. Although gvp genes encoding gas vesicle proteins are occurred in many bacteria, the formation of gas vesicles in streptomycetes has not been reported yet. During the transcriptome analysis of Streptomyces sp. CB03234-S, a high producer of 10-membered enediynes tiancimycins (TNMs) generated in our previous studies, the notable activation of a gvp gene cluster (gvp3234) was detected, which may be related to the morphological change and yield increase in CB03234-S. Subsequent bioinformatic analysis revealed that gvp3234 possessed some unique features different from other gvp gene clusters, and its relative expression level was much higher than homologues in Streptomyces coelicolor. The overexpression of gvp3234 further improved the total titer of TNM-A and TNM-D from 12.3 ± 0.1 mg/L up to 21.4 ± 0.8 mg/L in CB03234-S, and TEM analysis clearly showed the emergence of gas vesicles, while the heterologous expression of gvp3234 in Streptomyces sp. CB09001 also resulted in the similar formation of fluffy mycelial pellets with long hyphae like in CB03234-S and enhanced the production of xanthone derivative. Our work reported a functionally expressed gvp gene cluster and the corresponding formation of gas vesicles in streptomycetes for the first time. The revealed potential effects of gas vesicles on morphological and metabolic changes shall provide an alternative strategy for strain improvement of industrial streptomycetes to obtain better performances in liquid cultivation and enhance production of secondary metabolites.

A structural analysis of the natural egress of Toxoplasma gondii.

Previous studies have analysed the process of Toxoplasma gondii egress with the aid of inducers, such as calcium ionophores. Although calcium transients have been successful in triggering T. gondii egress, the structural panorama of "natural" and artificial events should match. The present study approaches the natural egress of this parasite using super-resolution and electron microscopy and reveals lytic and non-lytic events of individual egress; this corroborates the use of calcium ionophore as a reliable tool to trigger parasite egress. Altogether, our data suggest that different signalling routes can converge to similar structural aspects in natural and induced egress.

Chlamydia trachomatis regulates growth and development in response to host cell fatty acid availability in the absence of lipid droplets.

Chlamydia trachomatis (Ct) is a Gram-negative obligate intracellular pathogen of humans that causes significant morbidity from sexually transmitted and ocular diseases globally. Ct acquires host fatty acids (FA) to meet the metabolic and growth requirements of the organism. Lipid droplets (LDs) are storehouses of FAs in host cells and have been proposed to be a source of FAs for the parasitophorous vacuole, termed inclusion, in which Ct replicates. Previously, cells devoid of LDs were shown to produce reduced infectious progeny at 24 hr postinfection (hpi). Here, although we also found reduced progeny at 24 hpi, there were significantly more progeny at 48 hpi in the absence of LDs compared to the control wild-type (WT) cells. These findings were confirmed using transmission electron microscopy where cells without LDs were shown to have significantly more metabolically active reticulate bodies at 24 hpi and significantly more infectious but metabolically inert elementary bodies at 48 hpi than WT cells. Furthermore, by measuring basal oxygen consumption rates (OCR) using extracellular flux analysis, Ct infected cells without LDs had higher OCRs at 24 hpi than cells with LDs, confirming ongoing metabolic activity in the absence of LDs. Although the FA oleic acid is a major source of phospholipids for Ct and stimulates LD synthesis, treatment with oleic acid, but not other FAs, enhanced growth and led to an increase in basal OCR in both LD depleted and WT cells, indicating that FA transport to the inclusion is not affected by the loss of LDs. Our results show that Ct regulates inclusion metabolic activity and growth in response to host FA availability in the absence of LDs.

Renitence vacuoles facilitate protection against phagolysosomal damage in activated macrophages.

As professional phagocytes, macrophages are susceptible to endolysosomal membrane damage inflicted by the pathogens and noxious particles they ingest. Whether macrophages have mechanisms for limiting such damage is not well understood. Previously, we reported a phenomenon, termed "inducible renitence," in which lipopolysaccharide (LPS) activation of macrophages protected their endolysosomes against damage initiated by the phagocytosis of silica beads. To gain mechanistic insight into the process, we analyzed the kinetics of renitence and morphological features of LPS-activated versus resting macrophages following silica bead-mediated injury. We discovered novel vacuolar structures that form in LPS-activated but not resting macrophages following silica bead phagocytosis. Because of their correlation with renitence and damage-resistant nature, we termed these structures "renitence vacuoles" (RVs). RVs formed coincident with silica bead uptake in a process associated with membrane ruffling and macropinocytosis. However, unlike normal macropinosomes (MPs), which shrink within 20 min of formation, RVs persisted around bead-containing phagosomes. RVs fused with lysosomes, whereas associated phagosomes typically did not. These findings are consistent with a model in which RVs, as persistent MPs, prevent fusion between damaged phagosomes and intact lysosomes and thereby preserve endolysosomal integrity.

Physiological and transcriptome response to cadmium in cosmos (Cosmos bipinnatus Cav.) seedlings.

To date, several species of Asteraceae have been considered as Cd-accumulators. However, little information on the Cd tolerance and associated mechanisms of Asteraceae species Cosmos bipinnatus, is known. Presently, several physiological indexes and transcriptome profiling under Cd stress were investigated. C. bipinnatus exhibited strong Cd tolerance and recommended as a Cd-accumulator, although the biomasses were reduced by Cd. Meanwhile, Cd stresses reduced Zn and Ca uptake, but increased Fe uptake. Subcellular distribution indicated that the vacuole sequestration in root mainly detoxified Cd under lower Cd stress. Whilst, cell wall binding and vacuole sequestration in root co-detoxified Cd under high Cd exposure. Meanwhile, 66,407 unigenes were assembled and 41,674 (62.75%) unigenes were annotated in at least one database. 2,658 DEGs including 1,292 up-regulated unigenes and 1,366 down-regulated unigenes were identified under 40 µmol/L Cd stress. Among of these DEGs, ZIPs, HMAs, NRAMPs and ABC transporters might participate in Cd uptake, translocation and accumulation. Many DEGs participating in several processes such as cell wall biosynthesis, GSH metabolism, TCA cycle and antioxidant system probably play critical roles in cell wall binding, vacuole sequestration and detoxification. These results provided a novel insight into the physiological and transcriptome response to Cd in C. bipinnatus seedlings.

Contribution of VMA5 to vacuolar function, stress response, ion homeostasis and autophagy in Candida albicans.

AIM: V-ATPase is a conservative multi-subunit enzyme in eukaryotes and modulates several cellular responses. This study aimed to illustrate the roles of Vma5 in vacuolar function, oxidative stress response, calcium homeostasis, autophagy and virulence. MATERIALS & METHODS: The vma5Δ/Δ mutant was obtained using PCR-mediated homologous recombination. The functions of Vma5 were investigated by a series of biochemical and systemic infection methods. RESULTS: Disruption of VMA5 led to growth inhibition, vacuolar dysfunction, disturbance of calcium homeostasis and inhibition of calcium-related oxidative stress response. Furthermore, its deletion caused defects in autophagy completion and hyphal development, and resulted in attenuated Candida albicans virulence. CONCLUSION: Our findings provide new insights into V-ATPase functions in C. albicans, and reveal a potential candidate for development of antifungal drugs.

Precision targeting by phosphoinositides: how PIs direct endomembrane trafficking in plants.

Each phosphoinositide (PI, also known as phosphatidylinositol phosphate, polyphosphoinositide, PtdInsP or PIP) species is partitioned in the endomembrane system and thereby contributes to the identity of membrane compartments. However, membranes are in constant flux within this system, which raises the questions of how the spatiotemporal pattern of phosphoinositides is established and maintained within the cell. Here, we review the general mechanisms by which phosphoinositides and membrane trafficking feedbacks on each other to regulate cellular patterning. We then use the specific examples of polarized trafficking, endosomal sorting and vacuolar biogenesis to illustrate these general concepts.

Buoyancy Limitation of Filamentous Cyanobacteria under Prolonged Pressure due to the Gas Vesicles Collapse.

Freshwater cyanobacterium Pseudanabaena galeata were cultured in chambers under artificially generated pressures, which correspond to the hydrostatic pressures at deep water. Variations occurred in gas vesicles volume, and buoyancy state of cells under those conditions were analyzed at different time intervals (5 min, 1 day, and 5 days). Variations in gas vesicles morphology of cells were observed by transmission electron microscopy images. Settling velocity (Vs) of cells which governs the buoyancy was observed with the aid of a modified optical microscope. Moreover, effects of the prolonged pressure on cell ballast composition (protein and polysaccharides) were examined. Elevated pressure conditions reduced the cell ballast and caused a complete disappearance of gas vesicles in Pseudanabaena galeata cells. Hence cyanobacteria cells were not able to float within the study period. Observations and findings of the study indicate the potential application of hydrostatic pressure, which naturally occurred in hypolimnion of lakes, to inhibit the re-suspension of cyanobacteria cells.

Cadmium-induced changes in vacuolar aspects of Arabidopsis thaliana.

We have examined the changes due to Cd treatment in the vacuolar form in root tip cortical cells in Arabidopsis thaliana employing a transformant with GFP fused to a tonoplast protein. A Cd-induced enhancement in complexity with general expansion of vacuolar system within 24 h was evident. The changes in the vacuolar form were dependent on the applied Cd concentrations. Concomitantly, as revealed through dithizone staining, Cd accumulated in the seedling roots exhibiting abundance of Cd-dithizone complexes in root tip, root hairs and vasculature. To get insight into the involvement of SNARE protein-mediated vesicle fusion in Cd detoxification, the magnitude of Cd toxicity in a couple of knock out mutants of the vacuolar Qa-SNARE protein VAM3/SYP22 was compared with that in the wild type. The Cd toxicity appeared to be comparable in the mutants and the wild type. In order to analyze the Cd effects at cellular level, we treated the Arabidopsis suspension-cultured cells with Cd. Cd, however, did not induce a change in the vacuolar form in suspension-cultured cells although Cd measured with ICP-MS was obviously taken up into the cell. The V-ATPase activity in the microsomal fractions from vacuoles isolated from A. thaliana suspension cultured cells remained unaffected by Cd. Changes in the levels of certain metabolites of Cd-treated cells were also not so distinct except for those of glutathione. The significance of findings is discussed.

Naloxonazine, an Amastigote-Specific Compound, Affects Leishmania Parasites through Modulation of Host-Encoded Functions.

Host-directed therapies (HDTs) constitute promising alternatives to traditional therapy that directly targets the pathogen but is often hampered by pathogen resistance. HDT could represent a new treatment strategy for leishmaniasis, a neglected tropical disease caused by the obligate intracellular parasite Leishmania. This protozoan develops exclusively within phagocytic cells, where infection relies on a complex molecular interplay potentially exploitable for drug targets. We previously identified naloxonazine, a compound specifically active against intracellular but not axenic Leishmania donovani. We evaluated here whether this compound could present a host cell-dependent mechanism of action. Microarray profiling of THP-1 macrophages treated with naloxonazine showed upregulation of vATPases, which was further linked to an increased volume of intracellular acidic vacuoles. Treatment of Leishmania-infected macrophages with the vATPase inhibitor concanamycin A abolished naloxonazine effects, functionally demonstrating that naloxonazine affects Leishmania amastigotes indirectly, through host cell vacuolar remodeling. These results validate amastigote-specific screening approaches as a powerful way to identify alternative host-encoded targets. Although the therapeutic value of naloxonazine itself is unproven, our results further demonstrate the importance of intracellular acidic compartments for host defense against Leishmania, highlighting the possibility of targeting this host cell compartment for anti-leishmanial therapy.

Type II Secretion Is Necessary for Optimal Association of the Legionella-Containing Vacuole with Macrophage Rab1B but Enhances Intracellular Replication Mainly by Rab1B-Independent Mechanisms.

Previously, we documented that type II secretion (T2S) promotes intracellular infection of macrophages by Legionella pneumophila In the present study, we identified infection events that are modulated by T2S by comparing the behaviors of wild-type and T2S mutant bacteria in murine bone marrow-derived macrophages and human U937 cells. Although the two strains behaved similarly for entry into the host cells and evasion of lysosomal fusion, the mutant was impaired in the ability to initiate replication between 4 and 8 h postentry and to grow to large numbers in the Legionella-containing vacuole (LCV), as evident at 12 h. At 4 h postinoculation, mutant LCVs had a significantly reduced association with Rab1B, a host GTPase that facilitates the tethering of endoplasmic reticulum (ER)-derived vesicles to LCVs. The mutant did not lose expression or translocation of six type IV secretion effectors (e.g., SidM) that are well known for mediating Rab1B association with the LCV, indicating that T2S promotes the interaction between the LCV and Rab1B via a novel mechanism. Interestingly, the mutant's growth defect was exacerbated in macrophages that had been depleted of Rab1B by short hairpin RNA (shRNA) treatment, indicating that T2S also potentiates events beyond Rab1B association. In support of this, a sidM lspF double mutant had an intracellular growth defect that was more dramatic than that of the lspF mutant (and a sidM mutant) and showed a growth difference of as much as a 400-fold compared to the wild type. Together, these data reveal a new role for T2S in intracellular infection that involves both Rab1B-dependent and Rab1B-independent processes.

The morphological analysis of autophagy in primary skeletal muscle cells infected with Toxoplasma gondii.

Toxoplasma gondii is an obligate intracellular protozoan parasite, the causative agent of toxoplasmosis, one of the most widespread zoonoses in the world. During the host immune response, tissue cysts are formed, allowing the maintenance of the parasite within the host cell. Autophagy, a degradation process of cellular components, is critical for cellular homeostasis. Recently, it has been proposed that autophagy participates in host-pathogen interactions. Autophagic inducers (rapamycin or glucose plus serum deprivation) inhibited infection and parasite proliferation in a clinically relevant model of primary skeletal muscle cells (SkMC). The ultrastructural analysis showed in SkMC submitted to autophagic stimuli the presence of structures suggestive of autophagosomes close to the parasitophorous vacuole containing degraded parasites. Fluorescence microscopy results pointed out the increase in LC3 puncta in these cells after incubation with autophagic inducers. In the present study, SkMC autophagy controlled the proliferation of tachyzoites inside the cell, data reinforced by ultrastructural evidences and increased LC3 expression.

The position of lysosomes within the cell determines their luminal pH.

We examined the luminal pH of individual lysosomes using quantitative ratiometric fluorescence microscopy and report an unappreciated heterogeneity: peripheral lysosomes are less acidic than juxtanuclear ones despite their comparable buffering capacity. An increased passive (leak) permeability to protons, together with reduced vacuolar H(+)-adenosine triphosphatase (V-ATPase) activity, accounts for the reduced acidifying ability of peripheral lysosomes. The altered composition of peripheral lysosomes is due, at least in part, to more limited access to material exported by the biosynthetic pathway. The balance between Rab7 and Arl8b determines the subcellular localization of lysosomes; more peripheral lysosomes have reduced Rab7 density. This in turn results in decreased recruitment of Rab-interacting lysosomal protein (RILP), an effector that regulates the recruitment and stability of the V1G1 component of the lysosomal V-ATPase. Deliberate margination of lysosomes is associated with reduced acidification and impaired proteolytic activity. The heterogeneity in lysosomal pH may be an indication of a broader functional versatility.

Yersinia pestis Requires Host Rab1b for Survival in Macrophages.

Yersinia pestis is a facultative intracellular pathogen that causes the disease known as plague. During infection of macrophages Y. pestis actively evades the normal phagosomal maturation pathway to establish a replicative niche within the cell. However, the mechanisms used by Y. pestis to subvert killing by the macrophage are unknown. Host Rab GTPases are central mediators of vesicular trafficking and are commonly targeted by bacterial pathogens to alter phagosome maturation and killing by macrophages. Here we demonstrate for the first time that host Rab1b is required for Y. pestis to effectively evade killing by macrophages. We also show that Rab1b is specifically recruited to the Yersinia containing vacuole (YCV) and that Y. pestis is unable to subvert YCV acidification when Rab1b expression is knocked down in macrophages. Furthermore, Rab1b knockdown also altered the frequency of association between the YCV with the lysosomal marker Lamp1, suggesting that Rab1b recruitment to the YCV directly inhibits phagosome maturation. Finally, we show that Rab1b knockdown also impacts the pH of the Legionella pneumophila containing vacuole, another pathogen that recruits Rab1b to its vacuole. Together these data identify a novel role for Rab1b in the subversion of phagosome maturation by intracellular pathogens and suggest that recruitment of Rab1b to the pathogen containing vacuole may be a conserved mechanism to control vacuole pH.