Differential Intracellular Localization Research Articles

Design a dual-response two-photon fluorescent probe for simultaneous imaging of mitochondrial viscosity and peroxynitrite in a thrombosis model

BackgroundVenous thromboembolism is a sudden cardiovascular disease that can lead to death, and its pathologic development is closely related to vascular viscosity and inflammation. However, direct evidence from in vivo is really scarce. The key limitation is that the combined probes cannot detect multiple markers simultaneously, which may lead to unreliable results. Therefore, to develop a single probe that can simultaneously monitor the variations of viscosity in the vascular microenvironment as well as inflammation level during venous thrombosis. ResultsA dual-responsive two-photon fluorescent probe, Cou-ONOO, was designed and synthesized. Cou-ONOO provides a visualization tool for monitoring the viscosity of the vascular as well as the inflammatory marker ONOO‾ during thromboembolism via dual-channel simultaneous imaging. As a single probe that can recognize dual targets, Cou-ONOO effectively avoids the problems from unreliable results caused by complex synthesis and differences in intracellular localization, diffusion, and metabolism of different dyes as using combinatorial probes. Using Cou-ONOO, simultaneous imaging the variations of viscosity and ONOO‾at the cellular and tissue levels was successfully performed. In addition, Cou-ONOO also successfully visualized and tracked the viscosity of the vascular microenvironment and ONOO‾ during venous embolism in mice. SignificanceExperimental results show that both viscosity and inflammation are abnormally overexpressed in the microenvironment at the thrombus site during venous thrombosis. An intuitive visualization tool to elucidate the variations of viscosity as well as inflammation level in the vascular microenvironment during thrombosis was provided, which will facilitate a better clinical understanding of the pathological process of thrombosis.

Intracellular Salmonella Paratyphi A is motile and differs in the expression of flagella-chemotaxis, SPI-1 and carbon utilization pathways in comparison to intracellular S. Typhimurium.

Although Salmonella Typhimurium (STM) and Salmonella Paratyphi A (SPA) belong to the same phylogenetic species, share large portions of their genome and express many common virulence factors, they differ vastly in their host specificity, the immune response they elicit, and the clinical manifestations they cause. In this work, we compared their intracellular transcriptomic architecture and cellular phenotypes during human epithelial cell infection. While transcription induction of many metal transport systems, purines, biotin, PhoPQ and SPI-2 regulons was similar in both intracellular SPA and STM, we identified 234 differentially expressed genes that showed distinct expression patterns in intracellular SPA vs. STM. Surprisingly, clear expression differences were found in SPI-1, motility and chemotaxis, and carbon (mainly citrate, galactonate and ethanolamine) utilization pathways, indicating that these pathways are regulated differently during their intracellular phase. Concurring, on the cellular level, we show that while the majority of STM are non-motile and reside within Salmonella-Containing Vacuoles (SCV), a significant proportion of intracellular SPA cells are motile and compartmentalized in the cytosol. Moreover, we found that the elevated expression of SPI-1 and motility genes by intracellular SPA results in increased invasiveness of SPA, following exit from host cells. These findings demonstrate unexpected flagellum-dependent intracellular motility of a typhoidal Salmonella serovar and intriguing differences in intracellular localization between typhoidal and non-typhoidal salmonellae. We propose that these differences facilitate new cycles of host cell infection by SPA and may contribute to the ability of SPA to disseminate beyond the intestinal lamina propria of the human host during enteric fever.

PFKFB2 regulates glycolysis and proliferation in pancreatic cancer cells.

Tumor cells increase glucose metabolism through glycolysis and pentose phosphate pathways to meet the bioenergetic and biosynthetic demands of rapid cell proliferation. The family of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases (PFKFB1-4) are key regulators of glucose metabolism via their synthesis of fructose-2,6-bisphosphate (F2,6BP), a potent activator of glycolysis. Previous studies have reported the co-expression of PFKFB isozymes, as well as the mRNA splice variants of particular PFKFB isozymes, suggesting non-redundant functions. Majority of the evidence demonstrating a requirement for PFKFB activity in increased glycolysis and oncogenic properties in tumor cells comes from studies on PFKFB3 and PFKFB4 isozymes. In this study, we show that the PFKFB2 isozyme is expressed in tumor cell lines of various origin, overexpressed and localizes to the nucleus in pancreatic adenocarcinoma, relative to normal pancreatic tissue. We then demonstrate the differential intracellular localization of two PFKFB2 mRNA splice variants and that, when ectopically expressed, cytoplasmically localized mRNA splice variant causes a greater increase in F2,6BP which coincides with an increased glucose uptake, as compared with the mRNA splice variant localizing to the nucleus. We then show that PFKFB2 expression is required for steady-state F2,6BP levels, glycolytic activity, and proliferation of pancreatic adenocarcinoma cells. In conclusion, this study may provide a rationale for detailed investigation of PFKFB2's requirement for the glycolytic and oncogenic phenotype of pancreatic adenocarcinoma cells.

Contractile Behavior of Mouse Aorta Depends on SERCA2 Isoform Distribution: Effects of Replacing SERCA2a by SERCA2b.

The Sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA) actively pumps Ca2+ into the sarco/endoplasmic reticulum, thereby regulating intracellular Ca2+ concentrations and associated physiological processes. Different SERCA isoforms have been described (SERCA1, 2, and 3) with SERCA2 playing a pivotal role in Ca2+ homeostasis in cardiovascular tissues. In the heart, SERCA2a is the dominant isoform and has been proposed as therapeutic target in patients with heart failure. In the vasculature, both SERCA2a and SERCA2b are expressed with SERCA2b being the predominant isoform. The physiological role of SERCA2a in the vasculature, however, remains incompletely understood. In the present study, we used gene-modified mice in which the alternative splicing of the SERCA2-encoding gene (Atp2a2), underlying the expression of SERCA2a, is prevented and SERCA2a is replaced by SERCA2b. The resulting SERCA2b/b mice provide a unique opportunity to investigate the specific contribution of SERCA2a versus SERCA2b to vascular physiology. Aortic segments of SERCA2b/b (SERCA2a-deficient) and SERCA2a/b (control) mice were mounted in organ baths to evaluate vascular reactivity. SERCA2b/b aortic rings displayed higher contractions induced by phenylephrine (1 μM). Surprisingly, the initial inositol-3-phosphate mediated phasic contraction showed a faster decay of force in SERCA2b/b mice, while the subsequent tonic contraction was larger in SERCA2b/b segments. Moreover, in the presence of the calcium channel blocker diltiazem (35 μM) SERCA2b/b aortic rings showed higher contractions compared to SERCA2a/b, suggesting that SERCA2a (deficiency) modulates the activity of non-selective cation channels. Additionally, in endothelial cell (EC)-denuded aortic segments, the SERCA-inhibitor cyclopiazonic acid (CPA) caused markedly larger contractions in SERCA2b/b mice, while the increases of cytosolic Ca2+ were similar in both strains. Hence, aortas of SERCA2b/b mice appear to have a stronger coupling of intracellular Ca2+ to contraction, which may be in agreement with the reported difference in intracellular localization of SERCA2a versus SERCA2b. Finally, EC-mediated relaxation by acetylcholine and ATP was assessed. Concentration-response-curves for ATP showed a higher sensitivity of aortic segments of SERCA2b/b mice, while no difference in potency between strains were observed for acetylcholine. In summary, despite the relative low expression of SERCA2a in the murine aorta, our results point toward a distinct role in vascular physiology.

Insights into the limitations of transient expression systems for the functional study of p53 acetylation site and oncogenic mutants

Tumor suppressor protein p53 protects cells against malignant transformation mostly through transcriptional activation. Lysine acetylation is required to mediate activation of p53. The protein displays eight lysine residues and their evolutionary conservation argues for an essential role. The aim of this study was to investigate the significance of individual acetylation sites in mediating p53 functions. Differences in intracellular localization, protein expression levels, and transcriptional activity were investigated by overexpressing acetylation-deficient p53 variants in the colon carcinoma-derived p53 knock-out cell line HCT 116 p53(−/−). We found that not all lysine residues are equally capable of promoting p53’s functions. Individual amino acid mutations or combinations thereof led to altered p53 expression levels, intracellular distribution, or transcriptional transactivation capacity, as compared to the wild-type protein. However, we observed that the choice of protein tag and expression vector could significantly alter obtained results on certain aspects of p53 function.

Differential intracellular localization and dynamic nucleocytoplasmic shuttling of homeodomain-interacting protein kinase family members

The three canonical members of the family of homeodomain-interacting protein (HIP) kinases fulfill overlapping and distinct roles in cellular stress response pathways. Here we systematically compared all three endogenous HIPKs for their intracellular distribution and mutual interactions. The endogenous HIPKs are contained in high molecular weight complexes of ~700 kDa but do not directly interact physically. Under basal conditions, HIPK1 was mostly cytoplasmic, while HIPK3 was found in the nucleus and HIPK2 occurred in both compartments. Inhibition of nuclear export by leptomycin B resulted in the nuclear accumulation of mainly HIPK1 and HIPK2, indicating constitutive dynamic nucleocytoplasmic shuttling. The carcinogenic chemical stressor sodium arsenite caused the induction of HIPK2-dependent cell death and also resulted in a rapid and complete nuclear translocation of HIPK2, showing that the intracellular distribution of this kinase can undergo dynamic regulation.

Differential intracellular localization of Hsp70 in the gill and heart tissue of fresh water prawn Macrobrachium malcolmsonii during thermal stress.

Exposure of organisms to heat stress induces the expression of evolutionarily conserved proteins called the stress proteins or heat shock proteins (HSPs). At the cellular level, HSPs by acting as molecular chaperone prevents the heat induced aggregation of denatured proteins and play a significant role in adaptation to temperature. Among different HSP family members, Hsp70 is the highly conserved and ubiquitously expressed protein. The present study is carried out to detect changes in the localization of Hsp70/Hsc70 in gill and heart tissues of control and heat shocked juveniles of Macrobrachium malcolmsonii that could be correlated with the functional significance of these two isoforms. Two groups of prawn acclimated at 30°C were exposed to reported optimum Hsp70 induction temperatures of 36°C and 38°C for heart and gill, respectively, for a duration of 48h. These tissues were processed by immunocytochemical methods to detect intra-cellular localization of Hsp70/Hsc70. Western blotting analysis was performed to determine the cytoplasmic or nuclear localization of Hsp70/Hsc70 and band intensity was detected in total lysate, cytosolic and nuclear extracts of gill and heart tissue. The present investigation clearly shows that there are alterations in the intracellular localization of Hsp70/Hsc70 in the cells of the gill and heart tissues of M. malcolmsonii following heat stress. The western blotting results corroborate the results obtained by immunohistochemical localisation. The differential intracellular localization of Hsp70/Hsc70 appears to indicate the functional roles of this stress protein during exposure to thermal stress.

Comparison of ATP sulfurylase 2 from selenium hyperaccumulator Stanleya pinnata and non-accumulator Stanleya elata reveals differential intracellular localization and enzyme activity levels

BackgroundThe plant Stanleya pinnata hyperaccumulates Se up to 0.5% of its dry weight in organic forms, whereas the closely related Stanleya elata does not hyperaccumulate Se. ATP sulfurylase (ATPS) can catalyze the formation of adenosine 5′-phosphoselenate (APSe) from ATP and selenate. We investigated the S. pinnata ATPS2 isoform (SpATPS2) to assess its possible role in Se hyperaccumulation. MethodsATPS expression and activity was compared in the two Stanleya species. The ATPS2 protein sequences were modeled. Sub-cellular locations were analyzed using GFP fusions. Enzyme activity of purified recombinant SpATPS2 was measured. ResultsATPS2 transcript levels were six-fold higher in roots of S. pinnata relative to S. elata. Overall root ATPS enzyme activity was two-fold elevated in S. pinnata. Cloning and sequencing of SpATPS2 and S. elata ATPS2 (SeATPS2) showed the predicted SeATPS2 to be canonical, while SpATPS2, although very similar in its core structure, has unique features, including an interrupted plastid targeting signal due to a stop codon in the 5′ region of the coding sequence. Indeed GFP fusions revealed that SpATPS2 had exclusive cytosolic localization, while SeATPS2 showed dual localization in plastids and cytosol. SpATPS2 activity was inhibited by both sulfate and selenate, indicating that the enzyme acts on both substrates. ConclusionsThe ATPS2 from S. pinnata differs from non-accumulator ATPS2 in its elevated expression and sub-cellular localization. It likely acts on both selente and sulfate substrates. General significanceThese observations shed new light on the role of ATPS2 in the evolution of Se hyperaccumulation in plants. This article is part of a Special Issue entitled Selenium research in biochemistry and biophysics - 200 year anniversary issue, edited by Dr. Elias Arnér and Dr. Regina Brigelius-Flohe.

TRAP1 regulates autophagy in lung cancer cells.

Expression of TRAP1, a member of the HSP90 chaperone family, has been implicated in tumour protective effects, based on its differential mitochondrial localization and function. This work was designed to provide new insights into the pathways involved in TRAP1-provided cytoprotection on NSCLC. For this, TRAP1-depleted A549 human NSCLC cells and MRC-5 normal lung fibroblasts were produced using a siRNA approach and main cellular quality control mechanisms were investigated. TRAP1-depleted A549 cells displayed decreased cell viability likely due to impaired mitochondrial function including decreased ATP/AMP ratio, oxygen consumption and membrane potential, as well as increased apoptotic indicators. Furthermore, the negative impact of TRAP1 depletion on mitochondrial function was not observed in normal MRC-5 lung cells, which might be due to the differential intracellular localization of the chaperone in tumour versus normal cells. Additionally, A549 TRAP1-depleted cells showed increased autophagic flux. Functionally, autophagy inhibition resulted in decreased cell viability in both TRAP1-expressing and TRAP1-depleted tumour cells with minor effects on MRC-5 cells. Conversely, autophagy stimulation decreased cell viability of both A549 and MRC-5 TRAP1-expressing cells while in A549 TRAP1-depleted cells, increased autophagy augmented viability. Our results show that even though TRAP1 depletion affects both normal MRC-5 and tumour A549 cell proliferation, inhibition of autophagy per se led to a decrease in tumour cell mass, while having a reduced effect on the normal cell line. The strategy of targeting TRAP1 in NSCLC shows future potential therapeutic applications.

Retrograde nuclear transport from the cytoplasm is required for tRNATyr maturation in T. brucei

ABSTRACTRetrograde transport of tRNAs from the cytoplasm to the nucleus was first described in Saccharomyces cerevisiae and most recently in mammalian systems. Although the function of retrograde transport is not completely clear, it plays a role in the cellular response to changes in nutrient availability. Under low nutrient conditions tRNAs are sent from the cytoplasm to nucleus and presumably remain in storage there until nutrient levels improve. However, in S. cerevisiae tRNA retrograde transport is constitutive and occurs even when nutrient levels are adequate. Constitutive transport is important, at least, for the proper maturation of tRNAPhe, which undergoes cytoplasmic splicing, but requires the action of a nuclear modification enzyme that only acts on a spliced tRNA. A lingering question in retrograde tRNA transport is whether it is relegated to S. cerevisiae and multicellular eukaryotes or alternatively, is a pathway with deeper evolutionary roots. In the early branching eukaryote Trypanosoma brucei, tRNA splicing, like in yeast, occurs in the cytoplasm. In the present report, we have used a combination of cell fractionation and molecular approaches that show the presence of significant amounts of spliced tRNATyr in the nucleus of T. brucei. Notably, the modification enzyme tRNA-guanine transglycosylase (TGT) localizes to the nucleus and, as shown here, is not able to add queuosine (Q) to an intron-containing tRNA. We suggest that retrograde transport is partly the result of the differential intracellular localization of the splicing machinery (cytoplasmic) and a modification enzyme, TGT (nuclear). These findings expand the evolutionary distribution of retrograde transport mechanisms to include early diverging eukaryotes, while highlighting its importance for queuosine biosynthesis.

Photodynamic performance of zinc phthalocyanine in HeLa cells: A comparison between DPCC liposomes and BSA as delivery systems

Comparable intracellular concentrations (≈30pmol/106 cells) of bovine serum albumin-ZnPc (BSA) adduct outperformed dipalmitoyl-phosphatidyl-choline (DPPC) liposomes containing ZnPc at photodynamic-killing of human cervical cancer cells (HeLa) after only 15min of irradiation using red light (λ>620nm, 30W/cm2). This result could not be simply explained in terms of dye aggregation within the carrier, since in the liposomes the dye was considerably less aggregated than in bovine serum albumin, formulation that was capable to induce cell apoptosis upon red light exposure. Thus, using specific organelle staining, our cumulative data points towards intrinsic differences in intra-cellular localization depending on the cargo vehicle used, being ZnPc:BSA preferentially located in the near vicinity of the nucleus and in the Golgi structures, while the liposomal formulation ZnPc:DPPC was preferentially located in cellular membrane and cytoplasm. In addition to those differences, using real-time advanced fluorescence lifetime imaging of HeLa cells loaded with the photosensitizer contained in the different vehicles, we have found that only for the ZnPc:BSA formulation, there was no significant changes in the fluorescence lifetime of the photosensitizer inside the cells. This contrasts with the in situ≈two-fold reduction of the fluorescence lifetime measured for the liposomal ZnPc formulation. Those observations point towards the superiority of the protein to preserve dye aggregation, and its photochemical activity, post-cell uptake, demonstrating the pivotal role of the delivery vehicle at determining the ultimate fate of a photosensitizer.

Effects of a novel porphyrin-based photosensitizer on sensitive and multidrug-resistant human gastric cancer cell lines

Photodynamic therapy (PDT) has been considered to be a possible candidate approach in combating multidrug resistance (MDR) phenomenon during the treatment of cancer. To investigate the photocytotoxicity of a novel porphyrin-based photosensitizer, meso-5-[ρ-DTPA-aminophenyl]-10, 15, 20-triphenyl-porhyrin (DTP) (Fig. 1A), on MDR cells, the intracellular DTP uptake, phototoxicity and subcellular DTP localization were studied by using a human gastric cancer MGC803 cell line and its paclitaxel selected subline MGC803/PA expressing MDR phenotype. No significant difference was observed in intracellular DTP accumulation between sensitive and resistant cell lines after exposure to 1.56μM concentration for 6h. DTP-PDT induced significant photocytotoxicity on both MGC803 and MGC803/PA cell lines and the photokilling was greater in MGC803 cell line in comparison to MGC803/PA. The fluence that caused 50% cell death was 4.42 and 6.29J/cm2 in MGC803 and MGC803/PA cell lines, respectively. The presence of Pgp inhibitors verapamil and cyclosporin A could not modify the intracellular DTP level in MGC803/PA cell line and the phototoxic effects. DTP was localized at lysosomes of MGC803 cell line but at lysosomes and mitochondria of MGC803/PA. Our results indicated that DTP-mediated PDT could eradicate gastric cancer cells whether or not they express MDR although the efficacy is slightly reduced in the MDR cells. The photokilling in MDR cells could not be altered by MDR inhibitor verapamil. The slightly different photocytotoxicity between sensitive and resistant cell lines could not explained by classical Pgp MDR and might be attributed to the differential intracellular DTP localization sites.

Using concatenated subunits to investigate the functional consequences of heterotetrameric inositol 1,4,5-trisphosphate receptors.

Inositol 1,4,5-trisphosphate receptors (IP3Rs) are a family of ubiquitous, ER localized, tetrameric Ca2+ release channels. There are three subtypes of the IP3Rs (R1, R2, R3), encoded by three distinct genes, that share ∼60-70% sequence identity. The diversity of Ca2+ signals generated by IP3Rs is thought to be largely the result of differential tissue expression, intracellular localization and subtype-specific regulation of the three subtypes by various cellular factors, most significantly InsP3, Ca2+ and ATP. However, largely unexplored is the notion of additional signal diversity arising from the assembly of both homo and heterotetrameric InsP3Rs. In the present article, we review the biochemical and functional evidence supporting the existence of homo and heterotetrameric populations of InsP3Rs. In addition, we consider a strategy that utilizes genetically concatenated InsP3Rs to study the functional characteristics of heterotetramers with unequivocally defined composition. This approach reveals that the overall properties of IP3R are not necessarily simply a blend of the constituent monomers but that specific subtypes appear to dominate the overall characteristics of the tetramer. It is envisioned that the ability to generate tetramers with defined wild typeand mutant subunits will be useful in probing fundamental questions relating to IP3R structure and function.

Pulmonary vascular smooth muscle cells remodeling and dysfunction in cystic fibrosis

Background Pulmonary hypertension (PH) is a major feature of advanced cystic fibrosis (CF) and may lead to right ventricular dysfunction and cor pulmonale. Although PH is usually moderate in CF, its severity varies greatly across patients and its presence may negatively affect the prognosis. Extensive pulmonary vessel remodeling with muscularisation of the small arterioles are major pathological features of PH in CF. Although these structural changes are considered the major cause of the increase in pulmonary vascular resistance, their pathogenesis remains uncertain. The aim of this study was to determine whether the CFTR channel is present and affects functions of human pulmonary arteries smooth muscle cells (SMCs). Methods Lung and pulmonary arteries from CF and control patients were obtained from Hospital Foch, surgical department. Primary vascular smooth muscle cells (VSMCs) were isolated from the pulmonary artery (PA) of patients. Cytosolic Ca 2+ was measured using Fura-2 and dual-wavelength microfluorimetry. Protein expression was analyzed by immunofluorescence, immunoblot and RT-PCR analysis. Results Histological analysis revealed increased muscularisation of small pulmonary arteries from CF patients comparing to control patients (75 vs. 45%, n = 10, P 2+ ATPase 2a (SERCA2a). CFTR was detected in both control and CF PA-SMCs but exhibited differential intracellular localization. Particularly, in control PA-SMCs CFTR was localized within plasma membrane (PM) whereas in CF PA-SMCs CFTR was localized within the sarcoplasmic reticulum (SR), in line with previous observations in human airway epithelial cells (Front Pharmacol 2011;2:67). Accordingly, major alterations of calcium handling were detected in primary CF PA-SMCs. Particularly, total SR calcium content was increased in CF VSMCs, whereas PM Ca 2+ influx (store operated Ca 2+ entry [SOCE]) was decreased. Furthermore, primary CF PA-SMCs were poorly sensitive to Thrombin (inductor of proliferation) but highly sensitive to serotonin (inductor of contraction). In opposite to the brief increase in cytosolic Ca 2+ observed in control cells, in CF PA-SMCs serotonin-induced a sustained long-lasting oscillatory Ca 2+ transient, suggesting establishment of “membrane oscillator” during whole period of occupancy of serotonin receptor. Conclusions Relocalization of ΔCFTR to RS in PA-SMCs results in major alterations of Ca 2+ handling favoring hypercontractility of PA-SMCs in CF patients.

Gemcitabine Induces Poly (ADP-Ribose) Polymerase-1 (PARP-1) Degradation through Autophagy in Pancreatic Cancer

Poly (ADP-ribose) polymerase-1 (PARP-1) and autophagy play increasingly important roles in DNA damage repair and cell death. Gemcitabine (GEM) remains the first-line chemotherapeutic drug for pancreatic cancer (PC). However, little is known about the relationship between PARP-1 expression and autophagy in response to GEM. Here we demonstrate that GEM induces DNA-damage response and degradation of mono-ADP ribosylated PARP-1 through the autophagy pathway in PC cells, which is rescued by inhibiting autophagy. Hypoxia and serum starvation inhibit autophagic activity due to abrogated GEM-induced mono-ADP-ribosylated PARP-1 degradation. Activation of extracellular regulated protein kinases (ERK) induced by serum starvation shows differences in intracellular localization as well as modulation of autophagy and PARP-1 degradation in GEM-sensitive KLM1 and -resistant KLM1-R cells. Our study has revealed a novel role of autophagy in PARP-1 degradation in response to GEM, and the different impacts of MEK/ERK signaling pathway on autophagy between GEM-sensitive and -resistant PC cells.

Photochemical internalization (PCI)-mediated enhancement of bleomycin cytotoxicity by liposomal mTHPC formulations in human head and neck cancer cells.

Photodynamic therapy (PDT) with photosensitizers that locate in endocytic vesicles of cancer cells can be exploited to promote the intracellular release of anticancer drugs entrapped in endolysosomal vesicles. This new approach is commonly referred to as Photochemical Internalization (PCI). Here we report on the PCI effects of three different formulations (Foscan, Foslip, and Fospeg) of the clinically approved photosensitizer, meta-tetrahydroxyphenyl chlorin (mTHPC) on the anticancer drug bleomycin (BLM) in the head and neck cancer cell lines. Uptake and localization of Foscan, Foslip, and Fospeg in head and neck cancer cells were evaluated by fluorescence spectrophotometry and fluorescence microscopy. Photodynamic efficacy of Foscan, Foslip, and Fospeg were compared with cell proliferation assay. Moreover, PCI effects of Foscan, Foslip, and Fospeg on BLM were compared using protocols in which PDT was applied after or before BLM treatment. Cellular uptake of Foscan, Foslip, and Fospeg increased in a dose-dependent fashion with consistent higher uptakes of Foslip and Fospeg than Foscan. Fluorescence microscopy showed diffuse intracellular localization pattern for Foscan, Foslip, and Fospeg similar to that of a specific ER probe. However, the subcellular localization pattern of the Rhodamine-labelled same type of pegylated liposomes as Fospeg was similar to that of a specific endolysosomal probe, suggesting that Fospeg uptake appeared to initially proceed via endolysosomal trafficking. Foscan, Foslip, and Fospeg showed no apparent PCI-mediated cytotoxicity when PDT was performed after BLM treatment. However, significantly increased cytotoxicity of BLM (P < 0.05) was observed for both Foslip and Fospeg when PDT was performed before BLM treatment. The observed differences of PCI-mediated cytotoxicity between these two timing protocols appears to be related to the differential intracellular trafficking and localization of Foscan, Foslip, and Fospeg. Liposomal formulations of mTHPC may be candidates for developing mTHPC-based PCI protocols to enhance the efficacy of anticancer drugs entrapped in endolysosomal vesicles.

Highly Multiplexed Phenotypic Imaging for Cell Proliferation Studies

The application of multiplexed imaging technologies in phenotypic drug discovery (PDD) enables profiling of complex cellular perturbations in response to drug treatment. High-content analysis (HCA) is among the most pursued approaches in PDD, with a proven capability to identify compounds with a given cellular mechanism of action (MOA), as well as to unveil unexpected drug cellular activities. The ability of fluorescent image-based cytometric techniques to dissect the phenotypic heterogeneity of cell populations depends on the degree of multiplexing achievable. At present, most high-content assays employ up to four cellular markers separately detected in distinct fluorescence channels. We explored the possibility to increase HCA multiplexing through analysis of multiple proliferation markers in the same fluorescence channel by taking advantage of the different timing of antigen appearance during the cell cycle, or differential intracellular localization. Simultaneous analysis of DAPI staining and five immunofluorescence markers (BrdU incorporation, active caspase-3, phospho-histone H3, phospho-S6, and Ki-67) resulted in the first six-marker high-content assay readily applicable to compound MOA studies. This approach allows detection of rare cell subpopulations, unveiling a high degree of phenotypic heterogeneity in exponentially growing cell cultures and variability in the individual cell response to antiproliferative drugs.

Characterization of two pathogenic mutations in cystathionine beta-synthase: Different intracellular locations for wild-type and mutant proteins

Cystathionine β-synthase (CBS) is a pyridoxal 5′-phosphate (PLP)-dependent enzyme that catalyzes the condensation of homocysteine with serine to generate cystathionine. Homocystinuria is an autosomal recessive disorder commonly caused by a deficiency of CBS activity. Here, we characterized a novel CBS mutation (c.260C>A (p.T87N)) and a previously reported variant (c.700G>A (p.D234N)) found in Venezuelan homocystinuric patients, one nonresponsive and one responsive to vitamin B6. Both mutant proteins were expressed in vitro in prokaryotic and eukaryotic cells, finding lower soluble expression in HEK-293 cells (19% T87N and 23% D234N) compared to wild-type CBS. Residual activities obtained for the mutant proteins were 3.5% T87N and 43% D234N. Gel exclusion chromatography demonstrated a tendency of the T87N mutant to aggregate while the distribution of the D234N mutant was similar to wild-type enzyme. Using immunofluorescence microscopy, an unexpected difference in intracellular localization was observed between the wild-type and mutant proteins. While the T87N mutant exhibited a punctate appearance, the wild-type protein was homogeneously distributed inside the cell. Interestingly, the D234N protein showed both distributions. This study demonstrates that the pathogenic CBS mutations generate unstable proteins that are unable (T87N) or partially unable (D234N) to assemble into a functional enzyme, implying that these mutations might be responsible for the homocystinuria phenotype.

Metabolic Changes in Synechocystis PCC6803 upon Nitrogen-Starvation: Excess NADPH Sustains Polyhydroxybutyrate Accumulation

Polyhydroxybutyrate (PHB) is a common carbon storage polymer among heterotrophic bacteria. It is also accumulated in some photoautotrophic cyanobacteria; however, the knowledge of how PHB accumulation is regulated in this group is limited. PHB synthesis in Synechocystis sp. PCC 6803 is initiated once macronutrients like phosphorus or nitrogen are limiting. We have previously reported a mutation in the gene sll0783 that impairs PHB accumulation in this cyanobacterium upon nitrogen starvation. In this study we present data which explain the observed phenotype. We investigated differences in intracellular localization of PHB synthase, metabolism, and the NADPH pool between wild type and mutant. Localization of PHB synthase was not impaired in the sll0783 mutant; however, metabolome analysis revealed a difference in sorbitol levels, indicating a more oxidizing intracellular environment than in the wild type. We confirmed this by directly measuring the NADPH/NADP ratio and by altering the intracellular redox state of wild type and sll0783 mutant. We were able to physiologically complement the mutant phenotype of diminished PHB synthase activity by making the intracellular environment more reducing. Our data illustrate that the NADPH pool is an important factor for regulation of PHB biosynthesis and metabolism, which is also of interest for potential biotechnological applications.

Differential Localization of the Two T. brucei Poly(A) Binding Proteins to the Nucleus and RNP Granules Suggests Binding to Distinct mRNA Pools

The number of paralogs of proteins involved in translation initiation is larger in trypanosomes than in yeasts or many metazoan and includes two poly(A) binding proteins, PABP1 and PABP2, and four eIF4E variants. In many cases, the paralogs are individually essential and are thus unlikely to have redundant functions although, as yet, distinct functions of different isoforms have not been determined. Here, trypanosome PABP1 and PABP2 have been further characterised. PABP1 and PABP2 diverged subsequent to the differentiation of the Kinetoplastae lineage, supporting the existence of specific aspects of translation initiation regulation. PABP1 and PABP2 exhibit major differences in intracellular localization and distribution on polysome fractionation under various conditions that interfere with mRNA metabolism. Most striking are differences in localization to the four known types of inducible RNP granules. Moreover, only PABP2 but not PABP1 can accumulate in the nucleus. Taken together, these observations indicate that PABP1 and PABP2 likely associate with distinct populations of mRNAs. The differences in localization to inducible RNP granules also apply to paralogs of components of the eIF4F complex: eIF4E1 showed similar localization pattern to PABP2, whereas the localisation of eIF4E4 and eIF4G3 resembled that of PABP1. The grouping of translation initiation as either colocalizing with PABP1 or with PABP2 can be used to complement interaction studies to further define the translation initiation complexes in kinetoplastids.