Extent Of Colocalization Research Articles

Rab27a Contributes to Cathepsin S Secretion in Lacrimal Gland Acinar Cells.

Altered lacrimal gland (LG) secretion is a feature of autoimmune dacryoadenitis in Sjögren’s syndrome (SS). Cathepsin S (CTSS) is increased in tears of SS patients, which may contribute to disease. Rab3D and Rab27a/b isoforms are effectors of exocytosis in LG, but Rab27a is poorly studied. To investigate whether Rab27a mediates CTSS secretion, we utilized quantitative confocal fluorescence microscopy of LG from SS-model male NOD and control male BALB/c mice, showing that Rab27a-enriched vesicles containing CTSS were increased in NOD mouse LG. Live-cell imaging of cultured lacrimal gland acinar cells (LGAC) transduced with adenovirus encoding wild-type (WT) mCFP-Rab27a revealed carbachol-stimulated fusion and depletion of mCFP-Rab27a-enriched vesicles. LGAC transduced with dominant-negative (DN) mCFP-Rab27a exhibited significantly reduced carbachol-stimulated CTSS secretion by 0.5-fold and β-hexosaminidase by 0.3-fold, relative to stimulated LGAC transduced with WT mCFP-Rab27a. Colocalization of Rab27a and endolysosomal markers (Rab7, Lamp2) with the apical membrane was increased in both stimulated BALB/c and NOD mouse LG, but the extent of colocalization was much greater in NOD mouse LG. Following stimulation, Rab27a colocalization with endolysosomal membranes was decreased. In conclusion, Rab27a participates in CTSS secretion in LGAC though the major regulated pathway, and through a novel endolysosomal pathway that is increased in SS.

Exploring new potential role of DDB2 by host cell reactivation assay in human tumorigenic cells

BackgroundThe Host Cell Reactivation assay (HCR) allows studying the DNA repair capability in different types of human cells. This assay was carried out to assess the ability in removing UV-lesions from DNA, thus verifying NER efficiency. Previously we have shown that DDB2, a protein involved in the Global Genome Repair, interacts directly with PCNA and, in human cells, the loss of this interaction affects DNA repair machinery. In addition, a mutant form unable to interact with PCNA (DDB2PCNA-), has shown a reduced ability to interact with a UV-damaged DNA plasmid in vitro.MethodsIn this work, we have investigated whether DDB2 protein may influence the repair of a UV-damaged DNA plasmid into the cellular environment by applying the HCR method. To this end, human kidney 293 stable clones, expressing DDB2Wt or DDB2PCNA-, were co-transfected with pmRFP-N2 and UV-irradiated pEGFP-reported plasmids. Moreover, the co-localization between DDB2 proteins and different NER factors recruited at DNA damaged sites was analysed by immunofluorescence and confocal microscopy.ResultsThe results have shown that DDB2Wt recognize and repair the UV-induced lesions in plasmidic DNA transfected in the cells, whereas a delay in these processes were observed in the presence of DDB2PCNA-, as also confirmed by the different extent of co-localization of DDB2Wt and some NER proteins (such as XPG), vs the DDB2 mutant form.ConclusionThe HCR confirms itself as a very helpful approach to assess in the cellular context the effect of expressing mutant vs Wt NER proteins on the DNA damage response. Loss of interaction of DDB2 and PCNA affects negatively DNA repair efficiency.

Distribution and Co-localization of endosome markers in cells

Clathrin mediated endocytosis is one pathway for internalization of extracellular nano materials into cells [1, 2]. In this pathway, proteins attached to receptors and the internalized materials are encapsulated in clathrin coated membrane vesicles that subsequently fuse with or transform into intracellular compartments (early and late endosomes) as their contents are being directed to the lysosomes for degradation. The following proteins are commonly used to mark the pathway at various stages: Rab5 (early endosome), Rab7 (late endosome), and LAMP-1 (lysosome). In this work, we studied the distribution and co-localization of these marker proteins in two cell lines (C2C12 and A549) to determine whether these markers are unique for specific endosome types or whether they can co-exist with other markers. We estimate the densities and sizes of the endosomes containing the three markers, as well as the number of marker antibodies attached to each endosome. We determine that the markers are not unique to one endosome type but that the extent of co-localization is different for the two cell types. In fact, we find endosomes that contain all three markers simultaneously. Our results suggest that the use of these proteins as specific markers for specific endosome types should be reevaluated. This was the first successful use of triple image cross correlation spectroscopy to qualitatively and quantitatively study the extent of interaction among three different species in cells and also the first experimental study of three-way interactions of clathrin mediated endocytic markers.

Electron microscopy combined with spatial analysis: quantitative mapping of the nano-assemblies of plasma membrane-associating proteins and lipids

The plasma membrane (PM) is a complex environment consisting of > 700 species of lipids and many different types of membrane-associating proteins. These lipids and membrane proteins are distributed heterogeneously into nanometer-sized domains, called nanoclusters. The lateral spatial segregation in the PM gives rise to different curvature and lipid composition, which determines the efficiency of effector binding and signal transmission. Here, we describe an electron microscopy (EM)-spatial mapping technique to quantify the extent of nanoclusters formation in the PM. The nano-assemblies in the PM are quantified via expressing the GFP-tagged proteins or lipid-binding domains in the cells, which are then immunolabeled with the gold nanoparticles pre-coupled to the anti-GFP antibody. The gold nanoparticles are visualized via the transmission EM at high magnification. The statistical analysis of the Ripley’s K-function calculates the spatial distribution of the gold nanoparticles. Important spatial parameters, such as the extent of nanoclustering, the clustered fraction, the number of proteins per cluster, the optimal size of a nanocluster, and the number of proteins localized to the PM, can be calculated. Further detailed aggregation pattern, such as the populations of monomers, dimers, trimers, and higher ordered oligomers, can also be extracted from the spatial analysis. The EM-bivariate analysis quantifies the extent of co-localization between two different components in the PM and provides key information on the protein–protein and the protein–lipid interactions over a long-distance scale from 8 to 240 nm.

Significant Synteny and Colocalization of Ecologically Relevant Quantitative Trait Loci Within and Across Species of Salmonid Fishes.

The organization of functional regions within genomes has important implications for evolutionary potential. Considerable research effort has gone toward identifying the genomic basis of phenotypic traits of interest through quantitative trait loci (QTL) analyses. Less research has assessed the arrangement of QTL in the genome within and across species. To investigate the distribution, extent of colocalization, and the synteny of QTL for ecologically relevant traits, we used a comparative genomic mapping approach within and across a range of salmonid species. We compiled 943 QTL from all available species [lake whitefish (Coregonus clupeaformis), coho salmon (Oncorhynchus kisutch), rainbow trout (O. mykiss), Chinook salmon (O. tshawytscha), Atlantic salmon (Salmo salar), and Arctic charr (Salvelinus alpinus)]. We developed a novel analytical framework for mapping and testing the distribution of these QTL. We found no correlation between QTL density and gene density at the chromosome level but did at the fine-scale. Two chromosomes were significantly enriched for QTL. We found multiple synteny blocks for morphological, life history, and physiological traits across species, but only morphology and physiology had significantly more than expected. Two or three pairs of traits were significantly colocalized in three species (lake whitefish, coho salmon, and rainbow trout). Colocalization and fine-scale synteny suggest genetic linkage between traits within species and a conserved genetic basis across species. However, this pattern was weak overall, with colocalization and synteny being relatively rare. These findings advance our understanding of the role of genomic organization in the renowned ecological and phenotypic variability of salmonid fishes.

Involvement of autophagy in connexin 40 reduction in the late phase of traumatic brain injury in rats.

Brain trauma can activate an attenuation of connexin gap junction that is implicated in neuronal injury, but the underlying cellular mechanisms remain incompletely understood. Here, we aimed to study whether autophagy, a stress-response process for recycling of intracellular proteins and organelles, is involved in the reduction of connexin 40 (Cx40) during the late phase of traumatic brain injury (TBI). In a rat model of TBI induced by controlled cortical impact (CCI), we found that Cx40 protein in the brain started to decline at post-surgery day 2 and the decrease continued for up to day 6. Such a relatively late response of Cx40 following TBI was found to be coincident with the substantial induction of neuron degeneration and autophagy, elevated autophagic vacuole numbers, and induced LC3-II and p62 levels. At day 4 post-injury, the extent of co-localization between LC3 and Cx40 was greatly enhanced, and the reduction of Cx40 was rescued by the administration of an autophagy inhibitor chloroquine. Thus, autophagy stimulated in the injured brains may act as a suppressing mechanism to decrease gap junction protein Cx40 in the late phase of TBI.

Multiparameter Microscopy Analysis of the Follicular Lymphoma Microenvironment and Normal Germinal Centers: In Vivo evidence That Follicular Helper T Cells Form Synapses with Neoplastic B Cells and Are Associated with Proliferation and Expression of Activation Induced Cytidine Deaminase

The tumor microenvironment plays a central role in the pathogenesis of follicular lymphoma (FL) and has been shown to influence prognosis. The biological basis for this and the contribution of individual cell types however, remain unclear. In this study we compared the cellular content and structure of neoplastic follicles in FL with their normal counterparts in reactive lymph nodes (LNs). We specifically focused on follicular helper T cells (TFH) which, in normal germinal centers (GCs), form immune synapses with antigen responsive B cells triggering B cell proliferation and expression of activation induced cytidine deaminase (AID), the enzyme required for somatic hypermutation and class switch recombination. This is of relevance because off-target AID activity is thought to play a role in generating the mutations that characterize progressive FL.A limitation of previous studies of the FL microenvironment is the use of either single parameter immunohistochemistry which fails to accurately define the complex populations of cells involved, or flow cytometry on disaggregated cells which results in the loss of architectural information. In this study we used multiparameter confocal immunofluorescent (IF) microscopy to investigate in vivo the phenotype, distribution and interaction of CD4+ T cells in FL and to determine to what extent these are similar to normal GCs.Confocal IF microscopy was performed on multiple sections of formalin fixed paraffin embedded LN biopsy specimens from 20 patients with untreated FL, comparison was made with reactive LNs (n=5) and chronic lymphocytic leukemia (CLL) LN biopsies (n=5). Each section was stained with a combination of up to 4 simultaneously applied primary antibodies against CD3, CD4, CD20, PD1, ICOS, BCL6, AID, and Ki67, and fluorescently labelled secondary antibodies. Microscopy was performed using a Nikon TiE fluorescent microscope equipped with A1R Si Confocal imaging system; images were analyzed using NIS software.Results show that CD4+ T cells in FL are mainly located in the inter-follicular regions but they were also identified within the follicles in all cases. Combination staining with anti-CD4, PD1, and ICOS revealed that 23% (95%CI 18-27) of CD4+ T cells within follicles co-express PD1 and ICOS consistent with a TFH phenotype which is significantly higher than in inter-follicular areas where only 5% (95% CI 3-7) of CD4+ cells had this phenotype (p<0.001). PD1+ ICOS+ T cells were positive for the transcription factor BCL6, further confirming the TFH phenotype. There was no significant difference in the proportion of CD4+ cells that were TFH in FL follicles and reactive LN GCs. In CLL cases, 54% of CD4+ cells expressed PD1 but only 9% co-expressed PD1 and ICOS, significantly lower than either FL follicles or GCs (p<0.001). Automated analysis of 3D z-stacks demonstrated a very close spatial relationship between proliferating tumor cells and TFH in FL with a mean of 42% (95%CI 35-48) Ki67+ tumor cells in direct contact with TFH cells. No association was seen between the extent of co-localization and histological grade. A similar pattern of co-localization of TFH cells next to proliferating B cells was also identified in the light zones of reactive GCs. Of note, we also identified features of synapse formation between TFH cells and proliferating tumor cells; TFH cells demonstrated projections that encompass the tumor cell with distortion of the T cell nucleus and increased CD4 and PD1 expression at sites of cell contact (Figure 1). These findings were similarly present in reactive GCs. Finally, AID was expressed in proliferating GC B cells and in proliferating tumor cells in FL. AID expressing cells were found to be in close contact with PD1+ T cells in both GCs and FL.Our findings show many parallels between the follicles of FL and normal GCs. In particular the proportion of CD4+ T cells with a TFH phenotype and their localization in direct contact with proliferating AID+ B cells were very similar. Of note, features of immune synapses were observed in both GCs and FL. Taken together, the data suggest that TFH cells have an important role in the pathogenesis of FL just as they are vital in the normal GC reaction. Interruption of this interaction is a potential therapeutic target. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Electron microscopy methods for studying plasma membranes.

Electron microscopy allows direct visualization of the underlying organization of cell surface components on a nano-scale. Immuno-gold labelling of isolated plasma membranes generates point patterns that enable mapping of protein and lipid distributions. 2D spatial statistics reveals the extent to which these distributions are clustered or dispersed and allows the extent of co-localization between different cell surface components to be precisely determined. This approach has been successfully applied to the study of signalling network organization and the consequences of physiological changes in modulating cell surface function.

Arresting a Torsin ATPase Reshapes the Endoplasmic Reticulum

Torsins are membrane-tethered AAA+ ATPases residing in the nuclear envelope (NE) and endoplasmic reticulum (ER). Here, we show that the induction of a conditional, dominant-negative TorsinB variant provokes a profound reorganization of the endomembrane system into foci containing double membrane structures that are derived from the ER. These double-membrane sinusoidal structures are formed by compressing the ER lumen to a constant width of 15 nm, and are highly enriched in the ATPase activator LULL1. Further, we define an important role for a highly conserved aromatic motif at the C terminus of Torsins. Mutations in this motif perturb LULL1 binding, reduce ATPase activity, and profoundly limit the induction of sinusoidal structures.

AP-3 and Rabip4’ Coordinately Regulate Spatial Distribution of Lysosomes

The RUN and FYVE domain proteins rabip4 and rabip4’ are encoded by RUFY1 and differ in a 108 amino acid N-terminal extension in rabip4’. Their identical C terminus binds rab5 and rab4, but the function of rabip4s is incompletely understood. We here found that silencing RUFY1 gene products promoted outgrowth of plasma membrane protrusions, and polarized distribution and clustering of lysosomes at their tips. An interactor screen for proteins that function together with rabip4’ yielded the adaptor protein complex AP-3, of which the hinge region in the β3 subunit bound directly to the FYVE domain of rabip4’. Rabip4’ colocalized with AP-3 on a tubular subdomain of early endosomes and the extent of colocalization was increased by a dominant negative rab4 mutant. Knock-down of AP-3 had an ever more dramatic effect and caused accumulation of lysosomes in protrusions at the plasma membrane. The most peripheral lysosomes were localized beyond microtubules, within the cortical actin network. Our results uncover a novel function for AP-3 and rabip4’ in regulating lysosome positioning through an interorganellar pathway.

Regional distribution and molecular interaction of caveolins in bladder smooth muscle

What's known on the subject? and What does the study add? Caveolae are specialised regions of bladder smooth muscle (BSM) cell membranes where specific signalling pathways are regulated. Caveolin proteins are involved in caveolar biogenesis and function as signal transduction regulators. Expression of caveolin-1, -2, and -3 has been previously identified in the bladder; however, the distribution and relative expression of these proteins have not been defined. The present data show significant differences in the spatial distribution of caveolin proteins throughout the bladder wall. Region dependent variations in the co-localisation of caveolin subtypes in detrusor SM were also detected. These findings support the premise that the unique spatial pattern of caveolin proteins associated with BSM cells may enable regionally distinct functional responses to common stimuli. • To determine the regional expression profile of caveolin isoforms (integral membrane proteins abundant in caveolae), the spatial relationships among caveolin proteins within specific smooth muscle (SM) regions and the extent of their molecular interactions in bladder SM (BSM). • Regional differences in the expression of caveolin family members were determined by quantitative reverse transcriptase-polymerase chain reaction and Western blot of RNA and protein extracted from the base, body and dome of rat bladders. • To evaluate the distribution of caveolin-1 (Cav-1), Cav-2 and Cav-3 within the bladder, longitudinal tissue sections from the base to dome were processed for confocal microscopy and quantified for intensity of immunoreactivity (IR) and extent of co-localisation. • Interactions among Cav-1, Cav-2 and Cav-3 were determined by co-immunoprecipitation. • Differential expression of Cav-1 and Cav-3 was detected among bladder regions, with lowest expression in the bladder base relative to the dome. • Cav-1 was highly expressed in all regions, although an increase in IR from submucosa to serosa was detected in each region. • The distribution of Cav-2 IR generally paralleled Cav-1, but progressively decreased from submucosa to serosa in each region. • Cav-3 expression predominated in the medial region of BSM increasing progressively from base to dome, but was poorly expressed in the outer SM layer particularly in the dome. • Cav-1 co-precipitated extensively with both Cav-2 and Cav-3. Co-precipitation between Cav-3 and Cav-2 was also detected. • The isoform-specific spatial distribution and distinct molecular interactions among caveolins in BSM may contribute to the contractile heterogeneity of BSM cells and facilitate differential modulation of responses to local stimuli. • As BSM caveolae regulate key signalling processes involved in contraction, altered expression of caveolin proteins may generate a regional imbalance in contraction/relaxation responses, thus leading to bladder dysfunction.

Microdomains of muscarinic acetylcholine and InsP3 receptors create InsP3 junctions and sites of Ca2+ wave initiation in smooth muscle

SummaryIncreases in cytosolic Ca2+ concentration ([Ca2+]c) mediated by inositol (1,4,5)-trisphosphate [Ins(1,4,5)P3, hereafter InsP3] regulate activities that include division, contraction and cell death. InsP3-evoked Ca2+ release often begins at a single site, then regeneratively propagates through the cell as a Ca2+ wave. The Ca2+ wave consistently begins at the same site on successive activations. Here, we address the mechanisms that determine the Ca2+ wave initiation site in intestinal smooth muscle cells. Neither an increased sensitivity of InsP3 receptors (InsP3R) to InsP3 nor regional clustering of muscarinic receptors (mAChR3) or InsP3R1 explained the selection of an initiation site. However, examination of the overlap of mAChR3 and InsP3R1 localisation, by centre of mass analysis, revealed that there was a small percentage (∼10%) of sites that showed colocalisation. Indeed, the extent of colocalisation was greatest at the Ca2+ wave initiation site. The initiation site might arise from a selective delivery of InsP3 from mAChR3 activity to particular InsP3Rs to generate faster local [Ca2+]c increases at sites of colocalisation. In support of this hypothesis, a localised subthreshold ‘priming’ InsP3 concentration applied rapidly, but at regions distant from the initiation site, shifted the wave to the site of the priming. Conversely, when the Ca2+ rise at the initiation site was rapidly and selectively attenuated, the Ca2+ wave again shifted and initiated at a new site. These results indicate that Ca2+ waves initiate where there is a structural and functional coupling of mAChR3 and InsP3R1, which generates junctions in which InsP3 acts as a highly localised signal by being rapidly and selectively delivered to InsP3R1.

Ubiquitination of α5β1 Integrin Controls Fibroblast Migration through Lysosomal Degradation of Fibronectin-Integrin Complexes

Cell migration requires endocytosis and recycling of integrins, but it is not known whether degradation of these membrane proteins is involved. Here we demonstrate that in migrating cells, a fraction of the endocytosed fibronectin receptor, alpha 5 beta 1 integrin, is sorted into multivesicular endosomes together with fibronectin and degraded in lysosomes. This sorting requires fibronectin-induced ubiquitination of the alpha 5 subunit, and the activity of the endosomal sorting complex required for transport (ESCRT) machinery, which interacts with alpha 5 beta 1 integrin. Importantly, we demonstrate that both alpha 5 ubiquitination and ESCRT functions are required for proper migration of fibroblasts. We propose that ligand-mediated degradation of alpha 5 beta 1 integrin via the ESCRT pathway is required in order to prevent endosomal accumulation of ligand-bound integrins that might otherwise form nonproductive adhesion sites. Fibronectin and alpha 5 beta 1 integrin therefore are trafficked to lysosomes in a similar way to growth factors and their receptors.

Changes in Oestrogen Receptor α Expression in the Nucleus of the Solitary Tract of the Rat Over the Oestrous Cycle and Following Ovariectomy

Oestrogen is capable of modulating autonomic outflow and baroreflex function via actions on groups of neurones in the brainstem. We investigated the presence of oestrogen receptor (ER) alpha in a part of the nucleus of the solitary tract (NTS) associated with central cardiovascular control, aiming to determine whether ERalpha mRNA and protein expression is correlated with levels of circulating oestrogen during the oestrous cycle. Polymerase chain reaction (PCR) detected ERalpha mRNA in the NTS at each stage of the oestrous cycle, from ovariectomised, sham-operated and male rats. Real-time PCR showed variations in ERalpha mRNA expression during the oestrous cycle, with the highest levels seen in oestrus, and lowest levels in metoestrus (P < 0.05 versus oestrus) and proestrus (P < 0.05 versus oestrus). Expression in males was lower than in dioestrus and oestrus females (P < 0.05). After ovariectomy, ERalpha mRNA levels were decreased compared to sham-operated animals (P < 0.01). Confocal fluorescence immunohistochemistry with stereological analysis showed that numbers of ERalpha immunoreactive cell nuclei per mm(3) of tissue in the caudal NTS were significantly greater in proestrus than in other groups of rats (P < 0.05). There were also differences among the groups in the extent of colocalisation of ERalpha in neurones immunoreactive for tyrosine hydroxylase and nitric oxide synthase. These results imply a complex pattern of region-specific oestrogen signalling in the NTS and suggest that ERalpha expression in this important autonomic nucleus may be related to circulating oestrogen levels. This may have consequences for the regulation of autonomic tone and baroreflex sensitivity when oestrogen levels decline, for example following menopause.

Histochemical and Fluorescent Analyses of Mitochondrial Integrity in Chick Auditory Neurons following Deafferentation

Neurons rely exclusively on mitochondrial oxidative phosphorylation to meet cellular energy demands, and disruption of mitochondrial function often precipitates neuronal cell death. Auditory neurons in the chick brain stem (n. magnocellularis [NM]) receive glutamatergic innervation exclusively from ipsilateral eighth nerve afferents. Cochlea removal permanently disrupts afferent support and ultimately triggers apoptotic cell death in 30-50% of ipsilateral, deafferented neurons. Here, we evaluated whether disruption of mitochondrial function occurs during deafferentation-induced neuronal cell death. To determine whether mitochondrial dysfunction occurs preferentially within dying NM neurons. An experimental study. All birds underwent unilateral cochlea removal. Normally innervated neurons contralateral to surgery served as within-animal controls. Hatchling broiler chickens between 8 and 12 days of age served as subjects. A total of 62 birds were included in the study. Cochlea removal was performed to deafferent ipsilateral NM neurons and trigger neuronal cell death. Following unilateral cochlea removal, birds were sacrificed 12, 24, 48, or 168 hours later, and brain tissue was harvested. Brainstems were sectioned through NM and evaluated histochemically for oxidative enzyme reaction product accumulation or reacted for Mitotracker Red, an indicator of mitochondrial membrane potential (m) and cytoplasmic TdT-mediated dUTP Nick-End Labeling (TUNEL), an indicator of cell death. Histochemical staining intensities for three mitochondrial enzymes, succinate dehydrogenase (SDH), cytochrome c oxidase (CO), and ATP synthase (ATPase) were measured in individual neurons and compared in ipsilateral and contralateral NM. Comparisons were made using unpaired t-tests (CO) or Kruskal Wallis one way ANOVA followed by Dunn's post hoc pairwise comparisons (ATPase, SDH). Mitotracker Red tissue was examined qualitatively for the presence of and extent of colocalization between Mitotracker Red and TUNEL label in NM. RESULTS showed global upregulation of all three oxidative enzymes within deafferented NM neurons compared to contralateral, unperturbed NM neurons. In addition, differential SDH and ATPase staining intensities were detected across neurons within the ipsilateral nucleus, suggesting functional differences in mitochondrial metabolism across deafferented NM. Quantitative analyses revealed that deafferented neurons with preferentially elevated SDH and ATPase activities represent the subpopulation destined to die following cochlea removal. In addition, Mitotracker Red accumulated intensely within the subset of deafferented NM neurons that also exhibited cytoplasmic TdT-mediated dUTP Nick-End Labeling (TUNEL) and subsequently died. Taken together, our results demonstrate that a subset of deafferented NM neurons, presumably those that die, preferentially upregulates SDH, perhaps via the tricarboxylic acid (TCA) cycle. These same neurons undergo ATPase uncoupling and an eventual loss of Deltapsi(m).

Imaging and Organelle Distribution of Fluorescent Ingap/Zns Nanoparticles in Glial Cells

To assess the effects of oleic acid treatment on subcellular distribution of indium gallium phosphide-zinc sulfide (InGaP/ZnS) nanoparticles in microglia and astrocytes. The extent of colocalization between the nanoparticles and organelles was assessed by confocal microscopy, spectrofluorometry and cell sorting. Cell treatment with a common fatty acid (oleic acid) within the range of physiological concentrations markedly enhanced the InGaP/ZnS uptake by microglia and afforded their colocalization within lipid droplets/lysosomes but not with mitochondria. These results suggest that the availability of mono-unsaturated fatty acids, such as oleic acid, in different cells could significantly alter nanoparticle uptake and localization, which can in turn affect the functions of cells and tissues coexposed to nanoparticles.

DNA damage and homologous recombination signaling induced by thymidylate deprivation

DNA damage is accepted as a consequence of thymidylate deprivation induced by chemotherapeutic inhibitors of thymidylate synthase (TS), but the types of damage and signaling responses remain incompletely understood. Thymidylate deprivation increases dUTP and uracil in DNA, which is removed by base excision repair (BER). Because BER requires a synthesis step, strand break intermediates presumably accumulate. Thymidylate deprivation also induces cell cycle arrest during replication. Homologous recombination (HR) is a means of repairing persistent BER intermediates and collapsed replication forks. There are also intimate links between HR and S-phase checkpoint pathways. In this study, the goals were to determine the involvement of HR-associated proteins and DNA damage signaling responses to thymidylate deprivation. When RAD51, which is a central component of HR, was depleted by siRNA cells were sensitized to raltitrexed (RTX), which specifically inhibits TS. To our knowledge, this is the first demonstration in mammalian cells that depletion of RAD51 causes sensitivity to thymidylate deprivation. Activation of DNA damage signaling responses was examined following treatment with RTX. Phosphorylation of replication protein A (RPA2 subunit) and formation of damage-induced foci were strikingly evident following IC 50 doses of RTX. Induction was much more striking following RTX treatment than with hydroxyurea, which is commonly used to inhibit replication. RTX treatment also induced foci of RAD51, γ-H2AX, phospho-Chk1, and phospho-NBS1, although the extent of co-localization with RPA2 foci varied. Collectively, the results suggest that HR and S-phase checkpoint signaling processes are invoked by thymidylate deprivation and influence cellular resistance to thymidylate deprivation.

Leptin Increases Adult Hippocampal Neurogenesis in Vivo and in Vitro

Leptin, an adipose-derived hormone, has been implicated in several physiological processes involving the hippocampus. However, the role of leptin in adult hippocampal neurogenesis remains unknown. Here we show that leptin regulates neurogenesis in the dentate gyrus of adult mice as well as in cultured adult hippocampal progenitor cells. Chronic administration of leptin to adult mice increased cell proliferation without significant effects on the differentiation and the survival of newly proliferated cells in the dentate gyrus. The expression of the long form leptin receptor, LepRb, was detected in hippocampal progenitor cells by reverse transcription-PCR and immunohistochemistry. Leptin treatment also increased proliferation of cultured adult hippocampal progenitor cells. Analysis of signal transduction pathways revealed that leptin stimulated phosphorylation of Akt and STAT3 but not ERK1/2. Furthermore, pre-treating the cells with specific inhibitors of Akt or STAT3 attenuated leptin-induced cell proliferation in a dose-dependent manner. Taken together, our results support a role for leptin in adult hippocampal neurogenesis and suggest the involvement of the Akt and STAT3 signaling pathways in mediating the actions of leptin on neurogenesis.

Differential dynamics of Rab3A and Rab27A on secretory granules

We have assessed the dynamics of the association of Rab3A and Rab27A with secretory granules at various stages of their life in PC12 cells. Endogenous Rab3A colocalised with the secretory granule marker secretogranin II (SGII) and expressed EGFP-Rab3A and ECFP-Rab27A colocalised with one another. The extent of colocalisation between EGFP-Rab3A or EGFP-Rab27 and SGII increased after longer times post transfection suggesting that these Rab proteins are preferentially recruited to newly synthesised granules. Following the release of immature secretory granules from the trans-Golgi network, Rab3A and Rab27A became associated with the immature granules after a lag period of around 20 minutes. Rab dynamics on granules were analysed in fluorescence recovery after photobleaching (FRAP) experiments. The recovery profile of EGFP-Rab27A was comparable to that of ppANF-EGFP, whereas the recovery profile of EGFP-Rab3A was significantly faster, indicating that Rab3A but not Rab27A might be rapidly exchanged between granules and cytosol. Inhibition of heat-shock protein 90 with 10 muM geldanamycin did not affect the exchange process or regulated exocytosis. Rab dynamics during stimulation with 300 muM ATP were analysed in live cells. Loss of granular ppANF-EGFP fluorescence was seen at the cell periphery after stimulation but only limited changes in EGFP-Rab3A and EGFP-Rab27A fluorescence was observed, indicating that the Rab proteins do not immediately dissociate or disperse on stimulation. The data suggest potentially distinct roles for Rab3A and Rab27A and we suggest that the finding that young secretory granules have a higher capacity for binding Rab3A and Rab27A is functionally important for preferential exocytosis from these granules.

Analysis of Lipolytic Protein Trafficking and Interactions in Adipocytes

This work examined the colocalization, trafficking, and interactions of key proteins involved in lipolysis during brief cAMP-dependent protein kinase A (PKA) activation. Double label immunofluorescence analysis of 3T3-L1 adipocytes indicated that PKA activation increases the translocation of hormonesensitive lipase (HSL) to perilipin A (Plin)-containing droplets and increases the colocalization of adipose tissue triglyceride lipase (Atgl) with its coactivator, Abhd5. Imaging of live 3T3-L1 preadipocytes transfected with Aquorea victoria-based fluorescent reporters demonstrated that HSL rapidly and specifically translocates to lipid droplets (LDs) containing Plin, and that this translocation is partially dependent on Plin phosphorylation. HSL closely, if not directly, interacts with Plin, as indicated by fluorescence resonance energy transfer (FRET) and bimolecular fluorescence complementation (BiFC) experiments. In contrast, tagged Atgl did not support FRET or BiFC with Plin, although it did modestly translocate to LDs upon stimulation. Abhd5 strongly interacted with Plin in the basal state, as indicated by FRET and BiFC. PKA activation rapidly (within minutes) decreased FRET between Abhd5 and Plin, and this decrease depended upon Plin phosphorylation. Together, these results indicate that Plin mediates hormone-stimulated lipolysis via direct and indirect mechanisms. Plin indirectly controls Atgl activity by regulating accessibility to its coactivator, Abhd5. In contrast, Plin directly regulates the access of HSL to substrate via close, if not direct, interactions. The differential interactions of HSL and Atgl with Plin and Abhd5 also explain the findings that following stimulation, HSL and Atgl are differentially enriched at specific LDs.