RESUMO
The ubiquitous second messenger 3',5'-cyclic adenosine monophosphate (cAMP) regulates cardiac excitation-contraction coupling (ECC) by signaling in discrete subcellular microdomains. Phosphodiesterase subfamilies 4B and 4D are critically involved in the regulation of cAMP signaling in mammalian cardiomyocytes. Alterations of PDE4 activity in human hearts has been shown to result in arrhythmias and heart failure. Here, we sought to systematically investigate specific roles of PDE4B and PDE4D in the regulation of cAMP dynamics in three distinct subcellular microdomains, one of them located at the caveolin-rich plasma membrane which harbors the L-type calcium channels (LTCCs), as well as at two sarco/endoplasmic reticulum (SR) microdomains centered around SR Ca2+-ATPase (SERCA2a) and cardiac ryanodine receptor type 2 (RyR2). Transgenic mice expressing Förster Resonance Energy Transfer (FRET)-based cAMP-specific biosensors targeted to caveolin-rich plasma membrane, SERCA2a and RyR2 microdomains were crossed to PDE4B-KO and PDE4D-KO mice. Direct analysis of the specific effects of both PDE4 subfamilies on local cAMP dynamics was performed using FRET imaging. Our data demonstrate that all three microdomains are differentially regulated by these PDE4 subfamilies. Whereas both are involved in cAMP regulation at the caveolin-rich plasma membrane, there are clearly two distinct cAMP microdomains at the SR formed around RyR2 and SERCA2a, which are preferentially controlled by PDE4B and PDE4D, respectively. This correlates with local cAMP-dependent protein kinase (PKA) substrate phosphorylation and arrhythmia susceptibility. Immunoprecipitation assays confirmed that PDE4B is associated with RyR2 along with PDE4D. Stimulated Emission Depletion (STED) microscopy of immunostained cardiomyocytes suggested possible co-localization of PDE4B with both sarcolemmal and RyR2 microdomains. In conclusion, our functional approach could show that both PDE4B and PDE4D can differentially regulate cardiac cAMP microdomains associated with calcium homeostasis. PDE4B controls cAMP dynamics in both caveolin-rich plasma membrane and RyR2 vicinity. Interestingly, PDE4B is the major regulator of the RyR2 microdomain, as opposed to SERCA2a vicinity, which is predominantly under PDE4D control, suggesting a more complex regulatory pattern than previously thought, with multiple PDEs acting at the same location.
Assuntos
Cálcio , Canal de Liberação de Cálcio do Receptor de Rianodina , Camundongos , Humanos , Animais , Cálcio/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , AMP Cíclico/metabolismo , Miócitos Cardíacos/metabolismo , Camundongos Transgênicos , Caveolinas/metabolismo , Mamíferos/metabolismoRESUMO
The glycocalyx regulates the interaction of mammalian cells with extracellular molecules, such as cytokines. However, it is unknown to which extend the glycocalyx of distinct cancer cells control the binding and uptake of nanoparticles. In the present study, exome sequencing data of cancer patients and analysis of distinct melanoma and bladder cancer cell lines suggested differences in cancer cell-exposed glycocalyx components such as heparan sulphate. Our data indicate that glycocalyx differences affected the binding of cationic chitosan nanocapsules (Chi-NCs). The pronounced glycocalyx of bladder cancer cells enhanced the internalisation of nanoencapsulated capsaicin. Consequently, capsaicin induced apoptosis in the cancer cells, but not in the less glycosylated benign urothelial cells. Moreover, we measured counterion condensation on highly negatively charged heparan sulphate chains. Counterion condensation triggered a cooperative binding of Chi-NCs, characterised by a weak binding rate at low Chi-NC doses and a strongly increased binding rate at high Chi-NC concentrations. Our results indicate that the glycocalyx of tumour cells controls the binding and biological activity of nanoparticles. This has to be considered for the design of tumour cell directed nanocarriers to improve the delivery of cytotoxic drugs. Differential nanoparticle binding may also be useful to discriminate tumour cells from healthy cells.
Assuntos
Antipruriginosos/administração & dosagem , Antipruriginosos/farmacocinética , Capsaicina/administração & dosagem , Capsaicina/farmacocinética , Quitosana/química , Glicocálix/metabolismo , Nanocápsulas/química , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Heparitina Sulfato/metabolismo , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/metabolismo , Especificidade de Órgãos , Ligação Proteica , Eletricidade Estática , Nanomedicina TeranósticaRESUMO
In living cells, processes such as adhesion formation involve extensive structural changes in the plasma membrane and the cell interior. In order to visualize these highly dynamic events, two complementary light microscopy techniques that allow fast imaging of live samples were combined: spinning disk microscopy (SD) for fast and high-resolution volume recording and total internal reflection fluorescence (TIRF) microscopy for precise localization and visualization of the plasma membrane. A comprehensive and complete imaging protocol will be shown for guiding through sample preparation, microscope calibration, image formation and acquisition, resulting in multi-color SD-TIRF live imaging series with high spatio-temporal resolution. All necessary image post-processing steps to generate multi-dimensional live imaging datasets, i.e. registration and combination of the individual channels, are provided in a self-written macro for the open source software ImageJ. The imaging of fluorescent proteins during initiation and maturation of adhesion complexes, as well as the formation of the actin cytoskeletal network, was used as a proof of principle for this novel approach. The combination of high resolution 3D microscopy and TIRF provided a detailed description of these complex processes within the cellular environment and, at the same time, precise localization of the membrane-associated molecules detected with a high signal-to-background ratio.
Assuntos
Células Cultivadas/ultraestrutura , Processamento de Imagem Assistida por Computador/métodos , Microscopia de Fluorescência/métodosRESUMO
The earliest intracellular signals that occur after T cell activation are local, subsecond Ca2+ microdomains. Here, we identified a Ca2+ entry component involved in Ca2+ microdomain formation in both unstimulated and stimulated T cells. In unstimulated T cells, spontaneously generated small Ca2+ microdomains required ORAI1, STIM1, and STIM2. Super-resolution microscopy of unstimulated T cells identified a circular subplasmalemmal region with a diameter of about 300 nm with preformed patches of colocalized ORAI1, ryanodine receptors (RYRs), and STIM1. Preformed complexes of STIM1 and ORAI1 in unstimulated cells were confirmed by coimmunoprecipitation and Förster resonance energy transfer studies. Furthermore, within the first second after T cell receptor (TCR) stimulation, the number of Ca2+ microdomains increased in the subplasmalemmal space, an effect that required ORAI1, STIM2, RYR1, and the Ca2+ mobilizing second messenger NAADP (nicotinic acid adenine dinucleotide phosphate). These results indicate that preformed clusters of STIM and ORAI1 enable local Ca2+ entry events in unstimulated cells. Upon TCR activation, NAADP-evoked Ca2+ release through RYR1, in coordination with Ca2+ entry through ORAI1 and STIM, rapidly increases the number of Ca2+ microdomains, thereby initiating spread of Ca2+ signals deeper into the cytoplasm to promote full T cell activation.
Assuntos
Cálcio/metabolismo , Ativação Linfocitária , Proteína ORAI1/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Molécula 1 de Interação Estromal/metabolismo , Molécula 2 de Interação Estromal/metabolismo , Linfócitos T/citologia , Animais , Sinalização do Cálcio , Membrana Celular , Células Cultivadas , Feminino , Transferência Ressonante de Energia de Fluorescência , Masculino , Microdomínios da Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
Understanding the cellular processes that occur between the cytosol and the plasma membrane is an important task for biological research. Till now, however, it was not possible to combine fast and high-resolution imaging of both the isolated plasma membrane and the surrounding intracellular volume. Here, we demonstrate the combination of fast high-resolution spinning disk (SD) and total internal reflection fluorescence (TIRF) microscopy for specific imaging of the plasma membrane. A customised SD-TIRF microscope was used with specific design of the light paths that allowed, for the first time, live SD-TIRF experiments at high acquisition rates. A series of experiments is shown to demonstrate the feasibility and performance of our setup.
Assuntos
Membrana Celular/ultraestrutura , Citoplasma/ultraestrutura , Microscopia Intravital/métodos , Microscopia de Fluorescência/métodos , Células Cultivadas , Humanos , Microscopia Intravital/instrumentação , Microscopia de Fluorescência/instrumentaçãoRESUMO
Reelin controls neuronal migration and layer formation. Previous studies in reeler mice deficient in Reelin focused on the result of the developmental process in fixed tissue sections. It has remained unclear whether Reelin affects the migratory process, migration directionality, or migrating neurons guided by the radial glial scaffold. Moreover, Reelin has been regarded as an attractive signal because newly generated neurons migrate toward the Reelin-containing marginal zone. Conversely, Reelin might be a stop signal because migrating neurons in reeler, but not in wild-type mice, invade the marginal zone. Here, we monitored the migration of newly generated proopiomelanocortin-EGFP-expressing dentate granule cells in slice cultures from reeler, reeler-like mutants and wild-type mice of either sex using real-time microscopy. We discovered that not the actual migratory process and migratory speed, but migration directionality of the granule cells is controlled by Reelin. While wild-type granule cells migrated toward the marginal zone of the dentate gyrus, neurons in cultures from reeler and reeler-like mutants migrated randomly in all directions as revealed by vector analyses of migratory trajectories. Moreover, live imaging of granule cells in reeler slices cocultured to wild-type dentate gyrus showed that the reeler neurons changed their directions and migrated toward the Reelin-containing marginal zone of the wild-type culture, thus forming a compact granule cell layer. In contrast, directed migration was not observed when Reelin was ubiquitously present in the medium of reeler slices. These results indicate that topographically administered Reelin controls the formation of a granule cell layer.SIGNIFICANCE STATEMENT Neuronal migration and the various factors controlling its onset, speed, directionality, and arrest are poorly understood. Slice cultures offer a unique model to study the migration of individual neurons in an almost natural environment. In the present study, we took advantage of the expression of proopiomelanocortin-EGFP by newly generated, migrating granule cells to analyze their migratory trajectories in hippocampal slice cultures from wild-type mice and mutants deficient in Reelin signaling. We show that the compartmentalized presence of Reelin is essential for the directionality, but not the actual migratory process or speed, of migrating granule cells leading to their characteristic lamination in the dentate gyrus.
Assuntos
Moléculas de Adesão Celular Neuronais/fisiologia , Movimento Celular/fisiologia , Giro Denteado/citologia , Proteínas da Matriz Extracelular/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Serina Endopeptidases/fisiologia , Animais , Movimento Celular/genética , Células Cultivadas , Córtex Cerebral/citologia , Grânulos Citoplasmáticos/fisiologia , Células Ependimogliais , Feminino , Cinética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes Neurológicos , Mutação , Neurônios/fisiologia , Pró-Opiomelanocortina/genética , Pró-Opiomelanocortina/metabolismo , Proteína ReelinaRESUMO
In reeler mutant mice, which are deficient in reelin (Reln), the lamination of the cerebral cortex is disrupted. Reelin signaling induces phosphorylation of LIM kinase 1, which phosphorylates the actin-depolymerizing protein cofilin in migrating neurons. Conditional cofilin mutants show neuronal migration defects. Thus, both reelin and cofilin are indispensable during cortical development. To analyze the effects of cofilin phosphorylation on neuronal migration we used in utero electroporation to transfect E14.5 wild-type cortical neurons with pCAG-EGFP plasmids encoding either a nonphosphorylatable form of cofilin 1 (cofilin(S3A)), a pseudophosphorylated form (cofilin(S3E)) or wild-type cofilin 1 (cofilin(WT)). Wild-type controls and reeler neurons were transfected with pCAG-EGFP. Real-time microscopy and histological analyses revealed that overexpression of cofilin(WT) and both phosphomutants induced migration defects and morphological abnormalities of cortical neurons. Of note, reeler neurons and cofilin(S3A)- and cofilin(S3E)-transfected neurons showed aberrant backward migration towards the ventricular zone. Overexpression of cofilin(S3E), the pseudophosphorylated form, partially rescued the migration defect of reeler neurons, as did overexpression of Limk1. Collectively, the results indicate that reelin and cofilin cooperate in controlling cytoskeletal dynamics during neuronal migration.
Assuntos
Moléculas de Adesão Celular Neuronais/metabolismo , Movimento Celular , Forma Celular , Córtex Cerebral/citologia , Cofilina 1/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/citologia , Serina Endopeptidases/metabolismo , Animais , Contagem de Células , Eletroporação , Embrião de Mamíferos/citologia , Feminino , Camundongos Endogâmicos C57BL , Neurônios/metabolismo , Proteína Reelina , TransfecçãoRESUMO
Many gram-negative bacteria including pathogenic Yersinia spp. employ type III secretion systems to translocate effector proteins into eukaryotic target cells. Inside the host cell the effector proteins manipulate cellular functions to the benefit of the bacteria. To better understand the control of type III secretion during host cell interaction, sensitive and accurate assays to measure translocation are required. We here describe the application of an assay based on the fusion of a Yersinia enterocolitica effector protein fragment (Yersinia outer protein; YopE) with TEM-1 beta-lactamase for quantitative analysis of translocation. The assay relies on cleavage of a cell permeant FRET dye (CCF4/AM) by translocated beta-lactamase fusion. After cleavage of the cephalosporin core of CCF4 by the beta-lactamase, FRET from coumarin to fluorescein is disrupted and excitation of the coumarin moiety leads to blue fluorescence emission. Different applications of this method have been described in the literature highlighting its versatility. The method allows for analysis of translocation in vitro and also in in vivo, e.g., in a mouse model. Detection of the fluorescence signals can be performed using plate readers, FACS analysis or fluorescence microscopy. In the setup described here, in vitro translocation of effector fusions into HeLa cells by different Yersinia mutants is monitored by laser scanning microscopy. Recording intracellular conversion of the FRET reporter by the beta-lactamase effector fusion in real-time provides robust quantitative results. We here show exemplary data, demonstrating increased translocation by a Y. enterocolitica YopE mutant compared to the wild type strain.
Assuntos
Yersinia enterocolitica/patogenicidade , beta-Lactamases/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Células Eucarióticas , Transferência Ressonante de Energia de Fluorescência/métodos , Células HeLa , Interações Hospedeiro-Patógeno , Humanos , Translocação Genética , Sistemas de Secreção Tipo III , Yersinia enterocolitica/enzimologia , Yersinia enterocolitica/genética , beta-Lactamases/análiseRESUMO
Virulence of the nosocomial pathogen Staphylococcus epidermidis is crucially linked to formation of adherent biofilms on artificial surfaces. Biofilm assembly is significantly fostered by production of a bacteria derived extracellular matrix. However, the matrix composition, spatial organization, and relevance of specific molecular interactions for integration of bacterial cells into the multilayered biofilm community are not fully understood. Here we report on the function of novel 18 kDa Small basic protein (Sbp) that was isolated from S. epidermidis biofilm matrix preparations by an affinity chromatographic approach. Sbp accumulates within the biofilm matrix, being preferentially deposited at the biofilm-substratum interface. Analysis of Sbp-negative S. epidermidis mutants demonstrated the importance of Sbp for sustained colonization of abiotic surfaces, but also epithelial cells. In addition, Sbp promotes assembly of S. epidermidis cell aggregates and establishment of multilayered biofilms by influencing polysaccharide intercellular-adhesin (PIA) and accumulation associated protein (Aap) mediated intercellular aggregation. While inactivation of Sbp indirectly resulted in reduced PIA-synthesis and biofilm formation, Sbp serves as an essential ligand during Aap domain-B mediated biofilm accumulation. Our data support the conclusion that Sbp serves as an S. epidermidis biofilm scaffold protein that significantly contributes to key steps of surface colonization. Sbp-negative S. epidermidis mutants showed no attenuated virulence in a mouse catheter infection model. Nevertheless, the high prevalence of sbp in commensal and invasive S. epidermidis populations suggests that Sbp plays a significant role as a co-factor during both multi-factorial commensal colonization and infection of artificial surfaces.
Assuntos
Aderência Bacteriana/fisiologia , Biofilmes/crescimento & desenvolvimento , Proteínas Periplásmicas de Ligação/metabolismo , Staphylococcus epidermidis/fisiologia , Animais , Camundongos , Proteínas Periplásmicas de Ligação/genéticaRESUMO
Inositol-1,4,5-trisphosphate-3-kinase-A (ITPKA) has been considered as an actin bundling protein because its N-terminal actin binding domain (ABD) induces formation of linear actin bundles. Since in many cancer cell lines ITPKA is essential for formation of lamellipodia, which consist of cross-linked actin filaments, here we analyzed if full length-ITPKA may induce formation of more complex actin structures. Indeed, we found that incubation of F-actin with ITPKA resulted in formation of dense, branched actin networks. Based on our result that ITPKA does not exhibit an additional C-terminal ABD, we exclude that ITPKA cross-links actin filaments by simultaneous F-actin binding with two different ABDs. Instead, stimulated-emission-depletion-microscopy and measurement of InsP3 Kinase activity give evidence that that N-terminal ABD-homodimers of ITPKA bind to F-actin while the monomeric C-termini insert between adjacent actin filaments. Thereby, they prevent formation of thick actin bundles but induce formation of thin branched actin structures. Interestingly, when embedded in this dense actin network, InsP3 Kinase activity is doubled and the product of InsP3 Kinase activity, Ins(1,3,4,5)P4 , inhibits spontaneous actin polymerization which may reflect a local negative feedback regulation of InsP3 Kinase activity. In conclusion, we demonstrate that not only the ABD of ITPKA modulates actin dynamics but reveal that the InsP3 Kinase domain substantially contributes to this process.
Assuntos
Actinas/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Domínio Catalítico , Fosfotransferases (Aceptor do Grupo Álcool)/química , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
The liver stage of the Plasmodium parasite remains one of the most promising targets for intervention against malaria as it is clinically silent, precedes the symptomatic blood stage and represents a bottleneck in the parasite life cycle. However, many aspects of the development of the parasite during this stage are far from understood. During the liver stage, the parasite undergoes extensive replication, forming tens of thousands of infectious merozoites from each invading sporozoite. This implies a very efficient and accurate process of cytokinesis and thus also of organelle development and segregation. We have generated for the first time Plasmodium berghei double-fluorescent parasite lines, allowing visualization of the apicoplast, mitochondria and nuclei in live liver stage parasites. Using these we have seen that in parallel with nuclear division, the apicoplast and mitochondrion become two extensively branched and intertwining structures. The organelles then undergo impressive morphological and positional changes prior to cell division. To form merozoites, the parasite undergoes cytokinesis and the complex process of organelle development and segregation into the forming daughter merozoites could be analysed in detail using the newly generated transgenic parasites.
Assuntos
Citocinese , Fígado/parasitologia , Merozoítos/fisiologia , Organelas/fisiologia , Plasmodium berghei/fisiologia , Genes Reporter , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Merozoítos/crescimento & desenvolvimento , Microscopia de Fluorescência , Organelas/ultraestrutura , Plasmodium berghei/crescimento & desenvolvimento , Coloração e Rotulagem/métodosRESUMO
BACKGROUND INFORMATION: The Plasmodium parasite, during its life cycle, undergoes three phases of asexual reproduction, these being repeated rounds of erythrocytic schizogony, sporogony within oocysts on the mosquito midgut wall and exo-erythrocytic schizogony within the hepatocyte. During each phase of asexual reproduction, the parasite must ensure that every new daughter cell contains an apicoplast, as this organelle cannot be formed de novo and is essential for parasite survival. To date, studies visualizing the apicoplast in live Plasmodium parasites have been restricted to the blood stages of Plasmodium falciparum. RESULTS: In the present study, we have generated Plasmodium berghei parasites in which GFP (green fluorescent protein) is targeted to the apicoplast using the specific targeting sequence of ACP (acyl carrier protein), which has allowed us to visualize this organelle in live Plasmodium parasites. During each phase of asexual reproduction, the apicoplast becomes highly branched, but remains as a single organelle until the completion of nuclear division, whereupon it divides and is rapidly segregated into newly forming daughter cells. We have shown that the antimicrobial agents azithromycin, clindamycin and doxycycline block development of the apicoplast during exo-erythrocytic schizogony in vitro, leading to impaired parasite maturation. CONCLUSIONS: Using a range of powerful live microscopy techniques, we show for the first time the development of a Plasmodium organelle through the entire life cycle of the parasite. Evidence is provided that interference with the development of the Plasmodium apicoplast results in the failure to produce red-blood-cell-infective merozoites.
Assuntos
Proteínas de Fluorescência Verde/metabolismo , Estágios do Ciclo de Vida , Malária/parasitologia , Plasmodium berghei/citologia , Plasmodium berghei/crescimento & desenvolvimento , Plastídeos/metabolismo , Animais , Linhagem Celular , Proteínas de Fluorescência Verde/genética , Humanos , Camundongos , Plasmodium berghei/genética , Plasmodium berghei/metabolismo , Plastídeos/genéticaRESUMO
In the brain the expression of transforming growth factor beta1 (TGF-beta1) is involved both in neuroprotective and neurodegenerative processes. Recently, we have established a transgenic mouse model with inducible neuron-specific expression of TGF-beta1 based on the tetracycline-regulated gene expression system. A long-term expression of TGF-beta1 results in persisting perivascular thioflavin-positive depositions, which did not disappear even though the transgene synthesis was repressed completely by administration of doxycycline. Formation and composition of these depositions are hardly elucidated. The aim of this study was to identify TGF-beta1 responding genes potentially participating in forming these depositions. To address this problem we have compared the cortical mRNA expression pattern of TGF-beta1 expressing mice with mice impeded to express the transgenic protein using oligonucleotide microarray analysis. Differential gene expression was further characterized by quantitative real-time reverse transcription-polymerase chain reaction including animals, where the long-lasting TGF-beta1 expression was repressed. While no change of amyloid precursor protein RNA expression level was detected, various genes strongly involved in calcium homeostasis, tissue mineralization or vascular calcification were identified differentially expressed. It is suggested, that these genes might contribute to the perivascular depositions in the TGF-beta1 expressing mice.