RESUMO
Basement membrane transmigration during embryonal development, tissue homeostasis and tumor invasion relies on invadosomes, a collective term for invadopodia and podosomes. An adequate structural framework for this process is still missing. Here, we reveal the modular actin nano-architecture that enables podosome protrusion and mechanosensing. The podosome protrusive core contains a central branched actin module encased by a linear actin module, each harboring specific actin interactors and actin isoforms. From the core, two actin modules radiate: ventral filaments bound by vinculin and connected to the plasma membrane and dorsal interpodosomal filaments crosslinked by myosin IIA. On stiff substrates, the actin modules mediate long-range substrate exploration, associated with degradative behavior. On compliant substrates, the vinculin-bound ventral actin filaments shorten, resulting in short-range connectivity and a focally protrusive, non-degradative state. Our findings redefine podosome nanoscale architecture and reveal a paradigm for how actin modularity drives invadosome mechanosensing in cells that breach tissue boundaries.
Assuntos
Actinas/química , Actinas/metabolismo , Podossomos/metabolismo , Actinas/genética , Animais , Adesão Celular , Membrana Celular/genética , Membrana Celular/metabolismo , Movimento Celular , Células Cultivadas , Células Dendríticas/química , Células Dendríticas/citologia , Células Dendríticas/metabolismo , Humanos , Mecanotransdução Celular , Camundongos , Podossomos/química , Podossomos/genéticaRESUMO
Subcellular partitioning of creatine kinase contributes to the formation of patterns in intracellular ATP distribution and the fuelling of cellular processes with a high and sudden energy demand. We have previously shown that brain-type creatine kinase (CK-B) accumulates at the phagocytic cup in macrophages where it is involved in the compartmentalized generation of ATP for actin remodeling. Here, we report that CK-B catalytic activity also helps in the formation of protrusive ruffle structures which are actin-dependent and abundant on the surface of both unstimulated and LPS-activated macrophages. Recruitment of CK-B to these structures occurred transiently and inhibition of the enzyme's catalytic activity with cyclocreatine led to a general smoothening of surface morphology as visualized by scanning electron microscopy. Comparison of the dynamics of distribution of YFP-tagged CK-mutants and isoforms by live imaging revealed that amino acid residues in the C-terminal segment (aa positions 323-330) that forms one of the protein's two mobile loops are involved in partitioning over inner regions of the cytosol and nearby sites where membrane protrusions occur during induction of phagocytic cup formation. Although wt CK-B, muscle-type CK (CK-M), and a catalytically dead CK-B-E232Q mutant with intact loop region were normally recruited from the cytosolic pool, no dynamic transition to the phagocytic cup area was seen for the CK-homologue arginine kinase and a CK-B-D326A mutant protein. Bioinformatics analysis helped us to predict that conformational flexibility of the C-terminal loop, independent of conformational changes induced by substrate binding or catalytic activity, is likely involved in exposing the enzyme for binding at or near the sites of membrane protrusion formation.
Assuntos
Membrana Celular/metabolismo , Extensões da Superfície Celular/metabolismo , Creatina Quinase Forma BB/metabolismo , Macrófagos/metabolismo , Actinas/metabolismo , Animais , Linhagem Celular , Extensões da Superfície Celular/efeitos dos fármacos , Biologia Computacional , Creatinina/análogos & derivados , Creatinina/farmacologia , Drosophila melanogaster , Inibidores Enzimáticos/farmacologia , Humanos , Macrófagos/ultraestrutura , Camundongos , Estrutura Terciária de ProteínaRESUMO
Functional morphodynamic behavior of differentiated macrophages is strongly controlled by actin cytoskeleton rearrangements, a process in which also metabolic cofactors ATP and NAD(H) (i.e. NAD+ and NADH) and NADP(H) (i.e. NADP+ and NADPH) play an essential role. Whereas the link to intracellular ATP availability has been studied extensively, much less is known about the relationship between actin cytoskeleton dynamics and intracellular redox state and NAD+-supply. Here, we focus on the role of nicotinamide phosphoribosyltransferase (NAMPT), found in extracellular form as a cytokine and growth factor, and in intracellular form as one of the key enzymes for the production of NAD+ in macrophages. Inhibition of NAD+ salvage synthesis by the NAMPT-specific drug FK866 caused a decrease in cytosolic NAD+ levels in RAW 264.7 and Maf-DKO macrophages and led to significant downregulation of the glycolytic flux without directly affecting cell viability, proliferation, ATP production capacity or mitochondrial respiratory activity. Concomitant with these differential metabolic changes, the capacity for phagocytic ingestion of particles and also substrate adhesion of macrophages were altered. Depletion of cytoplasmic NAD+ induced cell-morphological changes and impaired early adhesion in phagocytosis of zymosan particles as well as spreading performance. Restoration of NAD+ levels by NAD+, NMN, or NADP+ supplementation reversed the inhibitory effects of FK866. We conclude that direct coupling to local, actin-based, cytoskeletal dynamics is an important aspect of NAD+'s cytosolic role in the regulation of morphofunctional characteristics of macrophages.
Assuntos
Acrilamidas/farmacologia , Citoesqueleto de Actina/fisiologia , Trifosfato de Adenosina/metabolismo , Citocinas/metabolismo , Macrófagos/citologia , NAD/metabolismo , Nicotinamida Fosforribosiltransferase/metabolismo , Piperidinas/farmacologia , Western Blotting , Linhagem Celular , Citocinas/antagonistas & inibidores , Primers do DNA/genética , Fluorescência , Humanos , Macrófagos/fisiologia , Microscopia Eletrônica de Varredura , Nicotinamida Fosforribosiltransferase/antagonistas & inibidores , Oxirredução , Consumo de Oxigênio/fisiologia , Fagocitose/efeitos dos fármacos , TransfecçãoRESUMO
Macrophages constantly undergo morphological changes when quiescently surveying the tissue milieu for signs of microbial infection or damage, or after activation when they are phagocytosing cellular debris or foreign material. These morphofunctional alterations require active actin cytoskeleton remodeling and metabolic adaptation. Here we analyzed RAW 264.7 and Maf-DKO macrophages as models to study whether there is a specific association between aspects of carbohydrate metabolism and actin-based processes in LPS-stimulated macrophages. We demonstrate that the capacity to undergo LPS-induced cell shape changes and to phagocytose complement-opsonized zymosan (COZ) particles does not depend on oxidative phosphorylation activity but is fueled by glycolysis. Different macrophage activities like spreading, formation of cell protrusions, as well as phagocytosis of COZ, were thereby strongly reliant on the presence of low levels of extracellular glucose. Since global ATP production was not affected by rewiring of glucose catabolism and inhibition of glycolysis by 2-deoxy-D-glucose and glucose deprivation had differential effects, our observations suggest a non-metabolic role for glucose in actin cytoskeletal remodeling in macrophages, e.g. via posttranslational modification of receptors or signaling molecules, or other effects on the machinery that drives actin cytoskeletal changes. Our findings impute a decisive role for the nutrient state of the tissue microenvironment in macrophage morphodynamics.
Assuntos
Glucose/metabolismo , Lipopolissacarídeos/farmacologia , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Actinas/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Apoptose , Linhagem Celular , Proliferação de Células , Forma Celular/efeitos dos fármacos , Sobrevivência Celular , Proteínas do Sistema Complemento/metabolismo , Citoesqueleto/metabolismo , Desoxiglucose/metabolismo , Glicólise , Lactatos/metabolismo , Camundongos , Fosforilação Oxidativa , Fagocitose , Pseudópodes/metabolismo , Zimosan/metabolismoRESUMO
Dendritic cells (DCs) are key players in the initiation of adaptive immune responses and are currently exploited in immunotherapy against cancer and infectious diseases. The targeted delivery of nanovaccine particles (NPs) to DCs in vivo is a promising strategy to enhance immune responses. Here, targeted nanovaccine carriers were generated that allow multimodal imaging of nanocarrier-DC interactions from the subcellular to the organism level. These carriers were made of biodegradable poly(D,L-lactide-co-glycolide) harboring superparamagnetic iron oxide particles (SPIO) and fluorescently labeled antigen in a single particle. Targeted delivery was facilitated by coating the NPs with antibodies recognizing the DC-specific receptor DC-SIGN. The fluorescent label allowed for rapid analysis and quantification of specific versus nonspecific uptake of targeted NPs by DCs compared to other blood cells. In addition, it showed that part of the encapsulated antigen reached the lysosomal compartment of DCs within 24 h. Moreover, the presence of fluorescent label did not prevent the antigen from being presented to antigen-specific T cells. The incorporated SPIO was applied to track the NPs at subcellular cell organel level using transmission electron microscopy (TEM). NPs were found within endolysosomal compartments, where part of the SPIO was already released within 24 h. Furthermore, part of the NPs seemed to localize within the cytoplasm. Ex vivo loading of DCs with NPs resulted in efficient labeling and detection by MRI and did not abolish cell migration within collagen scaffolds. In conclusion, incorporation of two imaging agents within a single carrier allows tracking of targeted nanovaccines on a subcellular, cellular and possibly organism level, thereby facilitating rational design of in vivo targeted vaccination strategies.
Assuntos
Moléculas de Adesão Celular/imunologia , Células Dendríticas/imunologia , Portadores de Fármacos , Lectinas Tipo C/imunologia , Imageamento por Ressonância Magnética , Nanopartículas de Magnetita/administração & dosagem , Receptores de Superfície Celular/imunologia , Vacinas de Subunidades Antigênicas/imunologia , Apresentação de Antígeno , Células Sanguíneas/imunologia , Moléculas de Adesão Celular/metabolismo , Células Dendríticas/metabolismo , Compostos Férricos/química , Citometria de Fluxo , Humanos , Ácido Láctico/química , Lectinas Tipo C/metabolismo , Nanopartículas de Magnetita/química , Nanopartículas de Magnetita/ultraestrutura , Nanotecnologia , Fragmentos de Peptídeos/imunologia , Ácido Poliglicólico/química , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Receptores de Superfície Celular/metabolismo , Linfócitos T/imunologiaRESUMO
DMPK, the product of the mutated gene in myotonic dystrophy type 1, belongs to the subfamily of Rho-associated serine-threonine protein kinases, whose members play a role in actin-based cell morphodynamics. Not much is known about the physiological role of differentially localized individual DMPK splice isoforms. We report here that prominent stellar-shaped stress fibers are formed during early and late steps of differentiation in DMPK-deficient myoblast-myotubes upon complementation with the short cytosolic DMPK E isoform. Expression of DMPK E led to an increased phosphorylation status of MLC2. We found no such effects with vectors that encode a mutant DMPK E which was rendered enzymatically inactive or any of the long C-terminally anchored DMPK isoforms. Presence of stellar structures appears associated with changes in cell shape and motility and a delay in myogenesis. Our data strongly suggest that cytosolic DMPK participates in remodeling of the actomyosin cytoskeleton in developing skeletal muscle cells. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.
Assuntos
Actomiosina/metabolismo , Diferenciação Celular , Citosol/enzimologia , Mioblastos/citologia , Mioblastos/enzimologia , Proteínas Serina-Treonina Quinases/metabolismo , Actinas/química , Actinas/metabolismo , Animais , Movimento Celular , Polaridade Celular , Proliferação de Células , Forma Celular , Isoenzimas/metabolismo , Camundongos , Desenvolvimento Muscular , Miosina Tipo II/metabolismo , Miotonina Proteína Quinase , Fosforilação , Estrutura Quaternária de Proteína , Transporte Proteico , Fibras de Estresse/metabolismo , Fibras de Estresse/ultraestrutura , Frações Subcelulares/metabolismoRESUMO
BACKGROUND: Studies on the myotonic dystrophy protein kinase (DMPK) gene and gene products have thus far mainly concentrated on the fate of length mutation in the (CTG)n repeat at the DNA level and consequences of repeat expansion at the RNA level in DM1 patients and disease models. Surprisingly little is known about the function of DMPK protein products. METHODOLOGY/PRINCIPAL FINDINGS: We demonstrate here that transient expression of one major protein product of the human gene, the hDMPK A isoform with a long tail anchor, results in mitochondrial fragmentation and clustering in the perinuclear region. Clustering occurred in a variety of cell types and was enhanced by an intact tubulin cytoskeleton. In addition to morphomechanical changes, hDMPK A expression induces physiological changes like loss of mitochondrial membrane potential, increased autophagy activity, and leakage of cytochrome c from the mitochondrial intermembrane space accompanied by apoptosis. Truncation analysis using YFP-hDMPK A fusion constructs revealed that the protein's tail domain was necessary and sufficient to evoke mitochondrial clustering behavior. CONCLUSION/SIGNIFICANCE: Our data suggest that the expression level of the DMPK A isoform needs to be tightly controlled in cells where the hDMPK gene is expressed. We speculate that aberrant splice isoform expression might be a codetermining factor in manifestation of specific DM1 features in patients.
Assuntos
Apoptose , Autofagia , Mitocôndrias/metabolismo , Mutação , Proteínas Serina-Treonina Quinases/química , Animais , Proteínas de Bactérias/química , Citocromos c/metabolismo , DNA/genética , Células HeLa , Humanos , Proteínas Luminescentes/química , Potenciais da Membrana , Camundongos , Microtúbulos/metabolismo , Membranas Mitocondriais/metabolismo , Miotonina Proteína Quinase , Isoformas de Proteínas , Estrutura Terciária de ProteínaRESUMO
Mitochondria continuously change shape, position, and matrix configuration for optimal metabolite exchange. It is well established that changes in mitochondrial metabolism influence mitochondrial shape and matrix configuration. We demonstrated previously that inhibition of mitochondrial complex I (CI or NADH:ubiquinone oxidoreductase) by rotenone accelerated matrix protein diffusion and decreased the fraction and velocity of moving mitochondria. In the present study, we investigated the relationship between inherited CI deficiency, mitochondrial shape, mobility, and matrix protein diffusion. To this end, we analyzed fibroblasts of two children that represented opposite extremes in a cohort of 16 patients, with respect to their residual CI activity and mitochondrial shape. Fluorescence correlation spectroscopy (FCS) revealed no relationship between residual CI activity, mitochondrial shape, the fraction of moving mitochondria, their velocity, and the rate of matrix-targeted enhanced yellow fluorescent protein (mitoEYFP) diffusion. However, mitochondrial velocity and matrix protein diffusion in moving mitochondria were two to three times higher in patient cells than in control cells. Nocodazole inhibited mitochondrial movement without altering matrix EYFP diffusion, suggesting that both activities are mutually independent. Unexpectedly, electron microscopy analysis revealed no differences in mitochondrial ultrastructure between control and patient cells. It is discussed that the matrix of a moving mitochondrion in the CI-deficient state becomes less dense, allowing faster metabolite diffusion, and that fibroblasts of CI-deficient patients become more glycolytic, allowing a higher mitochondrial velocity.
Assuntos
Complexo I de Transporte de Elétrons/deficiência , Mitocôndrias Musculares/fisiologia , Mitocôndrias/fisiologia , Proteínas de Bactérias/genética , Linhagem Celular , Complexo I de Transporte de Elétrons/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/análise , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Fibroblastos/enzimologia , Fibroblastos/ultraestrutura , Genes Reporter , Humanos , Proteínas Luminescentes/genética , Mitocôndrias/enzimologia , Mitocôndrias/ultraestrutura , Mitocôndrias Musculares/enzimologia , Mitocôndrias Musculares/ultraestrutura , Fosforilação Oxidativa , Valores de Referência , Pele/enzimologia , Pele/ultraestruturaRESUMO
The small GTPase Rab6 is a key regulator in the retrograde transfer from endosomes via the Golgi to the ER. Three isoforms of Rab6 have been identified, the ubiquitously expressed Rab6A and Rab6A', and the brain specific Rab6B. Recent studies have shown that Rab6A' is the major isoform regulating this retrograde transport. Cytoplasmic dynein is the main motor protein complex for this transport. Dynein consists of two heavy chains, two intermediate chains, four light intermediate chains and several light chains, called roadblock/LC7 proteins or DYNLRB proteins. In mammalian cells two light chain isoforms have been identified, DYNLRB1 and DYNLRB2. We here show with yeast-two-hybrid, co-immunoprecipitation and pull down studies that DYNLRB1 specifically interacts with all three Rab6 isoforms and co-localises at the Golgi. This is the first example of a direct interaction between Rab6 isoforms and the dynein complex. Pull down experiments showed further preferred association of DYNLRB1 with GTP-bound Rab6A and interestingly GDP-bound Rab6A' and Rab6B. In addition DYNLRB1 was found in the Golgi apparatus where it co-localises with EYFP-Rab6 isoforms. DYNLRB is a putative modulator of the intrinsic GTPase activity of GTP-binding proteins. In vitro we were not able to reproduce this effect on Rab6 GTPase activity.
Assuntos
Dineínas/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Dineínas do Citoplasma , Dineínas/química , Dineínas/ultraestrutura , GTP Fosfo-Hidrolases/metabolismo , Complexo de Golgi/ultraestrutura , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Humanos , Camundongos , Dados de Sequência Molecular , Ligação Proteica , Conformação Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Transporte Proteico , Proteínas Recombinantes de Fusão/metabolismo , Transfecção , Proteínas rab de Ligação ao GTP/químicaRESUMO
Myotonic dystrophy protein kinase (DMPK) is a Ser/Thr-type protein kinase with unknown function, originally identified as the product of the gene that is mutated by triplet repeat expansion in patients with myotonic dystrophy type 1 (DM1). Alternative splicing of DMPK transcripts results in multiple protein isoforms carrying distinct C termini. Here, we demonstrate by expressing individual DMPKs in various cell types, including C(2)C(12) and DMPK(-/-) myoblast cells, that unique sequence arrangements in these tails control the specificity of anchoring into intracellular membranes. Mouse DMPK A and C were found to associate specifically with either the endoplasmic reticulum (ER) or the mitochondrial outer membrane, whereas the corresponding human DMPK A and C proteins both localized to mitochondria. Expression of mouse and human DMPK A-but not C-isoforms in mammalian cells caused clustering of ER or mitochondria. Membrane association of DMPK isoforms was resistant to alkaline conditions, and mutagenesis analysis showed that proper anchoring was differentially dependent on basic residues flanking putative transmembrane domains, demonstrating that DMPK tails form unique tail anchors. This work identifies DMPK as the first kinase in the class of tail-anchored proteins, with a possible role in organelle distribution and dynamics.
Assuntos
Processamento Alternativo/fisiologia , Retículo Endoplasmático/enzimologia , Mitocôndrias/enzimologia , Mioblastos/enzimologia , Proteínas Serina-Treonina Quinases/metabolismo , Sequência de Aminoácidos , Animais , Células COS , Células Cultivadas , Chlorocebus aethiops , Retículo Endoplasmático/ultraestrutura , Células HeLa , Humanos , Membranas Intracelulares/enzimologia , Membranas Intracelulares/ultraestrutura , Camundongos , Microscopia Imunoeletrônica , Mitocôndrias/ultraestrutura , Dados de Sequência Molecular , Mioblastos/ultraestrutura , Distrofia Miotônica/metabolismo , Miotonina Proteína Quinase , Células NIH 3T3 , Isoformas de Proteínas/metabolismo , Expansão das Repetições de Trinucleotídeos/fisiologiaRESUMO
1. In cells of epithelial origin the protein tyrosine phosphatase PTP-BL is predominantly localized at the apical membrane of polarized cells. This large submembranous multidomain PTP is also expressed in cells of neuronal origin. We studied the localization of PTP-BL in mouse neuroblastoma cells utilizing EGFP-tagged versions of the protein. 2. In proliferating Neuro-2a cells, immunofluorescence and immuno-electron microscopy revealed a submembranous FERM domain-dependent localization at cell-cell boundaries for EGFP-PTP-BL. Additionally, significant amounts of EGFP-PTP-BL are located in the cytoplasm as well as in nuclei. Upon serum depletion-induced differentiation of Neuro-2a cells, a partial shift of EGFP-PTP-BL from a cortical localization to cytoskeleton-like F-actin-positive structures is observed. Parallel biochemical studies corroborate this finding and reveal a serum depletion-induced shift of EFGP-PTP-BL from a membrane(-associated) fraction to an NP40-soluble cytoskeletal fraction. 3. Different pools of PTP-BL-containing protein complexes can be discerned in neuronal cells, reflecting distinct molecular microenvironments in which PTP-BL may exert its function.
Assuntos
Diferenciação Celular/fisiologia , Membrana Celular/enzimologia , Citoesqueleto/enzimologia , Neurônios/enzimologia , Proteínas Tirosina Fosfatases/metabolismo , Citoesqueleto de Actina/enzimologia , Citoesqueleto de Actina/ultraestrutura , Actinas/metabolismo , Animais , Compartimento Celular/efeitos dos fármacos , Compartimento Celular/fisiologia , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Membrana Celular/ultraestrutura , Núcleo Celular/enzimologia , Núcleo Celular/ultraestrutura , Células Cultivadas , Meios de Cultura Livres de Soro/farmacologia , Citoplasma/enzimologia , Citoplasma/ultraestrutura , Citoesqueleto/ultraestrutura , Proteínas de Fluorescência Verde , Substâncias Macromoleculares/metabolismo , Camundongos , Neuroblastoma , Neurônios/ultraestrutura , Estrutura Terciária de Proteína/fisiologia , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/fisiologia , Proteína Tirosina Fosfatase não Receptora Tipo 13 , Proteínas Tirosina Fosfatases/químicaRESUMO
Recently, we described the molecular identification of dendritic cell-specific TrAnsMembrane protein (DC-STAMP), a multimembrane-spanning protein preferentially expressed by human DC (hDC). In this report, we describe the identification and expression profile of the murine homologue of DC-STAMP (mDC-STAMP) as well as the characterization of the DC-STAMP protein. The results demonstrate that mDC-STAMP is over 90% homologous to hDC-STAMP and is also preferentially expressed by DC in vitro and ex vivo. mDC-STAMP expression is enhanced by interleukin-4 and down-regulated upon DC maturation. Analysis of differently tagged DC-STAMP proteins further demonstrates that hDC-STAMP and mDC-STAMP are glycosylated and primarily localize to an intracellular compartment. Applying confocal microscopy and electron microscopy, we demonstrate that hDC-STAMP localizes to the endoplasmic reticulum (ER) in human embryonic kidney 293 cells as well as hDC transduced with an adenovirus encoding hDC-STAMP-green fluorescent protein fusion protein. These data imply that DC-STAMP may exert its effect in the ER.
Assuntos
Células Dendríticas/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Clonagem Molecular , Perfilação da Expressão Gênica , Proteínas de Fluorescência Verde/genética , Humanos , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Homologia de Sequência de AminoácidosRESUMO
Genetic ablation of adenylate kinase 1 (AK1), a member of the AK family of phosphotransfer enzymes, disturbs muscle energetic economy and decreases tolerance to metabolic stress, despite rearrangements in alternative high energy phosphoryl transfer pathways. To define the mechanisms of this adaptive response, soleus and gastrocnemius muscles from AK1(-/-) mice were characterized by cDNA array profiling, Western blot and ultrastructural analysis. We demonstrate that AK1 deficiency induces fiber-type specific variation in groups of transcripts involved in glycolysis and mitochondrial metabolism and in gene products defining structural and myogenic events. This was associated with increased phosphotransfer capacities of the glycolytic enzymes pyruvate kinase and 3-phosphoglycerate kinase. Moreover, in AK1(-/-) mice, fast-twitch gastrocnemius, but not slow-twitch soleus, had an increase in adenine nucleotide translocator (ANT) and mitochondrial creatine kinase protein, along with a doubling of the intermyofibrillar mitochondrial volume. These results provide molecular evidence for wide-scale remodeling in AK1-deficient muscles aimed at preservation of efficient energetic communication between ATP producing and utilizing cellular sites.
Assuntos
Adenilato Quinase/deficiência , Adenilato Quinase/genética , Metabolismo Energético , Isoenzimas/deficiência , Isoenzimas/genética , Músculo Esquelético/metabolismo , Adenilato Quinase/metabolismo , Animais , Éxons , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Homozigoto , Isoenzimas/metabolismo , Camundongos , Camundongos Knockout , Proteínas Musculares/genética , Músculo Esquelético/ultraestrutura , Análise de Sequência com Séries de Oligonucleotídeos , Consumo de Oxigênio , RNA/genética , RNA/isolamento & purificação , RNA Mensageiro/genética , Transcrição GênicaRESUMO
The mouse gene Ptprr encodes the neuronal protein tyrosine phosphatases PTP-SL and PTPBR7. These proteins differ in their N-terminal domains, with PTP-SL being a cytosolic, membrane-associated phosphatase and PTPBR7 a type I transmembrane protein. In this study, we further explored the nature of the PTP-SL-associated vesicles in neuronal cells using a panel of organelle markers and noted a comparable subcellular distribution for PTP-SL and the beta4-adaptin subunit of the AP4 complex. PTP-SL, PTPBR7 and beta4-adaptin are localised at the Golgi apparatus and at vesicles throughout the cytoplasm. Immunohistochemical analysis demonstrated that PTP-SL, PTPBR7 and beta4-adaptin are all endogenously expressed in brain. Interestingly, coexpression of PTP-SL and beta4-adaptin leads to an altered subcellular localisation for PTP-SL. Instead of the Golgi and vesicle-type staining pattern, still observable for beta4-adaptin, PTP-SL is now distributed throughout the cytoplasm. Although beta4-adaptin was found to interact with the phosphatase domain of PTP-SL and PTPBR7 in the yeast two-hybrid system, it failed to do so in transfected neuronal cells. Our data suggest that the tyrosine phosphatases PTP-SL and PTPBR7 may be involved in the formation and transport of AP4-coated vesicles or in the dephosphorylation of their transmembrane cargo molecules at or near the Golgi apparatus.
Assuntos
Subunidades beta do Complexo de Proteínas Adaptadoras/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/enzimologia , Células PC12/enzimologia , Proteínas Tirosina Fosfatases/metabolismo , Animais , Biomarcadores/análise , Encéfalo/enzimologia , Células COS , Chlorocebus aethiops , Complexo de Golgi/enzimologia , Complexo de Golgi/ultraestrutura , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência , Microscopia Imunoeletrônica , Neurônios/ultraestrutura , Células PC12/ultraestrutura , Ratos , Proteínas Tirosina Fosfatases Classe 7 Semelhantes a Receptores , Transfecção , Vesículas Transportadoras/enzimologia , Vesículas Transportadoras/ultraestruturaRESUMO
Dendritic cells (DCs) capture Ags or viruses in peripheral tissue to transport them to lymphoid organs to induce cellular T cell responses. Recently, a DC-specific C-type lectin was identified, DC-specific ICAM-grabbing non-integrin (DC-SIGN), that functions as cell adhesion receptor mediating both DC migration and T cell activation. DC-SIGN also functions as an HIV-1R that captures HIVgp120 and facilitates DC-induced HIV transmission of T cells. Internalization motifs in the cytoplasmic tail of DC-SIGN hint to a function of DC-SIGN as endocytic receptor. In this study we demonstrate that on DCs DC-SIGN is rapidly internalized upon binding of soluble ligand. Mutating a putative internalization motif in the cytoplasmic tail reduces ligand-induced internalization. Detailed analysis using ratio fluorescence imaging and electron microscopy showed that DC-SIGN-ligand complexes are targeted to late endosomes/lysosomes. Moreover, ligands internalized by DC-SIGN are efficiently processed and presented to CD4+ T cells. The distinct pattern of expression of C-type lectins on DCs in situ and their nonoverlapping Ag recognition profile hint to selective functions of these receptors to allow a DC to recognize a wide variety of Ags and to process these to induce T cell activation. These data point to a novel function of the adhesion receptor DC-SIGN as an efficient DC-specific Ag receptor that can be used as a target to induce viral and antitumor immunity.