RESUMO
For quality, interpretation, reproducibility and sharing value, microscopy images should be accompanied by detailed descriptions of the conditions that were used to produce them. Micro-Meta App is an intuitive, highly interoperable, open-source software tool that was developed in the context of the 4D Nucleome (4DN) consortium and is designed to facilitate the extraction and collection of relevant microscopy metadata as specified by the recent 4DN-BINA-OME tiered-system of Microscopy Metadata specifications. In addition to substantially lowering the burden of quality assurance, the visual nature of Micro-Meta App makes it particularly suited for training purposes.
Assuntos
Metadados , Microscopia Confocal/instrumentação , Microscopia Confocal/métodos , Microscopia de Fluorescência/instrumentação , Microscopia de Fluorescência/métodos , Aplicativos Móveis , Linguagens de Programação , Software , Animais , Linhagem Celular , Biologia Computacional/métodos , Humanos , Processamento de Imagem Assistida por Computador , Camundongos , Reconhecimento Automatizado de Padrão , Controle de Qualidade , Reprodutibilidade dos Testes , Interface Usuário-Computador , Fluxo de TrabalhoRESUMO
CONTEXT: Rapid on-site evaluation (ROSE) optimises the performance of cytology, but requires skilled handling, and smearing can make the material unavailable for some ancillary tests. There is a need to facilitate ROSE without sacrificing part of the sample. OBJECTIVE: We evaluated the image quality of inexpensive deconvolution fluorescence microscopy for optically sectioning non-smeared fine needle aspiration (FNA) tissue fragments. DESIGN: A portion of residual material from 14 FNA samples was stained for 3 min in Hoechst 33342 and Sypro™ Red to label DNA and protein respectively, transferred to an imaging chamber, and imaged at 200× or 400× magnification at 1 micron intervals using a GE DeltaVision inverted fluorescence microscope. A deconvolution algorithm was applied to remove out-of-plane signal, and the resulting images were inverted and pseudocoloured to resemble H&E sections. Five cytopathologists blindly diagnosed 2 to 4 representative image stacks per case (total 70 evaluations), and later compared them to conventional epifluorescent images. RESULTS: Accurate definitive diagnoses were rendered in 45 (64%) of 70 total evaluations; equivocal diagnoses (atypical or suspicious) were made in 21 (30%) of the 70. There were two false positive and two false negative "definite" diagnoses in three cases (4/70; 6%). Cytopathologists preferred deconvolved images compared to raw images (P < 0.01). The imaged fragments were recovered and prepared into a ThinPrep or cell block without discernible alteration. CONCLUSIONS: Deconvolution improves image quality of FNA fragments compared to epifluorescence, often allowing definitive diagnosis while enabling the ROSE material to be subsequently triaged.
Assuntos
Microscopia , Avaliação Rápida no Local , Biópsia por Agulha Fina/métodos , Citodiagnóstico , Técnicas Citológicas , HumanosRESUMO
Ion channels in myometrial cells play critical roles in spontaneous and agonist-induced uterine contraction during the menstrual cycle, pregnancy maintenance, and parturition; thus, identifying the genes of ion channels in these cells and determining their roles are essential to understanding the biology of reproduction. Previous studies with in vitro functional and pharmacological approaches have produced controversial results regarding the presence and role of TMEM16A Ca2+-activated Cl- channels in myometrial cells. To unambiguously determine the function of this channel in these cells, we employed a genetic approach by using smooth muscle cell-specific TMEM16A deletion (i.e. TMEM16ASMKO) mice. We found that myometrial cells from TMEM16ASMKO mice generated the same pattern and magnitude in Ca2+ signals upon stimulation with KCl, oxytocin, and PGF2α compared to the isogenic control myometrial cells. At the uterine tissue level, TMEM16A deletion also did not cause detectable changes in either spontaneous or agonist (i.e. KCl, oxytocin, and PGF2α)-induced contractions. Moreover, in vivo the TMEM16ASMKO mice gave birth at full term with the same litter size as genetically identical control mice. Finally, TMEM16A immunostaining in both control and TMEM16ASMKO mice revealed that this protein was highly expressed in the endometrial stroma, but did not co-localize with a smooth muscle specific marker MYH11. Collectively, these results unequivocally demonstrate that TMEM16A does not serve as a pacemaking channel for spontaneous uterine contraction, neither does it function as a depolarizing channel for agonist-evoked uterine contraction. Yet these two functions could underlie the normal gestation length and litter size in the TMEM16ASMKO mice.
Assuntos
Anoctamina-1/metabolismo , Sinalização do Cálcio/fisiologia , Miócitos de Músculo Liso/metabolismo , Contração Uterina/fisiologia , Animais , Anoctamina-1/genética , Feminino , Deleção de Genes , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Tamanho da Ninhada de Vivíparos , Camundongos , Cloreto de Potássio , Gravidez , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Contração Uterina/efeitos dos fármacosRESUMO
siRNAs are a new class of therapeutic modalities with promising clinical efficacy that requires modification or formulation for delivery to the tissue and cell of interest. Conjugation of siRNAs to lipophilic groups supports efficient cellular uptake by a mechanism that is not well characterized. Here we study the mechanism of internalization of asymmetric, chemically stabilized, cholesterol-modified siRNAs (sd-rxRNAs®) that efficiently enter cells and tissues without the need for formulation. We demonstrate that uptake is rapid with significant membrane association within minutes of exposure followed by the formation of vesicular structures and internalization. Furthermore, sd-rxRNAs are internalized by a specific class of early endosomes and show preferential association with epidermal growth factor (EGF) but not transferrin (Tf) trafficking pathways as shown by live cell TIRF and structured illumination microscopy (SIM). In fixed cells, we observe â¼25% of sd-rxRNA co-localizing with EGF and <5% with Tf, which is indicative of selective endosomal sorting. Likewise, preferential sd-rxRNA co-localization was demonstrated with EEA1 but not RBSN-containing endosomes, consistent with preferential EGF-like trafficking through EEA1-containing endosomes. sd-rxRNA cellular uptake is a two-step process, with rapid membrane association followed by internalization through a selective, saturable subset of the endocytic process. However, the mechanistic role of EEA1 is not yet known. This method of visualization can be used to better understand the kinetics and mechanisms of hydrophobic siRNA cellular uptake and will assist in further optimization of these types of compounds for therapeutic intervention.
Assuntos
Colesterol/química , Endossomos/metabolismo , Fator de Crescimento Epidérmico/metabolismo , RNA Interferente Pequeno/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Animais , Transporte Biológico , Células COS , Chlorocebus aethiops , Colesterol/metabolismo , Ciclofilinas/genética , Ciclofilinas/metabolismo , Endocitose , Fator de Crescimento Epidérmico/genética , Expressão Gênica , Células HeLa , Humanos , Interações Hidrofóbicas e Hidrofílicas , Microscopia de Fluorescência , RNA Interferente Pequeno/química , Transferrina/genética , Transferrina/metabolismo , Proteínas de Transporte Vesicular/genéticaRESUMO
The assembly and maintenance of most cilia and flagella rely on intraflagellar transport (IFT). Recent in vitro studies have suggested that, together, the calponin-homology domain within the IFT81 N-terminus and the highly basic N-terminus of IFT74 form a module for IFT of tubulin. By using Chlamydomonas mutants for IFT81 and IFT74, we tested this hypothesis in vivo. Modification of the predicted tubulin-binding residues in IFT81 did not significantly affect basic anterograde IFT and length of steady-state flagella but slowed down flagellar regeneration, a phenotype similar to that seen in a strain that lacks the IFT74 N-terminus. In both mutants, the frequency of tubulin transport by IFT was greatly reduced. A double mutant that combined the modifications to IFT81 and IFT74 was able to form only very short flagella. These results indicate that, together, the IFT81 and IFT74 N-termini are crucial for flagellar assembly, and are likely to function as the main module for IFT of tubulin.
Assuntos
Proteínas de Transporte/genética , Chlamydomonas reinhardtii/genética , Flagelos/genética , Tubulina (Proteína)/genética , Transporte Biológico/genética , Proteínas de Transporte/metabolismo , Chlamydomonas reinhardtii/metabolismo , Cílios/genética , Cílios/metabolismo , Flagelos/metabolismo , Fenótipo , Ligação Proteica , Tubulina (Proteína)/metabolismoRESUMO
Preterm birth (PTB) is the leading cause of neonatal mortality and morbidity, with few prevention and treatment options. Uterine contraction is a central feature of PTB, so gaining new insights into the mechanisms of this contraction and consequently identifying novel targets for tocolytics are essential for more successful management of PTB. Here we report that myometrial cells from human and mouse express bitter taste receptors (TAS2Rs) and their canonical signaling components (i.e., G-protein gustducin and phospholipase C ß2). Bitter tastants can completely relax myometrium precontracted by different uterotonics. In isolated single mouse myometrial cells, a phenotypical bitter tastant (chloroquine, ChQ) reverses the rise in intracellular Ca2+ concentration ([Ca2+]i) and cell shortening induced by uterotonics, and this reversal effect is inhibited by pertussis toxin and by genetic deletion of α-gustducin. In human myometrial cells, knockdown of TAS2R14 but not TAS2R10 inhibits ChQ's reversal effect on an oxytocin-induced rise in [Ca2+]i Finally, ChQ prevents mouse PTBs induced by bacterial endotoxin LPS or progesterone receptor antagonist mifepristone more often than current commonly used tocolytics, and this prevention is largely lost in α-gustducin-knockout mice. Collectively, our results reveal that activation of the canonical TAS2R signaling system in myometrial cells produces profound relaxation of myometrium precontracted by a broad spectrum of contractile agonists, and that targeting TAS2Rs is an attractive approach to developing effective tocolytics for PTB management.-Zheng, K., Lu, P., Delpapa, E., Bellve, K., Deng, R., Condon, J. C., Fogarty, K., Lifshitz, L. M., Simas, T. A. M., Shi, F., ZhuGe, R. Bitter taste receptors as targets for tocolytics in preterm labor therapy.
Assuntos
Regulação da Expressão Gênica/fisiologia , Miométrio/citologia , Trabalho de Parto Prematuro/tratamento farmacológico , Receptores Acoplados a Proteínas G/metabolismo , Albuterol , Animais , Cálcio/metabolismo , Cloroquina , Feminino , Humanos , Sulfato de Magnésio , Camundongos , Contração Muscular/efeitos dos fármacos , Contração Muscular/fisiologia , Músculo Liso/efeitos dos fármacos , Músculo Liso/fisiologia , Ocitocina/farmacologia , Fenantrolinas , Gravidez , Compostos de Amônio Quaternário , Receptores Acoplados a Proteínas G/genética , Transducina/genética , Transducina/metabolismoRESUMO
The dopamine (DA) transporter (DAT) facilitates high-affinity presynaptic DA reuptake that temporally and spatially constrains DA neurotransmission. Aberrant DAT function is implicated in attention-deficit/hyperactivity disorder and autism spectrum disorder. DAT is a major psychostimulant target, and psychostimulant reward strictly requires binding to DAT. DAT function is acutely modulated by dynamic membrane trafficking at the presynaptic terminal and a PKC-sensitive negative endocytic mechanism, or "endocytic brake," controls DAT plasma membrane stability. However, the molecular basis for the DAT endocytic brake is unknown, and it is unknown whether this braking mechanism is unique to DAT or common to monoamine transporters. Here, we report that the cdc42-activated, nonreceptor tyrosine kinase, Ack1, is a DAT endocytic brake that stabilizes DAT at the plasma membrane and is released in response to PKC activation. Pharmacologic and shRNA-mediated Ack1 silencing enhanced basal DAT internalization and blocked PKC-stimulated DAT internalization, but had no effects on SERT endocytosis. Both cdc42 activation and PKC stimulation converge on Ack1 to control Ack1 activity and DAT endocytic capacity, and Ack1 inactivation is required for stimulated DAT internalization downstream of PKC activation. Moreover, constitutive Ack1 activation is sufficient to rescue the gain-of-function endocytic phenotype exhibited by the ADHD DAT coding variant, R615C. These findings reveal a unique endocytic control switch that is highly specific for DAT. Moreover, the ability to rescue the DAT(R615C) coding variant suggests that manipulating DAT trafficking mechanisms may be a potential therapeutic approach to correct DAT coding variants that exhibit trafficking dysregulation.
Assuntos
Transtorno do Deficit de Atenção com Hiperatividade/genética , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Endocitose , Proteínas Tirosina Quinases/metabolismo , Membrana Celular/metabolismo , Clatrina/metabolismo , Neurônios Dopaminérgicos/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Modelos Biológicos , Mutação , Proteína Quinase C/metabolismo , Estabilidade Proteica , Transporte Proteico , RNA Interferente Pequeno/metabolismo , Serotonina/metabolismo , Proteína cdc42 de Ligação ao GTP/metabolismoRESUMO
Pompe disease is an autosomal recessive disorder caused by a deficiency of acid α-glucosidase (GAA), an enzyme responsible for hydrolyzing lysosomal glycogen. Deficiency of GAA leads to systemic glycogen accumulation in the lysosomes of skeletal muscle, motor neurons, and smooth muscle. Skeletal muscle and motor neuron pathology are known to contribute to respiratory insufficiency in Pompe disease, but the role of airway pathology has not been evaluated. Here we propose that GAA enzyme deficiency disrupts the function of the trachea and bronchi and this lower airway pathology contributes to respiratory insufficiency in Pompe disease. Using an established mouse model of Pompe disease, the Gaa-/- mouse, we compared histology, pulmonary mechanics, airway smooth muscle (ASM) function, and calcium signaling between Gaa-/- and age-matched wild-type (WT) mice. Lysosomal glycogen accumulation was observed in the smooth muscle of both the bronchi and the trachea in Gaa-/- but not WT mice. Furthermore, Gaa-/- mice had hyporesponsive airway resistance and bronchial ring contraction to the bronchoconstrictive agents methacholine (MCh) and potassium chloride (KCl) and to a bronchodilator (albuterol). Finally, calcium signaling during bronchiolar smooth muscle contraction was impaired in Gaa-/- mice indicating impaired extracellular calcium influx. We conclude that GAA enzyme deficiency leads to glycogen accumulation in the trachea and bronchi and impairs the ability of lower ASM to regulate calcium and respond appropriately to bronchodilator or constrictors. Accordingly, ASM dysfunction may contribute to respiratory impairments in Pompe disease.
Assuntos
Doença de Depósito de Glicogênio Tipo II/enzimologia , Doença de Depósito de Glicogênio Tipo II/fisiopatologia , Pulmão/enzimologia , Pulmão/patologia , Músculo Esquelético/enzimologia , Músculo Esquelético/fisiopatologia , alfa-Glucosidases/metabolismo , Albuterol/farmacologia , Animais , Brônquios/efeitos dos fármacos , Brônquios/fisiopatologia , Sinalização do Cálcio/efeitos dos fármacos , Espaço Extracelular/metabolismo , Glicogênio/metabolismo , Doença de Depósito de Glicogênio Tipo II/patologia , Pulmão/efeitos dos fármacos , Pulmão/fisiopatologia , Cloreto de Metacolina/farmacologia , Camundongos , Contração Muscular/efeitos dos fármacos , Músculo Esquelético/efeitos dos fármacos , Cloreto de Potássio/farmacologia , Traqueia/efeitos dos fármacos , Traqueia/fisiopatologiaRESUMO
The protein nephrocystin-4 (NPHP4) is widespread in ciliated organisms, and defects in NPHP4 cause nephronophthisis and blindness in humans. To learn more about the function of NPHP4, we have studied it in Chlamydomonas reinhardtii. NPHP4 is stably incorporated into the distal part of the flagellar transition zone, close to the membrane and distal to CEP290, another transition zone protein. Therefore, these two proteins, which are incorporated into the transition zone independently of each other, define different domains of the transition zone. An nphp4-null mutant forms flagella with nearly normal length, ultrastructure and intraflagellar transport. When fractions from isolated wild-type and nphp4 flagella were compared, few differences were observed between the axonemes, but the amounts of certain membrane proteins were greatly reduced in the mutant flagella, and cellular housekeeping proteins >50â kDa were no longer excluded from mutant flagella. Therefore, NPHP4 functions at the transition zone as an essential part of a barrier that regulates both membrane and soluble protein composition of flagella. The phenotypic consequences of NPHP4 mutations in humans likely follow from protein mislocalization due to defects in the transition zone barrier.
Assuntos
Chlamydomonas reinhardtii/metabolismo , Cílios/metabolismo , Flagelos/metabolismo , Proteínas de Membrana/metabolismo , Movimento Celular/fisiologia , Transporte Proteico/fisiologiaRESUMO
AIMS/HYPOTHESIS: During pregnancy, adipose tissue (AT) must expand to support the growing fetus and the future nutritional needs of the offspring. Limited expandability of AT is associated with insulin resistance, attributed to ectopic lipid deposition. This study aimed to investigate human AT expandability during pregnancy and its role in the pathogenesis of gestational diabetes mellitus (GDM). METHODS: This cross-sectional study of omental (OM) and subcutaneous (SQ) AT collected at Caesarean delivery included 11 pregnant and three non-pregnant women with normal glucose tolerance (NGT), five with GDM, three with type 2 diabetes mellitus. Adipocyte size, capillary density, collagen content and capillary growth were measured. Affymetrix arrays and real-time PCR studies of gene expression were performed. RESULTS: Mean OM adipocyte size was greater in women with GDM than in those with NGT (p = 0.004). Mean OM and SQ capillary density was lower in GDM compared with NGT (p = 0.015). Capillary growth did not differ significantly between groups. The most differentially expressed AT transcript when comparing non-pregnant and pregnant women corresponded to the IGF binding protein (IGFBP)-5, the expression levels of which was found by subsequent quantitative real-time PCR to be lower in women with GDM vs women with NGT (p < 0.0001). CONCLUSIONS/INTERPRETATION: The relative OM adipocyte hypertrophy and decreased OM and SQ capillary density are consistent with impaired AT expandability in GDM. The induction of adipose tissue IGFBP5 in pregnancy and its decrease in GDM point to the importance of the IGF-1 signalling pathway in AT expansion in pregnancy and GDM susceptibility.
Assuntos
Adipogenia , Tecido Adiposo/fisiopatologia , Diabetes Gestacional/fisiopatologia , Adipócitos/citologia , Adiposidade , Adulto , Glicemia/análise , Capilares/metabolismo , Estudos Transversais , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Teste de Tolerância a Glucose , Humanos , Proteína 5 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Fator de Crescimento Insulin-Like II/metabolismo , Neovascularização Fisiológica , Análise de Sequência com Séries de Oligonucleotídeos , Gravidez , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Cell surface receptors and other proteins internalize through diverse mechanisms at the plasma membrane and are sorted to different destinations. Different subpopulations of early endosomes have been described, raising the question of whether different internalization mechanisms deliver cargo into different subsets of early endosomes. To address this fundamental question, we developed a microscopy platform to detect the precise position of endosomes relative to the plasma membrane during the uptake of ligands. Axial resolution is maximized by concurrently applied total internal reflection fluorescence and epifluorescence-structured light. We found that transferrin receptors are delivered selectively from clathrin-coated pits on the plasma membrane into a specific subpopulation of endosomes enriched in the multivalent Rab GTPase and phosphoinositide-binding protein Rabenosyn-5. Depletion of Rabenosyn-5, but not of other early endosomal proteins such as early endosome antigen 1, resulted in impaired transferrin uptake and lysosomal degradation of transferrin receptors. These studies reveal a critical role for Rabenosyn-5 in determining the fate of transferrin receptors internalized by clathrin-mediated endocytosis and, more broadly, a mechanism whereby the delivery of cargo from the plasma membrane into specific early endosome subpopulations is required for its appropriate intracellular traffic.
Assuntos
Clatrina/metabolismo , Endocitose , Receptores da Transferrina/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Animais , Células COS , Membrana Celular/metabolismo , Chlorocebus aethiops , Endossomos/metabolismo , Humanos , Transporte Proteico , Fatores de TempoRESUMO
The type 1 ryanodine receptor (RyR1) is expressed widely in the brain, with high levels in the cerebellum, hippocampus, and hypothalamus. We have shown that L-type Ca(2+) channels in terminals of hypothalamic magnocellular neurons are coupled to RyRs, as they are in skeletal muscle, allowing voltage-induced Ca(2+) release (VICaR) from internal Ca(2+) stores without Ca(2+) influx. Here we demonstrate that RyR1 plays a role in VICaR in nerve terminals. Furthermore, in heterozygotes from the Ryr1(I4895T/WT) (IT/+) mouse line, carrying a knock-in mutation corresponding to one that causes a severe form of human central core disease, VICaR is absent, demonstrating that type 1 RyR mediates VICaR and that these mice have a neuronal phenotype. The absence of VICaR was shown in two ways: first, depolarization in the absence of Ca(2+) influx elicited Ca(2+)syntillas (scintilla, spark, in a nerve terminal, a SYNaptic structure) in WT, but not in mutant terminals; second, in the presence of extracellular Ca(2+), IT/+ terminals showed a twofold decrease in global Ca(2+) transients, with no change in plasmalemmal Ca(2+) current. From these studies we draw two conclusions: (i) RyR1 plays a role in VICaR in hypothalamic nerve terminals; and (ii) a neuronal alteration accompanies the myopathy in IT/+ mice, and, possibly in humans carrying the corresponding RyR1 mutation.
Assuntos
Canais de Cálcio Tipo L/metabolismo , Cálcio/metabolismo , Hipotálamo/citologia , Miopatia da Parte Central/genética , Neurônios/metabolismo , Terminações Pré-Sinápticas/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Animais , Fluorescência , Técnicas de Introdução de Genes , Hipotálamo/metabolismo , Camundongos , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismoRESUMO
Dopaminergic signaling profoundly impacts rewarding behaviors, movement, and executive function. The presynaptic dopamine (DA) transporter (DAT) recaptures released DA, thereby limiting synaptic DA availability and maintaining dopaminergic tone. DAT constitutively internalizes and PKC activation rapidly accelerates DAT endocytosis, resulting in DAT surface loss. Longstanding evidence supports PKC-stimulated DAT trafficking in heterologous expression studies. However, PKC-stimulated DAT internalization is not readily observed in cultured dopaminergic neurons. Moreover, conflicting reports implicate both classic and nonclassic endocytic mechanisms mediating DAT trafficking. Prior DAT trafficking studies relied primarily upon chronic gene disruption and dominant-negative protein expression, or were performed in cell lines and cultured neurons, yielding results difficult to translate to adult dopaminergic neurons. Here, we use newly described dynamin inhibitors to test whether constitutive and PKC-stimulated DAT internalization are dynamin-dependent in adult dopaminergic neurons. Ex vivo biotinylation studies in mouse striatal slices demonstrate that acute PKC activation drives native DAT surface loss, and that surface DAT surprisingly partitions between endocytic-willing and endocytic-resistant populations. Acute dynamin inhibition reveals that constitutive DAT internalization is dynamin-independent, whereas PKC-stimulated DAT internalization is dynamin-dependent. Moreover, total internal reflection fluorescence microscopy experiments demonstrate that constitutive DAT internalization occurs equivalently from lipid raft and nonraft microdomains, whereas PKC-stimulated DAT internalization arises exclusively from lipid rafts. Finally, DAT endocytic recycling relies on a dynamin-dependent mechanism that acts in concert with the actin cytoskeleton. These studies are the first comprehensive investigation of native DAT trafficking in ex vivo adult neurons, and reveal that DAT surface dynamics are governed by complex multimodal mechanisms.
Assuntos
Corpo Estriado/metabolismo , Citoesqueleto/metabolismo , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Neurônios Dopaminérgicos/metabolismo , Dinaminas/metabolismo , Endocitose/fisiologia , Animais , Linhagem Celular Tumoral , Corpo Estriado/citologia , Neurônios Dopaminérgicos/citologia , Humanos , Masculino , Camundongos , Transporte Proteico/fisiologiaRESUMO
Adrenal chromaffin cells (ACCs), stimulated by the splanchnic nerve, generate action potentials (APs) at a frequency near 0.5 Hz in the resting physiological state, at times described as 'rest and digest'. How such low frequency stimulation in turn elicits sufficient catecholamine exocytosis to set basal sympathetic tone is not readily explained by the classical mechanism of stimulus-secretion coupling, where exocytosis is synchronized to AP-induced Ca(2+) influx. By using simulated action potentials (sAPs) at 0.5 Hz in isolated patch-clamped mouse ACCs, we show here that less than 10% of all catecholaminergic exocytosis, measured by carbon fibre amperometry, is synchronized to an AP. The asynchronous phase, the dominant phase, of exocytosis does not require Ca(2+) influx. Furthermore, increased asynchronous exocytosis is accompanied by an AP-dependent decrease in frequency of Ca(2+) syntillas (i.e. transient, focal Ca(2+) release from internal stores) and is ryanodine sensitive. We propose a mechanism of disinhibition, wherein APs suppress Ca(2+) syntillas, which themselves inhibit exocytosis as they do in the case of spontaneous catecholaminergic exocytosis.
Assuntos
Glândulas Suprarrenais/citologia , Sinalização do Cálcio/fisiologia , Cálcio/metabolismo , Catecolaminas/metabolismo , Células Cromafins/metabolismo , Potenciais de Ação , Animais , Células Cultivadas , Masculino , Camundongos , Técnicas de Patch-Clamp , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismoRESUMO
Maintenance of fecal continence requires a continuous or basal tone of the internal anal sphincter (IAS). Paradoxically, the basal tone results largely from high-frequency rhythmic contractions of the IAS smooth muscle. However, the cellular and molecular mechanisms that initiate these contractions remain elusive. Here we show that the IAS contains multiple pacemakers. These pacemakers spontaneously generate propagating calcium waves that drive rhythmic contractions and establish the basal tone. These waves are myogenic and act independently of nerve, paracrine or autocrine signals. Using cell-specific gene knockout mice, we further found that TMEM16A Cl- channels in smooth muscle cells (but not in the interstitial cells of Cajal) are indispensable for pacemaking, rhythmic contractions, and basal tone. Our results identify TMEM16A in smooth muscle cells as a critical pacemaker channel that enables the IAS to contract rhythmically and continuously. This study provides cellular and molecular insights into fecal continence.
Assuntos
Canal Anal , Anoctamina-1 , Contração Muscular , Animais , Camundongos , Canal Anal/inervação , Canal Anal/fisiologia , Contração Muscular/fisiologia , Músculo Liso/fisiologia , Miócitos de Músculo Liso , Anoctamina-1/fisiologiaRESUMO
In addition to its role as a morphogen, Sonic hedgehog (Shh) has also been shown to function as a guidance factor that directly acts on the growth cones of various types of axons. However, the noncanonical signaling pathways that mediate the guidance effects of Shh protein remain poorly understood. We demonstrate that a novel signaling pathway consisting of protein kinase Cα (PKCα) and integrin-linked kinase (ILK) mediates the negative guidance effects of high concentration of Shh on retinal ganglion cell (RGC) axons. Shh rapidly increased Ca(2+) level and activated PKCα and ILK in the growth cones of RGC axons. By in vitro kinase assay, PKCα was found to directly phosphorylate ILK on threonine-173 and -181. Inhibition of PKCα or expression of a mutant ILK with the PKCα phosphorylation sites mutated (ILK-DM), abolished the Shh-induced macropinocytosis, growth cone collapse and repulsive axon turning. In vivo, expression of a dominant negative PKCα or ILK-DM disrupted RGC axon pathfinding at the optic chiasm but not the projection toward the optic disk, supporting that this signaling pathway plays a specific role in Shh-mediated negative guidance effects.
Assuntos
Axônios/enzimologia , Proteínas Hedgehog/metabolismo , Proteína Quinase C-alfa/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Acetofenonas/farmacologia , Animais , Axônios/fisiologia , Benzopiranos/farmacologia , Cálcio/metabolismo , Células Cultivadas , Embrião de Galinha , Inibidores Enzimáticos/farmacologia , Cones de Crescimento/enzimologia , Mutação , Fosforilação , Proteína Quinase C-alfa/antagonistas & inibidores , Proteína Quinase C-alfa/genética , Proteínas Serina-Treonina Quinases/genética , Células Ganglionares da Retina/citologia , Células Ganglionares da Retina/enzimologia , TreoninaRESUMO
Adenomyosis is a debilitating gynecological disease of the uterus with no medicinal cure. The tissue injury and repair hypothesis for adenomyosis suggests that uterine hyperperistalsis or dysperistalsis plays a pivotal role in establishing adenomyotic lesions. However, specific impairments in uterine peristalsis and the underlying cellular signals for these changes in adenomyosis remain elusive. Here, we report a precision-cut uterine slice preparation that preserves in vivo uterine architecture and generates peristalsis similar to that seen in the whole uterus. We found that uterine peristalsis in neonatal mice at day 14 and adult mice at day 55 presents as bursts with multiple peaks induced by intracellular Ca2+ oscillations. Using a mouse model of adenomyosis induced by tamoxifen, a selective estrogen receptor modulator, we discovered that uterine peristalsis and Ca2+ oscillations from adenomyotic uteri on days 14 and 55 become spikes (single peaks) with smaller amplitudes. The peak frequency of Ca2+ oscillations or peristalsis does not show a difference between control and adenomyotic mice. However, both the estimated force generated by uterine peristalsis and the total Ca2+ raised by Ca2+ oscillations are smaller in uteri from adenomyotic mice. Uteri from adenomyotic mice on day 14, but not on day 55, exhibit hyperresponsiveness to oxytocin. Embryo implantations are decreased in adenomyotic adult mice. Our results reveal a mode switch from bursts to spikes (rather than an increased peak frequency) of uterine Ca2+ oscillations and peristalsis and concurrent hyperresponsiveness to oxytocin in the neonatal stage are two characteristics of adenomyosis. These characteristics may contribute to embryo implantation impairments and decreased fertility in adenomyosis.
RESUMO
Macropinocytosis is a type of poorly characterized fluid-phase endocytosis that results in formation of relatively large vesicles. We report that Sonic hedgehog (Shh) protein induces macropinocytosis in the axons through activation of a noncanonical signaling pathway, including Rho GTPase and nonmuscle myosin II. Macropinocytosis induced by Shh is independent of clathrin-mediated endocytosis but dependent on dynamin, myosin II, and Rho GTPase activities. Inhibitors of macropinocytosis also abolished the negative effects of Shh on axonal growth, including growth cone collapse and chemorepulsive axon turning but not turning per se. Conversely, activation of myosin II or treatment of phorbol ester induces macropinocytosis in the axons and elicits growth cone collapse and repulsive axon turning. Furthermore, macropinocytosis is also induced by ephrin-A2, and inhibition of dynamin abolished repulsive axon turning induced by ephrin-A2. Macropinocytosis can be induced ex vivo by high Shh, correlating with axon retraction. These results demonstrate that macropinocytosis-mediated membrane trafficking is an important cellular mechanism involved in axon chemorepulsion induced by negative guidance factors.
Assuntos
Axônios/fisiologia , Cones de Crescimento/fisiologia , Pinocitose/fisiologia , Animais , Axônios/efeitos dos fármacos , Células Cultivadas , Embrião de Galinha , Dextranos/metabolismo , Relação Dose-Resposta a Droga , Interações Medicamentosas , Inibidores Enzimáticos/farmacologia , Proteínas de Fluorescência Verde/genética , Cones de Crescimento/efeitos dos fármacos , Proteínas Hedgehog/farmacologia , Técnicas In Vitro , Miosina Tipo II/metabolismo , Pinocitose/efeitos dos fármacos , Células Ganglionares da Retina/citologia , Acetato de Tetradecanoilforbol/farmacologia , Fatores de Tempo , Transfecção , Transferrina/metabolismo , Alcaloides de Veratrum/farmacologia , Proteínas rho de Ligação ao GTP/metabolismoRESUMO
Total internal reflection fluorescence (TIRF) microscopy reveals highly mobile structures containing enhanced green fluorescent protein-tagged glucose transporter 4 (GLUT4) within a zone about 100 nm beneath the plasma membrane of 3T3-L1 adipocytes. We developed a computer program (Fusion Assistant) that enables direct analysis of the docking/fusion kinetics of hundreds of exocytic fusion events. Insulin stimulation increases the fusion frequency of exocytic GLUT4 vesicles by approximately 4-fold, increasing GLUT4 content in the plasma membrane. Remarkably, insulin signaling modulates the kinetics of the fusion process, decreasing the vesicle tethering/docking duration prior to membrane fusion. In contrast, the kinetics of GLUT4 molecules spreading out in the plasma membrane from exocytic fusion sites is unchanged by insulin. As GLUT4 accumulates in the plasma membrane, it is also immobilized in punctate structures on the cell surface. A previous report suggested these structures are exocytic fusion sites (Lizunov et al., J. Cell Biol. 169:481-489, 2005). However, two-color TIRF microscopy using fluorescent proteins fused to clathrin light chain or GLUT4 reveals these structures are clathrin-coated patches. Taken together, these data show that insulin signaling accelerates the transition from docking of GLUT4-containing vesicles to their fusion with the plasma membrane and promotes GLUT4 accumulation in clathrin-based endocytic structures on the plasma membrane.