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1.
Physiol Rev ; 102(1): 7-60, 2022 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-33880962

RESUMO

The spermatozoon is a highly differentiated and polarized cell, with two main structures: the head, containing a haploid nucleus and the acrosomal exocytotic granule, and the flagellum, which generates energy and propels the cell; both structures are connected by the neck. The sperm's main aim is to participate in fertilization, thus activating development. Despite this common bauplan and function, there is an enormous diversity in structure and performance of sperm cells. For example, mammalian spermatozoa may exhibit several head patterns and overall sperm lengths ranging from ∼30 to 350 µm. Mechanisms of transport in the female tract, preparation for fertilization, and recognition of and interaction with the oocyte also show considerable variation. There has been much interest in understanding the origin of this diversity, both in evolutionary terms and in relation to mechanisms underlying sperm differentiation in the testis. Here, relationships between sperm bauplan and function are examined at two levels: first, by analyzing the selective forces that drive changes in sperm structure and physiology to understand the adaptive values of this variation and impact on male reproductive success and second, by examining cellular and molecular mechanisms of sperm formation in the testis that may explain how differentiation can give rise to such a wide array of sperm forms and functions.


Assuntos
Exocitose/fisiologia , Interações Espermatozoide-Óvulo/fisiologia , Espermatozoides/fisiologia , Testículo/citologia , Animais , Evolução Biológica , Humanos , Masculino , Mamíferos/fisiologia , Espermatozoides/citologia
2.
Int J Mol Sci ; 22(24)2021 Dec 17.
Artigo em Inglês | MEDLINE | ID: mdl-34948351

RESUMO

The polybasic juxtamembrane region (5RK) of the plasma membrane neuronal SNARE, syntaxin1A (Syx), was previously shown by us to act as a fusion clamp in PC12 cells, as charge neutralization of 5RK promotes spontaneous and inhibits Ca2+-triggered release. Using a Syx-based FRET probe (CSYS), we demonstrated that 5RK is required for a depolarization-induced Ca+2-dependent opening (close-to-open transition; CDO) of Syx, which involves the vesicular SNARE synaptobrevin2 and occurs concomitantly with Ca2+-triggered release. Here, we investigated the mechanism underlying the CDO requirement for 5RK and identified phosphorylation of Syx at Ser-14 (S14) by casein kinase 2 (CK2) as a crucial molecular determinant. Thus, following biochemical verification that both endogenous Syx and CSYS are constitutively S14 phosphorylated in PC12 cells, dynamic FRET analysis of phospho-null and phospho-mimetic mutants of CSYS and the use of a CK2 inhibitor revealed that the S14 phosphorylation confers the CDO requirement for 5RK. In accord, amperometric analysis of catecholamine release revealed that the phospho-null mutant does not support Ca2+-triggered release. These results identify a functionally important CK2 phosphorylation of Syx that is required for the 5RK-regulation of CDO and for concomitant Ca2+-triggered release. Further, also spontaneous release, conferred by charge neutralization of 5RK, was abolished in the phospho-null mutant.


Assuntos
Cálcio/metabolismo , Caseína Quinase II/metabolismo , Células Neuroendócrinas/metabolismo , Sintaxina 1/metabolismo , Animais , Células Cultivadas , Exocitose , Células Neuroendócrinas/citologia , Células PC12 , Fosforilação , Ratos , Sintaxina 1/química , Xenopus
3.
Artigo em Inglês | MEDLINE | ID: mdl-34620619

RESUMO

INTRODUCTION: Functional impairment of the stimulus secretion coupling in pancreatic beta cells is an essential component of type 2 diabetes. It is known that prolonged stimulation desensitizes the secretion of insulin and thus contributes to beta cell dysfunction. Beta cell rest, in contrast, was shown to enhance the secretory response. Here, the underlying mechanisms were investigated. RESEARCH DESIGN AND METHODS: To characterize the consequences of desensitization or rest for the number and mobility of submembrane granules, insulin-secreting MIN6 cells were desensitized by 18-hour culture with 500 µM tolbutamide or rested by 18-hour culture with 1 µM clonidine. The granules were labeled by hIns-EGFP or hIns-DsRed E5, imaged by TIRF microscopy of the cell footprint area and analyzed with an observer-independent program. Additionally, the insulin content and secretion were measured. RESULTS: Concurrent with the insulin content, submembrane granules were only slightly reduced after desensitization but markedly increased after rest. Both types of pretreatment diminished arrivals and departures of granules in the submembrane space and increased the proportion of immobile long-term resident granules, but desensitization lowered and rest increased the number of exocytoses, in parallel with the effect on insulin secretion. Labeling with hIns-DsRed E5 ('timer') showed that desensitization did not affect the proportion of aged granules, whereas rest increased it. Aged granules showed a high mobility and made up only a minority of long-term residents. Long-term resident granules were more numerous after rest and had a lower lateral mobility, suggesting a firmer attachment to the membrane. CONCLUSION: The number, mobility and age of submembrane granules reflect the preceding functional states of insulin-secreting cells. Representing the pool of releasable granules, their quantity and quality may thus form part of the beta cell memory on renewed stimulation.


Assuntos
Diabetes Mellitus Tipo 2 , Células Secretoras de Insulina , Idoso , Diabetes Mellitus Tipo 2/metabolismo , Exocitose , Humanos , Insulina/metabolismo , Secreção de Insulina , Células Secretoras de Insulina/metabolismo
4.
Int J Mol Sci ; 22(19)2021 Oct 06.
Artigo em Inglês | MEDLINE | ID: mdl-34639129

RESUMO

Multiple sclerosis (MS) is an inflammatory disease of the central nervous system that finally leads to demyelination. Demyelinating optic neuritis is a frequent symptom in MS. Recent studies also revealed synapse dysfunctions in MS patients and MS mouse models. We previously reported alterations of photoreceptor ribbon synapses in the experimental auto-immune encephalomyelitis (EAE) mouse model of MS. In the present study, we found that the previously observed decreased imunosignals of photoreceptor ribbons in early EAE resulted from a decrease in synaptic ribbon size, whereas the number/density of ribbons in photoreceptor synapses remained unchanged. Smaller photoreceptor ribbons are associated with fewer docked and ribbon-associated vesicles. At a functional level, depolarization-evoked exocytosis as monitored by optical recording was diminished even as early as on day 7 after EAE induction. Moreover compensatory, post-depolarization endocytosis was decreased. Decreased post-depolarization endocytosis in early EAE correlated with diminished synaptic enrichment of dynamin3. In contrast, basal endocytosis in photoreceptor synapses of resting non-depolarized retinal slices was increased in early EAE. Increased basal endocytosis correlated with increased de-phosphorylation of dynamin1. Thus, multiple endocytic pathways in photoreceptor synapse are differentially affected in early EAE and likely contribute to the observed synapse pathology in early EAE.


Assuntos
Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/patologia , Endocitose , Exocitose , Esclerose Múltipla/patologia , Células Fotorreceptoras Retinianas Bastonetes/patologia , Sinapses/patologia , Animais , Dinaminas/metabolismo , Encefalomielite Autoimune Experimental/etiologia , Encefalomielite Autoimune Experimental/metabolismo , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Esclerose Múltipla/etiologia , Esclerose Múltipla/metabolismo , Fosforilação , Retina/metabolismo , Retina/patologia , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Sinapses/metabolismo , Vesículas Sinápticas/metabolismo , Vesículas Sinápticas/patologia
5.
Nat Commun ; 12(1): 5740, 2021 09 30.
Artigo em Inglês | MEDLINE | ID: mdl-34593806

RESUMO

NG2 glia, also known as oligodendrocyte precursor cells (OPCs), play an important role in proliferation and give rise to myelinating oligodendrocytes during early brain development. In contrast to other glial cell types, the most intriguing aspect of NG2 glia is their ability to directly sense synaptic inputs from neurons. However, whether this synaptic interaction is bidirectional or unidirectional, or its physiological relevance has not yet been clarified. Here, we report that NG2 glia form synaptic complexes with hippocampal interneurons and that selective photostimulation of NG2 glia (expressing channelrhodopsin-2) functionally drives GABA release and enhances inhibitory synaptic transmission onto proximal interneurons in a microcircuit. The mechanism involves GAD67 biosynthesis and VAMP-2 containing vesicular exocytosis. Further, behavioral assays demonstrate that NG2 glia photoactivation triggers anxiety-like behavior in vivo and contributes to chronic social defeat stress.


Assuntos
Ansiedade/psicologia , Hipocampo/patologia , Células Precursoras de Oligodendrócitos/metabolismo , Estresse Psicológico/complicações , Ácido gama-Aminobutírico/metabolismo , Animais , Ansiedade/etiologia , Ansiedade/patologia , Diferenciação Celular , Modelos Animais de Doenças , Exocitose , Glutamato Descarboxilase/biossíntese , Hipocampo/citologia , Humanos , Interneurônios/patologia , Masculino , Camundongos , Camundongos Transgênicos , Técnicas de Patch-Clamp , Derrota Social , Estresse Psicológico/patologia , Estresse Psicológico/psicologia , Sinapses/patologia , Transmissão Sináptica/fisiologia , Proteína 2 Associada à Membrana da Vesícula/metabolismo
6.
Int J Mol Sci ; 22(19)2021 Oct 06.
Artigo em Inglês | MEDLINE | ID: mdl-34639144

RESUMO

Parkinson disease protein 7 (PARK7) is a multifunctional protein known to be involved in the regulation of sperm motility, mitochondrial function, and oxidative stress response in mammalian sperm. While ROS generation is needed to activate the downstream signaling pathways required for sperm to undergo capacitation, oxidative stress has detrimental effects for sperm cells and a precise balance between ROS levels and antioxidant activity is needed. Considering the putative antioxidant role of PARK7, the present work sought to determine whether this protein is related to the sperm ability to withstand in vitro capacitation. To this end, and using the pig as a model, semen samples were incubated in capacitation medium for 300 min; the acrosomal exocytosis was triggered by the addition of progesterone after 240 min of incubation. At each relevant time point (0, 120, 240, 250, and 300 min), sperm motility, acrosome and plasma membrane integrity, membrane lipid disorder, mitochondrial membrane potential, intracellular calcium and ROS were evaluated. In addition, localization and protein levels of PARK7 were also assessed through immunofluorescence and immunoblotting. Based on the relative content of PARK7, two groups of samples were set. As early as 120 min of incubation, sperm samples with larger PARK7 content showed higher percentages of viable and acrosome-intact sperm, lipid disorder and superoxide levels, and lower intracellular calcium levels when compared to sperm samples with lower PARK7. These data suggest that PARK7 could play a role in preventing sperm from undergoing premature capacitation, maintaining sperm viability and providing a better ability to keep ROS homeostasis, which is needed to elicit sperm capacitation. Further studies are required to elucidate the antioxidant properties of PARK7 during in vitro capacitation and acrosomal exocytosis of mammalian sperm, and the relationship between PARK7 and sperm motility.


Assuntos
Reação Acrossômica , Exocitose , Potencial da Membrana Mitocondrial , Proteína Desglicase DJ-1/metabolismo , Capacitação Espermática , Motilidade Espermática , Animais , Cálcio/metabolismo , Masculino , Lipídeos de Membrana/metabolismo , Progesterona/farmacologia , Proteína Desglicase DJ-1/genética , Transdução de Sinais , Superóxidos/metabolismo , Suínos
7.
Colloids Surf B Biointerfaces ; 208: 112140, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34597939

RESUMO

Semiconductor quantum dots (QDs) have been extensively explored for extensive bioapplications, yet their cellular fate, especially exocytosis, has not been thoroughly investigated. Herein, we systematically investigated the whole cellular process from the endocytosis, intercellular trafficking, to the exocytosis of a typical QD, core/shell CdSe/ZnS QD. Using confocal laser scanning microscopy and flow cytometry, and after carefully eliminating the effect of cell division, we found that the QDs were internalized by HeLa cells with a time-, dose-, and serum-dependent manner. The cellular uptake was inhibited by serum, but eventually peaked after 4-6 h incubation with or without serum. The primary endocytosis pathway was clathrin-mediated, and actin- and microtubule-dependent in the medium with serum, while the caveolae-mediated endocytosis and macropinocytosis were more important for the QDs in the serum-free medium. Inside cells, most QDs distributed in lysosomes, and some entered mitochondria, endoplasmic reticulum, and Golgi apparatus. The translocation of the QDs from other organelles to Golgi apparatus was observed. The exocytosis of QDs was faster than the endocytosis, reaching the maximum in about one hour after cultured in fresh culture medium, with around 60% of the internalized QDs remained undischarged. The exocytosis process was energy- and actin-dependent, and the lysosome exocytosis and endoplasmic reticulum/Golgi pathway were the main routes. This study provides a full picture of behavior and fate of QDs in cells, which may facilitate the design of ideal QDs applied in biomedical and other fields.


Assuntos
Compostos de Cádmio , Pontos Quânticos , Compostos de Selênio , Endocitose , Exocitose , Células HeLa , Humanos , Sulfetos , Compostos de Zinco
8.
J Vis Exp ; (175)2021 09 11.
Artigo em Inglês | MEDLINE | ID: mdl-34570110

RESUMO

Timelapse TIRF microscopy of pH-sensitive GFP (pHluorin) attached to vesicle SNARE proteins is an effective method to visualize single vesicle exocytic events in cell culture. To perform an unbiased, efficient identification and analysis of such events, a computer-vision based approach was developed and implemented in MATLAB. The analysis pipeline consists of a cell segmentation and exocytic-event identification algorithm. The computer-vision approach includes tools for investigating multiple parameters of single events, including the half-life of fluorescence decay and peak ΔF/F, as well as whole-cell analysis of the frequency of exocytosis. These and other parameters of fusion are used in a classification approach to distinguish distinct fusion modes. Here a newly built GUI performs the analysis pipeline from start to finish. Further adaptation of Ripley's K function in R Studio is used to distinguish between clustered, dispersed, or random occurrence of fusion events in both space and time.


Assuntos
Exocitose , Proteínas SNARE , Membrana Celular
9.
Elife ; 102021 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-34533135

RESUMO

Cilia are sensory organelles protruding from cell surfaces. Release of extracellular vesicles (EVs) from cilia was previously observed in mammals, Chlamydomonas, and in male Caenorhabditis elegans. Using the EV marker TSP-6 (an ortholog of mammalian CD9) and other ciliary receptors, we show that EVs are formed from ciliated sensory neurons in C. elegans hermaphrodites. Release of EVs is observed from two ciliary locations: the cilia tip and/or periciliary membrane compartment (PCMC). Outward budding of EVs from the cilia tip leads to their release into the environment. EVs' budding from the PCMC is concomitantly phagocytosed by the associated glial cells. To maintain cilia composition, a tight regulation of cargo import and removal is achieved by the action of intra-flagellar transport (IFT). Unbalanced IFT due to cargo overexpression or mutations in the IFT machinery leads to local accumulation of ciliary proteins. Disposal of excess ciliary proteins via EVs reduces their local accumulation and exports them to the environment and/or to the glia associated to these ciliated neurons. We suggest that EV budding from cilia subcompartments acts as a safeguard mechanism to remove deleterious excess of ciliary material.


Assuntos
Caenorhabditis elegans/genética , Caenorhabditis elegans/fisiologia , Cílios/metabolismo , Exocitose , Vesículas Extracelulares/fisiologia , Células Receptoras Sensoriais/fisiologia , Animais , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Transporte Proteico
10.
Elife ; 102021 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-34543184

RESUMO

Synaptotagmin 7 (SYT7) has emerged as a key regulator of presynaptic function, but its localization and precise role in the synaptic vesicle cycle remain the subject of debate. Here, we used iGluSnFR to optically interrogate glutamate release, at the single-bouton level, in SYT7KO-dissociated mouse hippocampal neurons. We analyzed asynchronous release, paired-pulse facilitation, and synaptic vesicle replenishment and found that SYT7 contributes to each of these processes to different degrees. 'Zap-and-freeze' electron microscopy revealed that a loss of SYT7 diminishes docking of synaptic vesicles after a stimulus and inhibits the recovery of depleted synaptic vesicles after a stimulus train. SYT7 supports these functions from the axonal plasma membrane, where its localization and stability require both γ-secretase-mediated cleavage and palmitoylation. In summary, SYT7 is a peripheral membrane protein that controls multiple modes of synaptic vesicle (SV) exocytosis and plasticity, in part, through enhancing activity-dependent docking of SVs.


Assuntos
Secretases da Proteína Precursora do Amiloide/metabolismo , Axônios/enzimologia , Membrana Celular/enzimologia , Hipocampo/enzimologia , Vesículas Sinápticas/enzimologia , Sinaptotagminas/metabolismo , Animais , Axônios/ultraestrutura , Membrana Celular/ultraestrutura , Células Cultivadas , Exocitose , Hipocampo/ultraestrutura , Lipoilação , Camundongos Knockout , Simulação de Acoplamento Molecular , Plasticidade Neuronal , Processamento de Proteína Pós-Traducional , Transporte Proteico , Proteólise , Ratos Sprague-Dawley , Transmissão Sináptica , Vesículas Sinápticas/ultraestrutura , Sinaptotagminas/genética , Fatores de Tempo
11.
Int J Mol Sci ; 22(17)2021 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-34502090

RESUMO

Polycystic ovary syndrome (PCOS) is the most common endocrine disorder in women. Previous studies have demonstrated the therapeutic efficacy of human bone marrow mesenchymal stem cells (BM-hMSCs) for PCOS; however, the regulatory mechanism remains unknown. Bone morphogenetic proteins (BMPs) secreted by BM-hMSCs may underlie the therapeutic effect of these cells on PCOS, based on the ability of BMPs to modulate androgen production and alter steroidogenesis pathway enzymes. In this study, we analyze the effect of BMP-2 on androgen production and steroidogenic pathway enzymes in H295R cells as a human PCOS in vitro cell model. In H295R cells, BMP-2 significantly suppressed cell proliferation, androgen production, and expression of androgen-synthesizing genes, as well as inflammatory gene expression. Furthermore, H295R cells treated with the BM-hMSCs secretome in the presence of neutralizing BMP-2 antibody or with BMP-2 gene knockdown showed augmented expression of androgen-producing genes. Taken together, these results indicate that BMP-2 is a key player mediating the favorable effects of the BM-hMSCs secretome in a human PCOS cell model. BMP-2 overexpression could increase the efficacy of BM-hMSC-based therapy, serving as a novel stem cell therapy for patients with intractable PCOS.


Assuntos
Androgênios/metabolismo , Proteína Morfogenética Óssea 2/farmacologia , Síndrome do Ovário Policístico/metabolismo , Células Tecais/metabolismo , Adulto , Animais , Linhagem Celular Tumoral , Proliferação de Células , Células Cultivadas , Meios de Cultivo Condicionados/farmacologia , Exocitose , Feminino , Humanos , Células-Tronco Mesenquimais/metabolismo , Camundongos , Células Tecais/efeitos dos fármacos , Células Tecais/fisiologia
13.
J Vis Exp ; (173)2021 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-34338673

RESUMO

Before neuronal degeneration, the cause of motor and cognitive deficits in patients with amyotrophic lateral sclerosis (ALS) and/or frontotemporal lobe dementia (FTLD) is dysfunction of communication between neurons and motor neurons and muscle. The underlying process of synaptic transmission involves membrane depolarization-dependent synaptic vesicle fusion and the release of neurotransmitters into the synapse. This process occurs through localized calcium influx into the presynaptic terminals where synaptic vesicles reside. Here, the protocol describes fluorescence-based live-imaging methodologies that reliably report depolarization-mediated synaptic vesicle exocytosis and presynaptic terminal calcium influx dynamics in cultured neurons. Using a styryl dye that is incorporated into synaptic vesicle membranes, the synaptic vesicle release is elucidated. On the other hand, to study calcium entry, Gcamp6m is used, a genetically encoded fluorescent reporter. We employ high potassium chloride-mediated depolarization to mimic neuronal activity. To quantify synaptic vesicle exocytosis unambiguously, we measure the loss of normalized styryl dye fluorescence as a function of time. Under similar stimulation conditions, in the case of calcium influx, Gcamp6m fluorescence increases. Normalization and quantification of this fluorescence change are performed in a similar manner to the styryl dye protocol. These methods can be multiplexed with transfection-based overexpression of fluorescently tagged mutant proteins. These protocols have been extensively used to study synaptic dysfunction in models of FUS-ALS and C9ORF72-ALS, utilizing primary rodent cortical and motor neurons. These protocols easily allow for rapid screening of compounds that may improve neuronal communication. As such, these methods are valuable not only for the study of ALS but for all areas of neurodegenerative and developmental neuroscience research.


Assuntos
Esclerose Amiotrófica Lateral , Exocitose , Humanos , Neurônios Motores , Terminações Pré-Sinápticas , Transmissão Sináptica , Vesículas Sinápticas
14.
J Cell Sci ; 134(15)2021 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-34342349

RESUMO

Regulated exocytosis is an essential process whereby specific cargo proteins are secreted in a stimulus-dependent manner. Cargo-containing secretory granules are synthesized in the trans-Golgi network (TGN); after budding from the TGN, granules undergo modifications, including an increase in size. These changes occur during a poorly understood process called secretory granule maturation. Here, we leverage the Drosophila larval salivary glands as a model to characterize a novel role for Rab GTPases during granule maturation. We find that secretory granules increase in size ∼300-fold between biogenesis and release, and loss of Rab1 or Rab11 reduces granule size. Surprisingly, we find that Rab1 and Rab11 localize to secretory granule membranes. Rab11 associates with granule membranes throughout maturation, and Rab11 recruits Rab1. In turn, Rab1 associates specifically with immature granules and drives granule growth. In addition to roles in granule growth, both Rab1 and Rab11 appear to have additional functions during exocytosis; Rab11 function is necessary for exocytosis, while the presence of Rab1 on immature granules may prevent precocious exocytosis. Overall, these results highlight a new role for Rab GTPases in secretory granule maturation.


Assuntos
Exocitose , Vesículas Secretórias , Animais , Grânulos Citoplasmáticos/metabolismo , Drosophila , Vesículas Secretórias/metabolismo , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo , Rede trans-Golgi/metabolismo
15.
Br J Anaesth ; 127(4): 587-599, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34384592

RESUMO

BACKGROUND: The cellular and molecular mechanisms by which general anaesthesia occurs is poorly understood. Hippocampal interneurone subpopulations, which are critical regulators of cognitive function, have diverse neurophysiological and synaptic properties, but their responses to anaesthetics are unclear. METHODS: We used live-cell imaging of fluorescent biosensors expressed in mouse hippocampal neurones to delineate interneurone subtype-specific effects of isoflurane on synaptic vesicle exocytosis. The role of voltage-gated sodium channel (Nav) subtype expression in determining isoflurane sensitivity was probed by overexpression or knockdown of specific Nav subtypes in identified interneurones. RESULTS: Clinically relevant concentrations of isoflurane differentially inhibited synaptic vesicle exocytosis: to 83.1% (11.7%) of control in parvalbumin-expressing interneurones, and to 58.6% (13.3%) and 64.5% (8.5%) of control in somatostatin-expressing interneurones and glutamatergic neurones, respectively. The relative expression of Nav1.1 (associated with lower sensitivity) and Nav1.6 (associated with higher sensitivity) determined the sensitivity of exocytosis to isoflurane. CONCLUSIONS: Isoflurane inhibits synaptic vesicle exocytosis from hippocampal glutamatergic neurones and GABAergic interneurones in a cell-type-specific manner depending on their expression of voltage-gated sodium channel subtypes.


Assuntos
Anestésicos Inalatórios/farmacologia , Hipocampo/efeitos dos fármacos , Isoflurano/farmacologia , Ácido gama-Aminobutírico/metabolismo , Animais , Exocitose/efeitos dos fármacos , Feminino , Técnicas de Silenciamento de Genes , Hipocampo/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Transmissão Sináptica/efeitos dos fármacos , Canais de Sódio Disparados por Voltagem/genética
16.
Int J Mol Sci ; 22(15)2021 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-34360666

RESUMO

The ability to fertilise an egg is acquired by the mammalian sperm during the complex biochemical process called capacitation. Capacitation is accompanied by the production of reactive oxygen species (ROS), but the mechanism of redox regulation during capacitation has not been elucidated. This study aimed to verify whether capacitation coincides with reversible oxidative post-translational modifications of proteins (oxPTMs). Flow cytometry, fluorescence microscopy and Western blot analyses were used to verify the sperm capacitation process. A fluorescent gel-based redox proteomic approach allowed us to observe changes in the level of reversible oxPTMs manifested by the reduction or oxidation of susceptible cysteines in sperm proteins. Sperm capacitation was accompanied with redox modifications of 48 protein spots corresponding to 22 proteins involved in the production of ROS (SOD, DLD), playing a role in downstream redox signal transfer (GAPDHS and GST) related to the cAMP/PKA pathway (ROPN1L, SPA17), acrosome exocytosis (ACRB, sperm acrosome associated protein 9, IZUMO4), actin polymerisation (CAPZB) and hyperactivation (TUBB4B, TUB1A). The results demonstrated that sperm capacitation is accompanied by altered levels of oxPTMs of a group of redox responsive proteins, filling gaps in our knowledge concerning sperm capacitation.


Assuntos
Reação Acrossômica , Exocitose , Processamento de Proteína Pós-Traducional , Proteoma/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Capacitação Espermática , Animais , Bovinos , Fertilização , Masculino , Oxirredução , Fosforilação , Proteoma/análise , Proteoma/química
17.
Neuron ; 109(19): 3119-3134.e5, 2021 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-34411513

RESUMO

Transformation of flat membrane into round vesicles is generally thought to underlie endocytosis and produce speed-, amount-, and vesicle-size-specific endocytic modes. Visualizing depolarization-induced exocytic and endocytic membrane transformation in live neuroendocrine chromaffin cells, we found that flat membrane is transformed into Λ-shaped, Ω-shaped, and O-shaped vesicles via invagination, Λ-base constriction, and Ω-pore constriction, respectively. Surprisingly, endocytic vesicle formation is predominantly from not flat-membrane-to-round-vesicle transformation but calcium-triggered and dynamin-mediated closure of (1) Ω profiles formed before depolarization and (2) fusion pores (called kiss-and-run). Varying calcium influxes control the speed, number, and vesicle size of these pore closures, resulting in speed-specific slow (more than ∼6 s), fast (less than ∼6 s), or ultrafast (<0.6 s) endocytosis, amount-specific compensatory endocytosis (endocytosis = exocytosis) or overshoot endocytosis (endocytosis > exocytosis), and size-specific bulk endocytosis. These findings reveal major membrane transformation mechanisms underlying endocytosis, diverse endocytic modes, and exocytosis-endocytosis coupling, calling for correction of the half-a-century concept that the flat-to-round transformation predominantly mediates endocytosis after physiological stimulation.


Assuntos
Células Cromafins/fisiologia , Células Cromafins/ultraestrutura , Endocitose/fisiologia , Células Neuroendócrinas/fisiologia , Células Neuroendócrinas/ultraestrutura , Animais , Sinalização do Cálcio , Bovinos , Fusão Celular , Membrana Celular/fisiologia , Membrana Celular/ultraestrutura , Sistemas Computacionais , Dinaminas/fisiologia , Exocitose/fisiologia , Fusão de Membrana , Cultura Primária de Células , Vesículas Sinápticas/metabolismo
18.
Biochem J ; 478(16): 3099-3123, 2021 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-34436540

RESUMO

Clathrin is best known for its contribution to clathrin-mediated endocytosis yet it also participates to a diverse range of cellular functions. Key to this is clathrin's ability to assemble into polyhedral lattices that include curved football or basket shapes, flat lattices or even tubular structures. In this review, we discuss clathrin structure and coated vesicle formation, how clathrin is utilised within different cellular processes including synaptic vesicle recycling, hormone desensitisation, spermiogenesis, cell migration and mitosis, and how clathrin's remarkable 'shapeshifting' ability to form diverse lattice structures might contribute to its multiple cellular functions.


Assuntos
Membrana Celular/metabolismo , Clatrina/metabolismo , Endocitose , Endossomos/metabolismo , Exocitose , Animais , Clatrina/química , Clatrina/ultraestrutura , Humanos , Microscopia Eletrônica/métodos , Modelos Biológicos , Conformação Proteica
19.
Cells ; 10(8)2021 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-34440773

RESUMO

The pancreatic islets of Langerhans secrete several hormones critical for glucose homeostasis. The ß-cells, the major cellular component of the pancreatic islets, secrete insulin, the only hormone capable of lowering the plasma glucose concentration. The counter-regulatory hormone glucagon is secreted by the α-cells while δ-cells secrete somatostatin that via paracrine mechanisms regulates the α- and ß-cell activity. These three peptide hormones are packed into secretory granules that are released through exocytosis following a local increase in intracellular Ca2+ concentration. The high voltage-gated Ca2+ channels (HVCCs) occupy a central role in pancreatic hormone release both as a source of Ca2+ required for excitation-secretion coupling as well as a scaffold for the release machinery. HVCCs are multi-protein complexes composed of the main pore-forming transmembrane α1 and the auxiliary intracellular ß, extracellular α2δ, and transmembrane γ subunits. Here, we review the current understanding regarding the role of all HVCC subunits expressed in pancreatic ß-cell on electrical activity, excitation-secretion coupling, and ß-cell mass. The evidence we review was obtained from many seminal studies employing pharmacological approaches as well as genetically modified mouse models. The significance for diabetes in humans is discussed in the context of genetic variations in the genes encoding for the HVCC subunits.


Assuntos
Glicemia/metabolismo , Canais de Cálcio/metabolismo , Sinalização do Cálcio , Diabetes Mellitus/metabolismo , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Ativação do Canal Iônico , Animais , Canais de Cálcio/genética , Diabetes Mellitus/sangue , Diabetes Mellitus/genética , Diabetes Mellitus/patologia , Exocitose , Humanos , Células Secretoras de Insulina/patologia , Potenciais da Membrana , Via Secretória
20.
Sci Rep ; 11(1): 17220, 2021 08 26.
Artigo em Inglês | MEDLINE | ID: mdl-34446743

RESUMO

Primary cilia protrude from the apical surface of many cell types and act as a sensory organelle that regulates diverse biological processes ranging from chemo- and mechanosensation to signaling. Ciliary dysfunction is associated with a wide array of genetic disorders, known as ciliopathies. Polycystic lesions are commonly found in the kidney, liver, and pancreas of ciliopathy patients and mouse models. However, the pathogenesis of the pancreatic phenotype remains poorly understood. Chibby1 (Cby1), a small conserved coiled-coil protein, localizes to the ciliary base and plays a crucial role in ciliogenesis. Here, we report that Cby1-knockout (KO) mice develop severe exocrine pancreatic atrophy with dilated ducts during early postnatal development. A significant reduction in the number and length of cilia was observed in Cby1-KO pancreta. In the adult Cby1-KO pancreas, inflammatory cell infiltration and fibrosis were noticeable. Intriguingly, Cby1-KO acinar cells showed an accumulation of zymogen granules (ZGs) with altered polarity. Moreover, isolated acini from Cby1-KO pancreas exhibited defective ZG secretion in vitro. Collectively, our results suggest that, upon loss of Cby1, concomitant with ciliary defects, acinar cells accumulate ZGs due to defective exocytosis, leading to cell death and progressive exocrine pancreatic degeneration after birth.


Assuntos
Proteínas de Transporte/genética , Cílios/metabolismo , Pâncreas Exócrino/metabolismo , Pâncreas/metabolismo , Pancreatite/genética , Células Acinares/metabolismo , Animais , Atrofia , Proteínas de Transporte/metabolismo , Ciliopatias/genética , Ciliopatias/metabolismo , Exocitose/genética , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica de Transmissão , Pâncreas/patologia , Pâncreas/ultraestrutura , Pâncreas Exócrino/patologia , Pancreatite/metabolismo , Vesículas Secretórias/metabolismo
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