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1.
Curr Biol ; 32(13): R748-R750, 2022 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-35820387

RESUMO

A new study uses reconstituted, functional octameric exocyst complex to provide new insights into the assembly of this tethering complex and reveal how the activity of the lipid kinase PIP5K1C stimulated by Arf6 on exocytic vesicles allows for exocyst-mediated tethering at the plasma membrane.


Assuntos
Exocitose , Proteínas de Transporte Vesicular , Transporte Biológico , Citoplasma/metabolismo , Vesículas Secretórias/metabolismo , Proteínas de Transporte Vesicular/metabolismo
2.
Int J Mol Sci ; 23(15)2022 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-35897641

RESUMO

The superfamily of Ras proteins comprises different molecules belonging to the GTPase family. They normally cycle between an active state bound to GTP which activates effectors while the protein is membrane-associated, and an inactive GDP-bound state. They regulate the intracellular trafficking and other cellular processes. The family of Rab proteins includes several members and they have been found, among other Ras proteins, to be fundamental for important biological processes, such as endocytosis and exocytosis. SNARE proteins control the fusion of vesicles by forming quaternary complexes which are divided into two small groups on the two different compartments. Generally, the association of three SNARE proteins on the donor compartment with the one on the target compartment determines the formation of the SNARE complex, the opening of the fusion pore and the formation of one single bigger vesicle. Interestingly, novel interactions between other molecules involved in intracellular trafficking, endosomal fusion and maturation have recently been found, such as the interaction between invariant chain and the Qb SNARE vti1b, and more functional connections between Rab proteins and SNAREs are supposed to be fundamental for the regulation of membrane fusion.


Assuntos
Fusão de Membrana , Proteínas SNARE , Exocitose/fisiologia , Fusão de Membrana/fisiologia , Proteínas Qb-SNARE/metabolismo , Proteínas SNARE/metabolismo , Proteínas ras/metabolismo
3.
Nat Commun ; 13(1): 4268, 2022 Jul 25.
Artigo em Inglês | MEDLINE | ID: mdl-35879332

RESUMO

Therapeutic targeting of angiogenesis in glioblastoma has yielded mixed outcomes. Investigation of tumor-associated angiogenesis has focused on the factors that stimulate the sprouting, migration, and hyperproliferation of the endothelial cells. However, little is known regarding the processes underlying the formation of the tumor-associated vessels. To address this issue, we investigated vessel formation in CD31+ cells isolated from human glioblastoma tumors. The results indicate that overexpression of integrin α3ß1 plays a central role in the promotion of tube formation in the tumor-associated endothelial cells in glioblastoma. Blocking α3ß1 function reduced sprout and tube formation in the tumor-associated endothelial cells and vessel density in organotypic cultures of glioblastoma. The data further suggest a mechanistic model in which integrin α3ß1-promoted calcium influx stimulates macropinocytosis and directed maturation of the macropinosomes in a manner that promotes lysosomal exocytosis during nascent lumen formation. Altogether, our data indicate that integrin α3ß1 may be a therapeutic target on the glioblastoma vasculature.


Assuntos
Glioblastoma , Integrina alfa3beta1 , Cálcio , Movimento Celular , Células Endoteliais/patologia , Exocitose , Glioblastoma/genética , Glioblastoma/patologia , Humanos , Lisossomos/patologia , Neovascularização Patológica/patologia
4.
Front Endocrinol (Lausanne) ; 13: 915509, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35774142

RESUMO

Insulin-stimulated glucose uptake in skeletal muscle is of fundamental importance to prevent postprandial hyperglycemia, and long-term deficits in insulin-stimulated glucose uptake underlie insulin resistance and type 2 diabetes. Skeletal muscle is responsible for ~80% of the peripheral glucose uptake from circulation via the insulin-responsive glucose transporter GLUT4. GLUT4 is mainly sequestered in intracellular GLUT4 storage vesicles in the basal state. In response to insulin, the GLUT4 storage vesicles rapidly translocate to the plasma membrane, where they undergo vesicle docking, priming, and fusion via the high-affinity interactions among the soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) exocytosis proteins and their regulators. Numerous studies have elucidated that GLUT4 translocation is defective in insulin resistance and type 2 diabetes. Emerging evidence also links defects in several SNAREs and SNARE regulatory proteins to insulin resistance and type 2 diabetes in rodents and humans. Therefore, we highlight the latest research on the role of SNAREs and their regulatory proteins in insulin-stimulated GLUT4 translocation in skeletal muscle. Subsequently, we discuss the novel emerging role of SNARE proteins as interaction partners in pathways not typically thought to involve SNAREs and how these atypical functions reveal novel therapeutic targets for combating peripheral insulin resistance and diabetes.


Assuntos
Diabetes Mellitus Tipo 2 , Resistência à Insulina , Diabetes Mellitus Tipo 2/metabolismo , Exocitose , Glucose/metabolismo , Transportador de Glucose Tipo 4/metabolismo , Humanos , Insulina/metabolismo , Proteínas de Transporte de Monossacarídeos/metabolismo , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Proteínas SNARE/metabolismo
5.
Cell Physiol Biochem ; 56(3): 282-292, 2022 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-35781358

RESUMO

BACKGROUND/AIMS: Besides their physiological properties, vitamins, such as vitamin C (ascorbic acid) and B6 (pyridoxine), ameliorate the symptoms of allergic disorders. Because exocytosis in mast cells can be detected electrophysiologically by the changes in the membrane capacitance (Cm), its continuous monitoring in the presence of these vitamins would determine their mast cell-stabilizing, anti-allergic properties. METHODS: Employing the whole-cell patch-clamp technique in rat peritoneal mast cells, we examined the effects of ascorbic acid and pyridoxine on the degranulation of mast cells and the increase in the Cm during exocytosis. RESULTS: Both ascorbic acid and pyridoxine dose-dependently suppressed the GTP-γ-S-induced increase in the Cm and inhibited the degranulation from mast cells. Surprisingly enough, relatively low concentrations of pyridoxine (1, 2 mM) synergistically enhanced the suppressive effect of 2 mM ascorbic acid on mast cell degranulation. CONCLUSION: These results provided electrophysiological evidence for the first time that ascorbic acid and pyridoxine inhibited the process of exocytosis in a dose-dependent manner. At relatively lower concentrations, these vitamins were not enough to stabilize mast cells. However, such concentrations of pyridoxine synergistically potentiated the mast cell-stabilizing property of ascorbic acid.


Assuntos
Mastócitos , Piridoxina , Animais , Ácido Ascórbico/farmacologia , Exocitose , Piridoxina/farmacologia , Ratos , Vitaminas
6.
Methods Mol Biol ; 2473: 89-100, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35819761

RESUMO

Whole-cell patch clamping is a standard method to monitor the secretion of synaptic vesicles. In this chapter, we describe the basic steps of whole-cell patch clamping for measuring synaptic exocytosis, aiming to provide reference for researchers who are new to this field.


Assuntos
Exocitose , Transmissão Sináptica , Técnicas de Patch-Clamp , Vesículas Sinápticas
7.
Methods Mol Biol ; 2473: 157-164, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35819765

RESUMO

Total internal reflection fluorescence microscopy (TIRFM) provides extremely thin optical sectioning with excellent signal-to-noise ratios, which allows for visualization of membrane dynamics at the cell surface with superb spatiotemporal resolution. In this chapter, TIRFM is used to record and analyze exocytosis of single glucose transporter-4 (GLUT4) containing vesicles in 3T3-L1 adipocytes.


Assuntos
Adipócitos , Exocitose , Células 3T3-L1 , Animais , Membrana Celular/metabolismo , Camundongos , Microscopia de Fluorescência/métodos
8.
Cancer Lett ; 543: 215765, 2022 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-35680072

RESUMO

Neuroendocrine tumors constitute a heterogeneous group of tumors arising from hormone-secreting cells and are generally associated with a dysfunction of secretion. Pheochromocytoma (Pheo) is a neuroendocrine tumor that develops from chromaffin cells of the adrenal medulla, and is responsible for an excess of catecholamine secretion leading to severe clinical symptoms such as hypertension, elevated stroke risk and various cardiovascular complications. Surprisingly, while the hypersecretory activity of Pheo is well known to pathologists and clinicians, it has never been carefully explored at the cellular and molecular levels. In the present study, we have combined catecholamine secretion measurement by carbon fiber amperometry on human tumor cells directly cultured from freshly resected Pheos, with the analysis by mass spectrometry of the exocytotic proteins differentially expressed between the tumor and the matched adjacent non-tumor tissue. In most patients, catecholamine secretion recordings from single Pheo cells revealed a higher number of exocytic events per cell associated with faster kinetic parameters. Accordingly, we unravel significant tumor-associated modifications in the expression of key proteins involved in different steps of the calcium-regulated exocytic pathway. Altogether, our findings indicate that dysfunction of the calcium-regulated exocytosis at the level of individual Pheo cell is a cause of the tumor-associated hypersecretion of catecholamines.


Assuntos
Neoplasias das Glândulas Suprarrenais , Medula Suprarrenal , Feocromocitoma , Neoplasias das Glândulas Suprarrenais/metabolismo , Medula Suprarrenal/metabolismo , Cálcio , Cálcio na Dieta , Catecolaminas/metabolismo , Exocitose , Humanos , Feocromocitoma/metabolismo
9.
J Chem Theory Comput ; 18(7): 4544-4554, 2022 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-35759758

RESUMO

The fusion pore controls the release of exocytotic vesicle contents through a precise orchestration of lipids from the fusing membranes and proteins. There is a major lipid reorganization during the different stages in life of the fusion pore (membrane fusion, nucleation, and expansion) that can be scrutinized thermodynamically. In this work, using umbrella sampling simulations we describe the expansion of the fusion pore. We have calculated free energy profiles to drive a nascent, just nucleated, fusion pore to its expanded configuration. We have quantified the effects on the free energy of one and two Synaptotagmin-1 C2B domains in the cytosolic space. We show that C2B domains cumulatively reduce the cost for expansion, favoring the system to evolve toward full fusion. Finally, by conducting thousands of unbiased molecular dynamics simulations, we show that C2B domains significantly decrease the probability of kiss-and-run events.


Assuntos
Cálcio , Exocitose , Cálcio/metabolismo , Fusão de Membrana
10.
Cell Mol Life Sci ; 79(6): 344, 2022 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-35660980

RESUMO

Weibel-Palade bodies (WPB) are elongated, rod-like secretory organelles unique to endothelial cells that store the pro-coagulant von-Willebrand factor (VWF) and undergo regulated exocytosis upon stimulation with Ca2+- or cAMP-raising agonists. We show here that WPB preferentially initiate fusion with the plasma membrane at their tips and identify synaptotagmin-like protein 2-a (Slp2-a) as a positive regulator of VWF secretion most likely mediating this topological selectivity. Following secretagogue stimulation, Slp2-a accumulates at one WPB tip before fusion occurs at this site. Depletion of Slp2-a reduces Ca2+-dependent secretion of highly multimeric VWF and interferes with the formation of actin rings at WPB-plasma membrane fusion sites that support the expulsion of the VWF multimers and most likely require a tip-end fusion topology. Phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P2] binding via the C2A domain of Slp2-a is required for accumulation of Slp2-a at the tip ends of fusing WPB, suggesting that Slp2-a mediates polar exocytosis by initiating contacts between WPB tips and plasma membrane PI(4,5)P2.


Assuntos
Corpos de Weibel-Palade , Fator de von Willebrand , Células Cultivadas , Exocitose/fisiologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Corpos de Weibel-Palade/metabolismo , Fator de von Willebrand/genética , Fator de von Willebrand/metabolismo
11.
Cells ; 11(11)2022 05 30.
Artigo em Inglês | MEDLINE | ID: mdl-35681487

RESUMO

Under physiological conditions, the widely expressed calcium-activated TRPM4 channel conducts sodium into cells. This sodium influx depolarizes the plasma membrane and reduces the driving force for calcium entry. The aberrant expression or function of TRPM4 has been reported in various diseases, including different types of cancer. TRPM4 is mainly localized in the plasma membrane, but it is also found in intracellular vesicles, which can undergo exocytosis. In this study, we show that calcium-induced exocytosis in the colorectal cancer cell line HCT116 is dependent on TRPM4. In addition, the findings from some studies of prostate cancer cell lines suggest a more general role of TRPM4 in calcium-induced exocytosis in cancer cells. Furthermore, calcium-induced exocytosis depends on TRPM4 ion conductivity. Additionally, an increase in intracellular calcium results in the delivery of TRPM4 to the plasma membrane. This process also depends on TRPM4 ion conductivity. TRPM4-dependent exocytosis and the delivery of TRPM4 to the plasma membrane are mediated by SNARE proteins. Finally, we provide evidence that calcium-induced exocytosis depends on TRPM4 ion conductivity, not within the plasma membrane, but rather in TRPM4-containing vesicles.


Assuntos
Exocitose , Canais de Cátion TRPM , Cálcio/metabolismo , Linhagem Celular Tumoral , Humanos , Sódio/metabolismo , Canais de Cátion TRPM/metabolismo
12.
Anal Chem ; 94(27): 9548-9556, 2022 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-35750055

RESUMO

For decades, carbon-fiber microelectrodes have been used in amperometric measurements of neurotransmitter release at a wide variety of cell types, providing a tremendous amount of valuable information on the mechanisms involved in dense-core vesicle fusion. The electroactive molecules that are released can be detected at the opposing microelectrode surface, allowing for precise quantification as well as detailed kinetic information on the stages of neurotransmitter release. However, it remains unclear how much of the catecholamine that is released into the artificial synapse escapes detection. This work examines two separate mechanisms by which released neurotransmitter goes undetected in a typical amperometric measurement. First, diffusional loss is assessed by monitoring exocytosis at single bovine chromaffin cells using carbon-fiber microelectrodes fabricated in a recessed (cavity) geometry. This creates a microsampling vial that minimizes diffusional loss of analyte prior to detection. More molecules were detected per exocytotic release event when using a recessed cavity sensor as compared to the conventional configuration. In addition, pharmacological inhibition of the norepinephrine transporter (NET), which serves to remove catecholamine from the extracellular space, increased both the size and the time course of individual amperometric events. Overall, this study characterizes distinct physical and biological mechanisms by which released neurotransmitter escapes detection at the opposing microelectrode surface, while also revealing an important role for the NET in "presynaptic" modulation of neurotransmitter release.


Assuntos
Células Cromafins , Exocitose , Animais , Fibra de Carbono , Catecolaminas/metabolismo , Bovinos , Células Cromafins/metabolismo , Exocitose/fisiologia , Microeletrodos , Neurotransmissores/metabolismo
13.
PLoS One ; 17(6): e0270299, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35767558

RESUMO

Weibel-Palade bodies (WPB) are unique secretory granules of endothelial cells that store the procoagulant von-Willebrand factor (VWF) in a highly compacted form. Upon exocytosis the densely packed VWF unfurls into long strands that expose binding sites for circulating platelets and thereby initiate the formation of a platelet plug at sites of blood vessel injury. Dense packing of VWF requires the establishment of an acidic pH in the lumen of maturing WPB but the mechanism responsible for this acidification has not yet been fully established. We show here that subunits of the vacuolar-type H+-ATPase are present on mature WPB and that interference with the proton pump activity of the ATPase employing inhibitors of different chemical nature blocks a reduction in the relative internal pH of WPB. Furthermore, depletion of the V-ATPase subunit V0d1 from primary endothelial cells prevents WPB pH reduction and the establishment of an elongated morphology of WPB that is dictated by the densely packed VWF tubules. Thus, the vacuolar-type H+-ATPase present on WPB is required for proper acidification and maturation of the organelle.


Assuntos
ATPases Vacuolares Próton-Translocadoras , Corpos de Weibel-Palade , Células Cultivadas , Células Endoteliais/metabolismo , Exocitose , Concentração de Íons de Hidrogênio , ATPases Vacuolares Próton-Translocadoras/metabolismo , Corpos de Weibel-Palade/metabolismo , Fator de von Willebrand/metabolismo
15.
Nat Commun ; 13(1): 3497, 2022 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-35715404

RESUMO

The balance between fast synchronous and delayed asynchronous release of neurotransmitters has a major role in defining computational properties of neuronal synapses and regulation of neuronal network activity. However, how it is tuned at the single synapse level remains poorly understood. Here, using the fluorescent glutamate sensor SF-iGluSnFR, we image quantal vesicular release in tens to hundreds of individual synaptic outputs from single pyramidal cells with 4 millisecond temporal and 75 nm spatial resolution. We find that the ratio between synchronous and asynchronous synaptic vesicle exocytosis varies extensively among synapses supplied by the same axon, and that the synchronicity of release is reduced at low release probability synapses. We further demonstrate that asynchronous exocytosis sites are more widely distributed within the release area than synchronous sites. Together, our results reveal a universal relationship between the two major functional properties of synapses - the timing and the overall efficacy of neurotransmitter release.


Assuntos
Ácido Glutâmico , Sinapses , Exocitose/fisiologia , Neurotransmissores , Sinapses/fisiologia , Transmissão Sináptica/fisiologia
16.
Int J Mol Sci ; 23(11)2022 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-35682799

RESUMO

Ellipticine is an indole alkaloid with proven antitumor activity against various tumors in vitro and a diverse mechanism of action, which includes topoisomerase II inhibition, intercalation, and cell cycle impact. Olivacine-ellipticine's isomer-shows similar properties. The objectives of this work were as follows: (a) to find a new path of olivacine synthesis, (b) to study the cytotoxic properties of olivacine and ellipticine in comparison to doxorubicin as well as their impact on the cell cycle, and (c) to investigate the cellular pharmacokinetics of the tested compounds to understand drug resistance in cancer cells better. SRB and MTT assays were used to study the anticancer activity of olivacine and ellipticine in vitro. Both compounds showed a cytotoxic effect on various cell lines, most notably on the doxorubicin-resistant LoVo/DX model, with olivacine's cytotoxicity approximately three times higher than doxorubicin. Olivacine proved to be less effective against cancer cells and less cytotoxic to normal cells than ellipticine. Olivacine proved to have fluorescent properties. Microscopic observation of cells treated with olivacine showed the difference in sensitivity depending on the cell line, with A549 cells visibly affected by a much lower concentration of olivacine than normal NHDF cells. An increased percentage of cells in G0/G1 was observed after treatment with olivacine and ellipticine, suggesting an impact on cell cycle progression, potentially via higher p53 protein expression, which blocks the transition from G0/G1 to the S phase. Ellipticine induced apoptosis at a concentration as low as 1 µM. It has been proved that the tested compounds (ellipticine and olivacine) undergo lysosomal exocytosis. Reducing exocytosis is possible through the use of compounds that inhibit the activity of the proton pump. Olivacine and ellipticine exhibited diverse cytotoxicity against a panel of cancer cells. Analysis of the lysosomal exocytosis of olivacine and ellipticine shows the need to look for derivatives with comparable anticancer activity but reduced weak base character.


Assuntos
Antineoplásicos , Elipticinas , Neoplasias , Antineoplásicos/farmacologia , Doxorrubicina/farmacologia , Resistência a Medicamentos , Elipticinas/farmacologia , Exocitose , Lisossomos
17.
Front Immunol ; 13: 883010, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35514977

RESUMO

Cytotoxic T cells (CTLs) are the main cellular mediators of the adaptive immune defenses against intracellular pathogens and malignant cells. Upon recognition of specific antigen on their cellular target, CTLs assemble an immunological synapse where they mobilise their killing machinery that is released into the synaptic cleft to orchestrate the demise of their cell target. The arsenal of CTLs is stored in lysosome-like organelles that undergo exocytosis in response to signals triggered by the T cell antigen receptor following antigen recognition. These organelles include lytic granules carrying a cargo of cytotoxic proteins packed on a proteoglycan scaffold, multivesicular bodies carrying the death receptor ligand FasL, and the recently discovered supramolecular attack particles that carry a core of cytotoxic proteins encased in a non-membranous glycoprotein shell. Here we will briefly review the main features of these killing entities and discuss their interrelationship and interplay in CTL-mediated killing.


Assuntos
Grânulos Citoplasmáticos , Linfócitos T Citotóxicos , Exocitose , Sinapses Imunológicas/metabolismo , Perforina/metabolismo
18.
J Immunol Res ; 2022: 1695802, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35571570

RESUMO

Extracellular vesicles (EVs) are vesicular bodies (40-1000 nm) with double-layer membrane structures released by different cell types into extracellular environments, including apoptosis bodies, microvesicles, and exosomes. Exosomes (30-100 nm) are vesicles enclosed by extracellular membrane and contain effective molecules of secretory cells. They are derived from intracellular multivesicular bodies (MVBs) that fuse with the plasma membrane and release their intracellular vesicles by exocytosis. Research has shown that almost all human cells could secrete exosomes, which have a certain relationship with corresponding diseases. In chronic liver diseases, exosomes release a variety of bioactive components into extracellular spaces, mediating intercellular signal transduction and materials transport. Moreover, exosomes play a role in the diagnosis, treatment, and prognosis of various chronic liver diseases as potential biomarkers and therapeutic targets. Previous studies have found that mesenchymal stem cell-derived exosomes (MSC-ex) could alleviate acute and chronic liver injury and have the advantages of high biocompatibility and low immunogenicity. In this paper, we briefly summarize the role of exosomes in the pathogenesis of different chronic liver diseases and the latest research progresses of MSC-ex as the clinical therapeutic targets.


Assuntos
Micropartículas Derivadas de Células , Exossomos , Vesículas Extracelulares , Hepatopatias , Micropartículas Derivadas de Células/metabolismo , Exocitose , Exossomos/metabolismo , Vesículas Extracelulares/metabolismo , Humanos , Hepatopatias/metabolismo , Hepatopatias/terapia
19.
Proc Natl Acad Sci U S A ; 119(20): e2111051119, 2022 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-35537054

RESUMO

Exocytosis and endocytosis are tightly coupled. In addition to initiating exocytosis, Ca2+ plays critical roles in exocytosis­endocytosis coupling in neurons and nonneuronal cells. Both positive and negative roles of Ca2+ in endocytosis have been reported; however, Ca2+ inhibition in endocytosis remains debatable with unknown mechanisms. Here, we show that synaptotagmin-1 (Syt1), the primary Ca2+ sensor initiating exocytosis, plays bidirectional and opposite roles in exocytosis­endocytosis coupling by promoting slow, small-sized clathrin-mediated endocytosis but inhibiting fast, large-sized bulk endocytosis. Ca2+-binding ability is required for Syt1 to regulate both types of endocytic pathways, the disruption of which leads to inefficient vesicle recycling under mild stimulation and excessive membrane retrieval following intense stimulation. Ca2+-dependent membrane tubulation may explain the opposite endocytic roles of Syt1 and provides a general membrane-remodeling working model for endocytosis determination. Thus, Syt1 is a primary bidirectional Ca2+ sensor facilitating clathrin-mediated endocytosis but clamping bulk endocytosis, probably by manipulating membrane curvature to ensure both efficient and precise coupling of endocytosis to exocytosis.


Assuntos
Endocitose , Transmissão Sináptica , Sinaptotagmina I , Cálcio/metabolismo , Endocitose/fisiologia , Exocitose/fisiologia , Neurônios/metabolismo , Sinaptotagmina I/metabolismo
20.
BMC Biol ; 20(1): 111, 2022 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-35549945

RESUMO

BACKGROUND: In vertebrate cells, the Golgi functional subunits, mini-stacks, are linked into a tri-dimensional network. How this "ribbon" architecture relates to Golgi functions remains unclear. Are all connections between mini-stacks equal? Is the local structure of the ribbon of functional importance? These are difficult questions to address, without a quantifiable readout of the output of ribbon-embedded mini-stacks. Endothelial cells produce secretory granules, the Weibel-Palade bodies (WPB), whose von Willebrand Factor (VWF) cargo is central to hemostasis. The Golgi apparatus controls WPB size at both mini-stack and ribbon levels. Mini-stack dimensions delimit the size of VWF "boluses" whilst the ribbon architecture allows their linear co-packaging, thereby generating WPBs of different lengths. This Golgi/WPB size relationship suits mathematical analysis. RESULTS: WPB lengths were quantized as multiples of the bolus size and mathematical modeling simulated the effects of different Golgi ribbon organizations on WPB size, to be compared with the ground truth of experimental data. An initial simple model, with the Golgi as a single long ribbon composed of linearly interlinked mini-stacks, was refined to a collection of mini-ribbons and then to a mixture of mini-stack dimers plus long ribbon segments. Complementing these models with cell culture experiments led to novel findings. Firstly, one-bolus sized WPBs are secreted faster than larger secretory granules. Secondly, microtubule depolymerization unlinks the Golgi into equal proportions of mini-stack monomers and dimers. Kinetics of binding/unbinding of mini-stack monomers underpinning the presence of stable dimers was then simulated. Assuming that stable mini-stack dimers and monomers persist within the ribbon resulted in a final model that predicts a "breathing" arrangement of the Golgi, where monomer and dimer mini-stacks within longer structures undergo continuous linking/unlinking, consistent with experimentally observed WPB size distributions. CONCLUSIONS: Hypothetical Golgi organizations were validated against a quantifiable secretory output. The best-fitting Golgi model, accounting for stable mini-stack dimers, is consistent with a highly dynamic ribbon structure, capable of rapid rearrangement. Our modeling exercise therefore predicts that at the fine-grained level the Golgi ribbon is more complex than generally thought. Future experiments will confirm whether such a ribbon organization is endothelial-specific or a general feature of vertebrate cells.


Assuntos
Células Endoteliais , Fator de von Willebrand , Células Cultivadas , Exocitose , Complexo de Golgi , Corpos de Weibel-Palade/fisiologia , Fator de von Willebrand/farmacologia , Fator de von Willebrand/fisiologia
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