RESUMO
InP/ZnS quantum dots (QDs) have received a large focus in recent years as a safer alternative to heavy metal-based QDs. Given their intrinsic fluorescent imaging capabilities, these QDs can be potentially relevant for in vivo platelet imaging. The InP/ZnS QDs are synthesized and their biocompatibility investigated through the use of different phase transfer agents. Analysis of platelet function indicates that platelet-QD interaction can occur at all concentrations and for all QD permutations tested. However, as the QD concentration increases, platelet aggregation is induced by QDs alone independent of natural platelet agonists. This study helps to define a range of concentrations and coatings (thioglycolic acid and penicillamine) that are biocompatible with platelet function. With this information, the platelet-QD interaction can be identified using multiple methods. Fluorescent lifetime imaging microscopy (FLIM) and confocal studies have shown QDs localize on the surface of the platelet toward the center while showing evidence of energy transfer within the QD population. It is believed that these findings are an important stepping point for the development of fluorescent probes for platelet imaging.
Assuntos
Pontos Quânticos , LigantesRESUMO
Investigating human platelet function in low-oxygen environments is important in multiple settings, including hypobaric hypoxia (e.g., high altitude), sea level hypoxia-related disease, and thrombus stability. These studies often involve drawing blood from which platelets are isolated and analysed at atmospheric conditions or re-exposed to low oxygen levels in hypoxia chambers before testing. However, it remains unknown how the in vitro handling of the samples itself changes their dissolved oxygen concentration, which might affect platelet function and experimental results. Here, we prepared healthy donor platelet-rich plasma and washed platelet (WP) suspensions and exposed them to 2% oxygen. We found that the use of hypoxia pre-equilibrated tubes, higher platelet concentrations (>2 × 108/mL versus 2 × 107/mL), smaller volumes (600 µL versus 3 mL), and presence of plasma reduced the time for samples to reach 2% oxygen. Notably, oxygen levels decreased below 2% in most suspensions, but also in WP maintained at atmospheric 21% oxygen. Additionally, platelet spreading on fibrinogen was decreased when using hypoxic fibrinogen-coated culture plates regardless of the oxygen percentage (2% or 21%) in which platelet incubation took place. Thus, sample handling and experimental conditions should be carefully monitored in platelet-hypoxia studies as they might compromise results interpretation and comparison across studies.
Assuntos
Plaquetas/fisiologia , Oxigênio/análise , Plasma Rico em Plaquetas/fisiologia , Atmosfera , Plaquetas/metabolismo , Hipóxia Celular , Hemostasia , Humanos , Oxigênio/farmacologia , Testes de Função Plaquetária , Plasma Rico em Plaquetas/metabolismoRESUMO
Megakaryocytes give rise to platelets via extension of proplatelet arms, which are released through the vascular sinusoids into the bloodstream. Megakaryocytes and their precursors undergo varying interactions with the extracellular environment in the bone marrow during their maturation and positioning in the vascular niche. We demonstrate that podosomes are abundant in primary murine megakaryocytes adherent on multiple extracellular matrix substrates, including native basement membrane. Megakaryocyte podosome lifetime and density, but not podosome size, are dependent on the type of matrix, with podosome lifetime dramatically increased on collagen fibers compared with fibrinogen. Podosome stability and dynamics depend on actin cytoskeletal dynamics but not matrix metalloproteases. However, podosomes degrade matrix and appear to be important for megakaryocytes to extend protrusions across a native basement membrane. We thus demonstrate for the first time a fundamental requirement for podosomes in megakaryocyte process extension across a basement membrane, and our results suggest that podosomes may have a role in proplatelet arm extension or penetration of basement membrane.
Assuntos
Membrana Basal/fisiologia , Extensões da Superfície Celular/fisiologia , Matriz Extracelular/metabolismo , Megacariócitos/fisiologia , Animais , Membrana Basal/metabolismo , Plaquetas/metabolismo , Plaquetas/fisiologia , Extensões da Superfície Celular/metabolismo , Células Cultivadas , Fibrinogênio/metabolismo , Células HEK293 , Humanos , Recém-Nascido , Metaloproteinases da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miosina Tipo II/metabolismoRESUMO
Quantum dots (QDs) are semi-conducting nanoparticles that have been developed for a range of biological and non-biological functions. They can be tuned to multiple different emission wavelengths and can have significant benefits over other fluorescent systems. Many studies have utilised QDs with a cadmium-based core; however, these QDs have since been shown to have poor biological compatibility. Therefore, other QDs, such as indium phosphide QDs, have been developed. These QDs retain excellent fluorescent intensity and tunability but are thought to have elevated biological compatibility. Herein we discuss the applicability of a range of QDs to the cardiovascular system. Key disease states such as myocardial infarction and stroke are associated with cardiovascular disease (CVD), and there is an opportunity to improve clinical imaging to aide clinical outcomes for these disease states. QDs offer potential clinical benefits given their ability to perform multiple functions, such as carry an imaging agent, a therapy, and a targeting motif. Two key cell types associated with CVD are platelets and immune cells. Both cell types play key roles in establishing an inflammatory environment within CVD, and as such aid the formation of pathological thrombi. However, it is unclear at present how and with which cell types QDs interact, and if they potentially drive unwanted changes or activation of these cell types. Therefore, although QDs show great promise for boosting imaging capability, further work needs to be completed to fully understand their biological compatibility.
Assuntos
Doenças Cardiovasculares , Nanopartículas , Pontos Quânticos , Humanos , Doenças Cardiovasculares/diagnóstico por imagemRESUMO
The actin cytoskeleton provides scaffolding and physical force to effect fundamental processes such as motility, cytokinesis and vesicle trafficking. The Arp2/3 complex nucleates actin structures and contributes to endocytic vesicle invagination and trafficking away from the plasma membrane. Internalisation and directed recycling of integrins are major driving forces for invasive cell motility and potentially for cancer metastasis. Here, we describe a direct requirement for WASH and Arp2/3-mediated actin polymerisation on the endosomal membrane system for α5ß1 integrin recycling. WASH regulates the trafficking of endosomal α5ß1 integrin to the plasma membrane and is fundamental for integrin-driven cell morphology changes and integrin-mediated cancer cell invasion. Thus, we implicate WASH and Arp2/3-driven actin nucleation in receptor recycling leading to invasive motility.
Assuntos
Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Movimento Celular , Integrina alfa5beta1/metabolismo , Neoplasias/fisiopatologia , Proteína da Síndrome de Wiskott-Aldrich/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/genética , Actinas/metabolismo , Animais , Linhagem Celular Tumoral , Membrana Celular/genética , Membrana Celular/metabolismo , Endossomos/genética , Endossomos/metabolismo , Humanos , Integrina alfa5beta1/genética , Invasividade Neoplásica , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Transporte Proteico , Proteína da Síndrome de Wiskott-Aldrich/genéticaRESUMO
BACKGROUND: Approximately 17.3% of the global population exhibits an element of zinc (Zn2+) deficiency. One symptom of Zn2+ deficiency is increased bleeding through impaired hemostasis. Platelets are crucial to hemostasis and are inhibited by endothelial-derived prostacyclin (prostaglandin I2 [PGI2]), which signals via adenylyl cyclase (AC) and cyclic adenosine monophosphate signaling. In other cell types, Zn2+ modulates cyclic adenosine monophosphate concentrations by changing AC and/or phosphodiesterase activity. OBJECTIVES: To investigate if Zn2+ can modulate platelet PGI2 signaling. METHODS: Platelet aggregation, spreading, and western blotting assays with Zn2+ chelators and cyclic nucleotide elevating agents were performed in washed platelets and platelet-rich plasma conditions. In vitro thrombus formation with various Zn2+ chelators and PGI2 was assessed in whole blood. RESULTS: Incubation of whole blood or washed platelets with Zn2+ chelators caused either embolization of preformed thrombi or reversal of platelet spreading, respectively. To understand this effect, we analyzed resting platelets and identified that incubation with Zn2+ chelators elevated pVASPser157, a marker of PGI2 signaling. In agreement that Zn2+ affects PGI2 signaling, addition of the AC inhibitor SQ22536 blocked Zn2+ chelation-induced platelet spreading reversal, while addition of Zn2+ blocked PGI2-mediated platelet reversal. Moreover, Zn2+ specifically blocked forskolin-mediated AC reversal of platelet spreading. Finally, PGI2 inhibition of platelet aggregation and in vitro thrombus formation was potentiated in the presence of low doses of Zn2+ chelators, increasing its effectiveness in inducing platelet inhibition. CONCLUSION: Zn2+ chelation potentiates platelet PGI2 signaling, elevating PGI2's ability to prevent effective platelet activation, aggregation, and thrombus formation.
Assuntos
Plaquetas , Trombose , Humanos , Plaquetas/metabolismo , Prostaglandinas/metabolismo , Prostaglandinas/farmacologia , Zinco/metabolismo , Agregação Plaquetária , Epoprostenol/farmacologia , AMP Cíclico , Adenilil Ciclases , Trombose/metabolismo , Quelantes/farmacologia , Monofosfato de Adenosina/farmacologiaRESUMO
Fluorescence imaging has gathered interest over the recent years for its real-time response and high sensitivity. Developing probes for this modality has proven to be a challenge. Quantum dots (QDs) are colloidal nanoparticles that possess unique optical and electronic properties due to quantum confinement effects, whose excellent optical properties make them ideal for fluorescence imaging of biological systems. By selectively controlling the synthetic methodologies it is possible to obtain QDs that emit in the first (650-950 nm) and second (1000-1400 nm) near infra-red (NIR) windows, allowing for superior imaging properties. Despite the excellent optical properties and biocompatibility shown by some NIR QDs, there are still some challenges to overcome to enable there use in clinical applications. In this review, we discuss the latest advances in the application of NIR QDs in preclinical settings, together with the synthetic approaches and material developments that make NIR QDs promising for future biomedical applications.
RESUMO
Vav proteins belong to the family of guanine-nucleotide-exchange factors for the Rho/Rac family of small G-proteins. In addition, they serve as important adapter proteins for the activation of PLCgamma (phospholipase Cgamma) isoforms by ITAM (immunoreceptor tyrosine-based activation motif) receptors, including the platelet collagen receptor GPVI (glycoprotein VI). Vav proteins are also regulated downstream of integrins, including the major platelet integrin alphaIIbbeta3, which has recently been shown to regulate PLCgamma2. In the present study, we have investigated the role of Vav family proteins in filopodia and lamellipodia formation on fibrinogen using platelets deficient in Vav1 and Vav3. Wild-type mouse platelets undergo a limited degree of spreading on fibrinogen, characterized by the formation of numerous filopodia and limited lamellipodia structures. Platelets deficient in Vav1 and Vav3 exhibit reduced filopodia and lamellipodia formation during spreading on fibrinogen. This is accompanied by reduced alphaIIbbeta3-mediated PLCgamma2 tyrosine phosphorylation and reduced Ca(2+) mobilization. In contrast, the G-protein agonist thrombin stimulates full spreading of control and Vav1/3-deficient platelets. Consistent with this, stimulation of F-actin (filamentous actin) formation and Rac activation by thrombin is not altered in Vav-deficient cells. These results demonstrate that Vav1 and Vav3 are required for optimal spreading and regulation of PLCgamma2 by integrin alphaIIbbeta3, but that their requirement is by-passed upon G-protein receptor activation.
Assuntos
Fatores de Troca do Nucleotídeo Guanina/metabolismo , Fosfolipase C gama/metabolismo , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Proteínas Proto-Oncogênicas c-vav/metabolismo , Animais , Plaquetas/metabolismo , Fibrinogênio/metabolismo , Deleção de Genes , Regulação da Expressão Gênica , Fatores de Troca do Nucleotídeo Guanina/genética , Camundongos , Proteínas Proto-Oncogênicas c-vav/genéticaRESUMO
BACKGROUND: The platelet cytoskeleton mediates the dramatic change in platelet morphology that takes place upon activation and stabilizes thrombus formation. The Arp2/3 complex plays a vital role in these processes, providing the protrusive force for lamellipodia formation. The Arp2/3 complex is highly regulated by a number of actin-binding proteins including the haematopoietic-specific protein HS1 and its homologue cortactin. The present study investigates the role of HS1 in platelets using HS1-/- mice. RESULTS: The present results demonstrate that HS1 is not required for platelet activation, shape change or aggregation. Platelets from HS1-/- mice spread normally on a variety of adhesion proteins and have normal F-actin and Arp2/3 complex distributions. Clot retraction, an actin-dependent process, is also normal in these mice. Platelet aggregation and secretion is indistinguishable between knock out and littermates and there is no increase in bleeding using the tail bleeding assay. CONCLUSION: This study concludes that HS1 does not play a major role in platelet function. It is possible that a role for HS1 is masked by the presence of cortactin.
Assuntos
Plaquetas/metabolismo , Proteínas dos Microfilamentos/metabolismo , Actinas/metabolismo , Animais , Plaquetas/citologia , Forma Celular , Genótipo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas dos Microfilamentos/deficiência , Proteínas dos Microfilamentos/genética , Ativação Plaquetária , Trombose/genética , Trombose/metabolismo , Trombose/patologiaRESUMO
Cells use various actin-based motile structures to allow them to move across and through matrix of varying density and composition. Podosomes are actin cytoskeletal structures that form in motile cells and that mediate adhesion to substrate, migration, and other specialized functions such as transmigration through cell and matrix barriers. The podosome is a unique and interesting entity, which appears in the light microscope as an individual punctum, but is linked to other podosomes like a node on a network of the underlying cytoskeleton. Here, we discuss the signals that control podosome assembly and dynamics in different cell types and the actin organising proteins that regulate both the inner actin core and integrin-rich surrounding ring structures. We review the structure and composition of podosomes and also their functions in various cell types of both myeloid and endothelial lineage. We also discuss the emerging idea that podosomes can sense matrix stiffness and enable cells to respond to their environment.
Assuntos
Citoesqueleto de Actina/metabolismo , Movimento Celular , Matriz Extracelular/metabolismo , Mecanotransdução Celular , Animais , Adesão Celular , Humanos , Transdução de SinaisRESUMO
Here, we present emerging ideas surrounding the interplay between the actin cytoskeleton and receptor transport and activation. The bulk of actin dynamics in cells is thought to contribute to architecture and mobility. Actin also contributes to trafficking, acting as a molecular scaffold, providing force to deform membranes, facilitating vesicle abscission or propelling a vesicle through the cytoplasm ( 1) (,) ( 2) and recent studies highlight important connections between the directed trafficking of receptors and the impact on cell migration and actin dynamics. Additionally, a number of newly described actin nucleation promoting factors, such as the vesicle associated protein WASH, reveal unexpected roles of actin in membrane traffic and suggest that the cell dedicates a significant proportion of its regulation of actin dynamics to controlling trafficking.
Assuntos
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Receptores ErbB/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Membrana Celular/metabolismo , Movimento Celular , Citoplasma/metabolismo , Endocitose , Endossomos/metabolismo , Complexo de Golgi/metabolismo , Humanos , Integrina alfa5beta1/metabolismo , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Mapeamento de Interação de Proteínas , Transporte ProteicoRESUMO
The development of a megakaryocyte lineage specific Cre deleter, using the Pf4 (CXCL4) promoter (Pf4-Cre), was a significant step forward in the specific analysis of platelet and megakaryocyte cell biology. However, in the present study we have employed a sensitive reporter-based approach to demonstrate that Pf4-Cre also recombines in a significant proportion of both fetal liver and bone marrow hematopoietic stem cells (HSCs), including the most primitive fraction containing the long-term repopulating HSCs. Consequently, we demonstrate that Pf4-Cre activity is not megakaryocyte lineage-specific but extends to other myeloid and lymphoid lineages at significant levels between 15-60%. Finally, we show for the first time that Pf4 transcripts are present in adult HSCs and primitive hematopoietic progenitor cells. These results have fundamental implications for the use of the Pf4-Cre mouse model and for our understanding of a possible role for Pf4 in the development of the hematopoietic lineage.
Assuntos
Células da Medula Óssea/citologia , Linhagem da Célula , Feto/citologia , Células-Tronco Hematopoéticas/citologia , Integrases/metabolismo , Fígado/citologia , Megacariócitos/citologia , Fator Plaquetário 4/fisiologia , Animais , Plaquetas/metabolismo , Células da Medula Óssea/metabolismo , Células Cultivadas , DNA/genética , Feto/metabolismo , Citometria de Fluxo , Células-Tronco Hematopoéticas/metabolismo , Fígado/metabolismo , Linfócitos/citologia , Linfócitos/metabolismo , Megacariócitos/metabolismo , Camundongos , Camundongos Transgênicos , Células Mieloides/citologia , Células Mieloides/metabolismo , Reação em Cadeia da Polimerase em Tempo RealRESUMO
We describe here the development and characterization of a conditionally inducible mouse model expressing Lifeact-GFP, a peptide that reports the dynamics of filamentous actin. We have used this model to study platelets, megakaryocytes and melanoblasts and we provide evidence that Lifeact-GFP is a useful reporter in these cell types ex vivo. In the case of platelets and megakaryocytes, these cells are not transfectable by traditional methods, so conditional activation of Lifeact allows the study of actin dynamics in these cells live. We studied melanoblasts in native skin explants from embryos, allowing the visualization of live actin dynamics during cytokinesis and migration. Our study revealed that melanoblasts lacking the small GTPase Rac1 show a delay in the formation of new pseudopodia following cytokinesis that accounts for the previously reported cytokinesis delay in these cells. Thus, through use of this mouse model, we were able to gain insights into the actin dynamics of cells that could only previously be studied using fixed specimens or following isolation from their native tissue environment.
Assuntos
Citoesqueleto de Actina/ultraestrutura , Regulação da Expressão Gênica , Actinas/genética , Actinas/metabolismo , Animais , Linhagem Celular , Movimento Celular , Citocinese , Genes Reporter , Proteínas de Fluorescência Verde/genética , Melanócitos/metabolismo , Melanócitos/ultraestrutura , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia de Fluorescência , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/ultraestrutura , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , Especificidade de Órgãos , Peptídeos/genética , Pseudópodes/metabolismo , Proteínas Recombinantes/genética , Imagem com Lapso de Tempo , Transcrição Gênica , Proteínas rac de Ligação ao GTP/genética , Proteínas rac de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTPRESUMO
It is now apparent that each of the known, naturally occurring polyphosphoinositides, the phosphatidylinositol monophosphates (PtdIns3P, PtdIns4P, PtdIns5P), phosphatidylinositol bisphosphates [PtdIns(3,4)P(2), PtdIns(3,5)P(2), PtdIns(4,5)P(2)], and phosphatidylinositol trisphosphate [PtdIns(3,4,5)P(3)], have distinct roles in regulating many cellular events, including intracellular signaling, migration, and vesicular trafficking. Traditional identification techniques require [(32)P]inorganic phosphate or [(3)H]inositol radiolabeling, acidified lipid extraction, deacylation, and ion-exchange head group separation, which are time-consuming and not suitable for samples in which radiolabeling is impractical, thus greatly restricting the study of these lipids in many physiologically relevant systems. Thus, we have developed a novel, high-efficiency, buffered citrate extraction methodology to minimize acid-induced phosphoinositide degradation, together with a high-sensitivity liquid chromatography-mass spectrometry (LC-MS) protocol using an acetonitrile-chloroform-methanol-water-ethylamine gradient with a microbore silica column that enables the identification and quantification of all phosphoinositides in a sample. The liquid chromatograph is sufficient to resolve PtdInsP(3) and PtdInsP(2) regioisomers; however, the PtdInsP regioisomers require a combination of LC and diagnostic fragmentation to MS(3). Data are presented using this approach for the analysis of phosphoinositides in human platelet and yeast samples.