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1.
Nature ; 591(7850): 438-444, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33627868

RESUMO

Stromal cells in adult bone marrow that express leptin receptor (LEPR) are a critical source of growth factors, including stem cell factor (SCF), for the maintenance of haematopoietic stem cells and early restricted progenitors1-6. LEPR+ cells are heterogeneous, including skeletal stem cells and osteogenic and adipogenic progenitors7-12, although few markers have been available to distinguish these subsets or to compare their functions. Here we show that expression of an osteogenic growth factor, osteolectin13,14, distinguishes peri-arteriolar LEPR+ cells poised to undergo osteogenesis from peri-sinusoidal LEPR+ cells poised to undergo adipogenesis (but retaining osteogenic potential). Peri-arteriolar LEPR+osteolectin+ cells are rapidly dividing, short-lived osteogenic progenitors that increase in number after fracture and are depleted during ageing. Deletion of Scf from adult osteolectin+ cells did not affect the maintenance of haematopoietic stem cells or most restricted progenitors but depleted common lymphoid progenitors, impairing lymphopoiesis, bacterial clearance, and survival after acute bacterial infection. Peri-arteriolar osteolectin+ cell maintenance required mechanical stimulation. Voluntary running increased, whereas hindlimb unloading decreased, the frequencies of peri-arteriolar osteolectin+ cells and common lymphoid progenitors. Deletion of the mechanosensitive ion channel PIEZO1 from osteolectin+ cells depleted osteolectin+ cells and common lymphoid progenitors. These results show that a peri-arteriolar niche for osteogenesis and lymphopoiesis in bone marrow is maintained by mechanical stimulation and depleted during ageing.


Assuntos
Arteríolas , Linfopoese , Osteogênese , Nicho de Células-Tronco , Tecido Adiposo/citologia , Envelhecimento , Animais , Células da Medula Óssea/citologia , Osso e Ossos/citologia , Feminino , Fatores de Crescimento de Células Hematopoéticas/metabolismo , Lectinas Tipo C/metabolismo , Linfócitos/citologia , Masculino , Camundongos , Receptores para Leptina/metabolismo , Fator de Células-Tronco , Células Estromais/citologia
2.
Arterioscler Thromb Vasc Biol ; 44(3): e82-e98, 2024 03.
Artigo em Inglês | MEDLINE | ID: mdl-38205640

RESUMO

BACKGROUND: Integrins mediate the adhesion, crawling, and migration of neutrophils during vascular inflammation. Thiol exchange is important in the regulation of integrin functions. ERp72 (endoplasmic reticulum-resident protein 72) is a member of the thiol isomerase family responsible for the catalysis of disulfide rearrangement. However, the role of ERp72 in the regulation of Mac-1 (integrin αMß2) on neutrophils remains elusive. METHODS: Intravital microscopy of the cremaster microcirculation was performed to determine in vivo neutrophil movement. Static adhesion, flow chamber, and flow cytometry were used to evaluate in vitro integrin functions. Confocal fluorescent microscopy and coimmunoprecipitation were utilized to characterize the interactions between ERp72 and Mac-1 on neutrophil surface. Cell-impermeable probes and mass spectrometry were used to label reactive thiols and identify target disulfide bonds during redox exchange. Biomembrane force probe was performed to quantitatively measure the binding affinity of Mac-1. A murine model of acute lung injury induced by lipopolysaccharide was utilized to evaluate neutrophil-associated vasculopathy. RESULTS: ERp72-deficient neutrophils exhibited increased rolling but decreased adhesion/crawling on inflamed venules in vivo and defective static adhesion in vitro. The defect was due to defective activation of integrin Mac-1 but not LFA-1 (lymphocyte function-associated antigen-1) using blocking or epitope-specific antibodies. ERp72 interacted with Mac-1 in lipid rafts on neutrophil surface leading to the reduction of the C654-C711 disulfide bond in the αM subunit that is critical for Mac-1 activation. Recombinant ERp72, via its catalytic motifs, increased the binding affinity of Mac-1 with ICAM-1 (intercellular adhesion molecule-1) and rescued the defective adhesion of ERp72-deficient neutrophils both in vitro and in vivo. Deletion of ERp72 in the bone marrow inhibited neutrophil infiltration, ameliorated tissue damage, and increased survival during murine acute lung injury. CONCLUSIONS: Extracellular ERp72 regulates integrin Mac-1 activity by catalyzing disulfide rearrangement on the αM subunit and may be a novel target for the treatment of neutrophil-associated vasculopathy.


Assuntos
Lesão Pulmonar Aguda , Antígeno de Macrófago 1 , Animais , Camundongos , Lesão Pulmonar Aguda/genética , Lesão Pulmonar Aguda/metabolismo , Adesão Celular , Dissulfetos , Molécula 1 de Adesão Intercelular/metabolismo , Antígeno-1 Associado à Função Linfocitária/metabolismo , Antígeno de Macrófago 1/genética , Antígeno de Macrófago 1/metabolismo , Infiltração de Neutrófilos , Neutrófilos/metabolismo , Compostos de Sulfidrila/metabolismo
3.
J Nanobiotechnology ; 22(1): 363, 2024 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-38910248

RESUMO

Fluorescence nanoscopy, also known as super-resolution microscopy, has transcended the conventional resolution barriers and enabled visualization of biological samples at nanometric resolutions. A series of super-resolution techniques have been developed and applied to investigate the molecular distribution, organization, and interactions in blood cells, as well as the underlying mechanisms of blood-cell-associated diseases. In this review, we provide an overview of various fluorescence nanoscopy technologies, outlining their current development stage and the challenges they are facing in terms of functionality and practicality. We specifically explore how these innovations have propelled forward the analysis of thrombocytes (platelets), erythrocytes (red blood cells) and leukocytes (white blood cells), shedding light on the nanoscale arrangement of subcellular components and molecular interactions. We spotlight novel biomarkers uncovered by fluorescence nanoscopy for disease diagnosis, such as thrombocytopathies, malignancies, and infectious diseases. Furthermore, we discuss the technological hurdles and chart out prospective avenues for future research directions. This review aims to underscore the significant contributions of fluorescence nanoscopy to the field of blood cell analysis and disease diagnosis, poised to revolutionize our approach to exploring, understanding, and managing disease at the molecular level.


Assuntos
Células Sanguíneas , Microscopia de Fluorescência , Animais , Humanos , Células Sanguíneas/ultraestrutura , Plaquetas/metabolismo , Eritrócitos , Hematologia/métodos , Leucócitos/metabolismo , Microscopia de Fluorescência/métodos , Nanotecnologia/métodos
4.
Eur Biophys J ; 51(2): 119-133, 2022 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-35171346

RESUMO

Mechanobiology is an emerging field at the interface of biology and mechanics, investigating the roles of mechanical forces within biomolecules, organelles, cells, and tissues. As a highlight, the recent advances of micropipette-based aspiration assays and dynamic force spectroscopies such as biomembrane force probe (BFP) provide unprecedented mechanobiological insights with excellent live-cell compatibility. In their classic applications, these assays measure force-dependent ligand-receptor-binding kinetics, protein conformational changes, and cellular mechanical properties such as cortical tension and stiffness. In recent years, when combined with advanced microscopies in high spatial and temporal resolutions, these biomechanical nanotools enable characterization of receptor-mediated cell mechanosensing and subsequent organelle behaviors at single-cellular and molecular level. In this review, we summarize the latest developments of these assays for live-cell mechanobiology studies. We also provide perspectives on their future upgrades with multimodal integration and high-throughput capability.


Assuntos
Fenômenos Mecânicos , Proteínas , Fenômenos Biomecânicos , Biofísica , Cinética , Ligantes , Proteínas/química
5.
Eur Biophys J ; 51(2): 135-146, 2022 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-35286429

RESUMO

Mechanical stimuli such as tension, compression, and shear stress play critical roles in the physiological functions of red blood cells (RBCs) and their homeostasis, ATP release, and rheological properties. Intracellular calcium (Ca2+) mobilization reflects RBC mechanosensing as they transverse the complex vasculature. Emerging studies have demonstrated the presence of mechanosensitive Ca2+ permeable ion channels and their function has been implicated in the regulation of RBC volume and deformability. However, how these mechanoreceptors trigger Ca2+ influx and subsequent cellular responses are still unclear. Here, we introduce a fluorescence-coupled micropipette aspiration assay to examine RBC mechanosensing at the single-cell level. To achieve a wide range of cell aspirations, we implemented and compared two negative pressure adjusting apparatuses: a homemade water manometer (- 2.94 to 0 mmH2O) and a pneumatic high-speed pressure clamp (- 25 to 0 mmHg). To visualize Ca2+ influx, RBCs were pre-loaded with an intensiometric probe Cal-520 AM, then imaged under a confocal microscope with concurrent bright-field and fluorescent imaging at acquisition rates of 10 frames per second. Remarkably, we observed the related changes in intracellular Ca2+ levels immediately after aspirating individual RBCs in a pressure-dependent manner. The RBC aspirated by the water manometer only displayed 1.1-fold increase in fluorescence intensity, whereas the RBC aspirated by the pneumatic clamp showed up to threefold increase. These results demonstrated the water manometer as a gentle tool for cell manipulation with minimal pre-activation, while the high-speed pneumatic clamp as a much stronger pressure actuator to examine cell mechanosensing directly. Together, this multimodal platform enables us to precisely control aspiration and membrane tension, and subsequently correlate this with intracellular calcium concentration dynamics in a robust and reproducible manner.


Assuntos
Cálcio , Deformação Eritrocítica , Cálcio/metabolismo , Eritrócitos , Canais Iônicos/metabolismo , Transdução de Sinais
6.
Analyst ; 147(6): 1222-1235, 2022 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-35212697

RESUMO

Microvascular thrombosis and inflammation (thromboinflammation) are major causes of morbidity and mortality in critically ill patients with limited therapeutic options. Platelets are central to thromboinflammation, and microvascular platelet thrombi are highly effective at recruiting and activating leukocytes at sites of endothelial injury. Whilst parallel-plate flow chambers, microslides and straight microchannel assays have been widely used to recapitulate leukocyte adhesive behavior on 2-dimensional (2D) surfaces, none of these methods achieve high fidelity 3-dimensional (3D) geometries emulating microvascular platelet thrombi. As a result, the role of hydrodynamic factors in regulating leukocyte interactions with platelet thrombi remains ill-defined. Here, we report a microfluidic post model that allows visualization and analysis of neutrophil-platelet interactions in a 3D flow field. We have utilized the unique mechanosensitive features of platelets to enable selective micropatterning of the 3D posts with human or mouse platelets. By modulating the activation status of platelets, our method enables precise control of platelet surface reactivity and neutrophil recruitment. In addition, our microfluidic post assay accurately recapitulated the rolling versus stationary adhesion behavior of single neutrophils and demonstrated the efficacy of the P-selectin and Mac-1 blocking antibodies to reduce neutrophil recruitment and stationary adhesion, respectively. Moreover, the geometry of posts had a major influence on the efficiency of neutrophil recruitment and adhesion stability. This new post method highlights the importance of platelet 3D geometries in facilitating efficient, localized neutrophil recruitment. These findings have potentially important implications for the potent proinflammatory function of microvascular platelet thrombi.


Assuntos
Plaquetas , Trombose , Animais , Adesão Celular , Humanos , Inflamação , Leucócitos , Camundongos , Microfluídica , Neutrófilos
7.
Phys Chem Chem Phys ; 24(24): 14857-14865, 2022 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-35698887

RESUMO

von Willebrand factor (VWF) senses and responds to the hemodynamic forces to interact with the circulatory system and platelets in hemostasis and thrombosis. The dark side of this mechanobiology is implicated in atherothrombosis, stroke, and, more recently, the COVID-19 thrombotic symptoms. The force-responsive element controlling VWF activation predominantly resides in the N terminal auto-inhibitory module (N-AIM) flanking its A1 domain. Nevertheless, the detailed mechano-chemistry of soluble VWF N-AIM is poorly understood at the sub-molecular level as it is assumed to be unstructured loops. Using the free molecular dynamics (MD) simulations, we first predicted a hairpin-like structure of the soluble A1 N-AIM derived polypeptide (Lp; sequences Q1238-E1260). Then we combined molecular docking and steered molecular dynamics (SMD) simulations to examine how Lp regulates the A1-GPIbα interaction under tensile forces. Our simulation results indicate that Lp suppresses the catch bond in a sandwich complex of A1-Lp-GPIbα yet contributes an additional catch-bond residue D1249. To experimentally benchmark the binding kinetics for A1-GPIbα in the absence or presence of Lp, we conducted the force spectroscopy-biomembrane force probe (BFP) assays. We found similar suppression on the A1-GPIbα catch bond with soluble Lp in presence. Clinically, as more and more therapeutic candidates targeting the A1-GPIbα axis have entered clinical trials to treat patients with TTP and acute coronary syndrome, our work represents an endeavor further towards an effective anti-thrombotic approach without severe bleeding side effects as most existing drugs suffer.


Assuntos
COVID-19 , Complexo Glicoproteico GPIb-IX de Plaquetas/metabolismo , Fator de von Willebrand , Plaquetas , Humanos , Simulação de Acoplamento Molecular , Ligação Proteica , Fator de von Willebrand/química , Fator de von Willebrand/metabolismo
8.
Nano Lett ; 20(7): 4775-4781, 2020 07 08.
Artigo em Inglês | MEDLINE | ID: mdl-32208705

RESUMO

Video-rate super-resolution imaging through biological tissue can visualize and track biomolecule interplays and transportations inside cellular organisms. Structured illumination microscopy allows for wide-field super resolution observation of biological samples but is limited by the strong extinction of light by biological tissues, which restricts the imaging depth and degrades its imaging resolution. Here we report a photon upconversion scheme using lanthanide-doped nanoparticles for wide-field super-resolution imaging through the biological transparent window, featured by near-infrared and low-irradiance nonlinear structured illumination. We demonstrate that the 976 nm excitation and 800 nm upconverted emission can mitigate the aberration. We found that the nonlinear response of upconversion emissions from single nanoparticles can effectively generate the required high spatial frequency components in the Fourier domain. These strategies lead to a new modality in microscopy with a resolution below 131 nm, 1/7th of the excitation wavelength, and an imaging rate of 1 Hz.

9.
Nano Lett ; 20(7): 5133-5140, 2020 07 08.
Artigo em Inglês | MEDLINE | ID: mdl-32530632

RESUMO

Immune checkpoint blockade with monoclonal antibodies (mAbs) that target programmed cell death protein-1 (PD-1) has remarkably revolutionized cancer therapy. Their binding kinetics measured by surface plasmon resonance does not always correlate well with their immunotherapeutic efficacies, mainly due to the lack of two-dimensional cell plasma membrane and the capability of force sensing and manipulation. In this regard, based on a more suitable and ultra-sensitive biomechanical nanotool, biomembrane force probe (BFP), we developed a Double-edge Smart Feedback control system as an ultra-stable platform to characterize ultra-long bond lifetimes of receptor-ligand binding on living cells. We further benchmarked the dissociation kinetics for three clinically approved PD-1 blockade mAbs (Nivolumab, Pembrolizumab, and Camrelizumab), intriguingly correlating well with the objective response rates in the hepatocellular carcinoma second-line treatment. This ultra-stable BFP potentially provides a compelling kinetic platform to direct the screening, optimization, and clinical selection of therapeutic antibodies in the future.


Assuntos
Antineoplásicos Imunológicos , Receptor de Morte Celular Programada 1 , Anticorpos Monoclonais , Antineoplásicos Imunológicos/farmacologia , Cinética , Nivolumabe
10.
Immunol Cell Biol ; 98(2): 93-113, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31698518

RESUMO

T lymphocytes utilize amoeboid migration to navigate effectively within complex microenvironments. The precise rearrangement of the actin cytoskeleton required for cellular forward propulsion is mediated by actin regulators, including the actin-related protein 2/3 (Arp2/3) complex, a macromolecular machine that nucleates branched actin filaments at the leading edge. The consequences of modulating Arp2/3 activity on the biophysical properties of the actomyosin cortex and downstream T cell function are incompletely understood. We report that even a moderate decrease of Arp3 levels in T cells profoundly affects actin cortex integrity. Reduction in total F-actin content leads to reduced cortical tension and disrupted lamellipodia formation. Instead, in Arp3-knockdown cells, the motility mode is dominated by blebbing migration characterized by transient, balloon-like protrusions at the leading edge. Although this migration mode seems to be compatible with interstitial migration in three-dimensional environments, diminished locomotion kinetics and impaired cytotoxicity interfere with optimal T cell function. These findings define the importance of finely tuned, Arp2/3-dependent mechanophysical membrane integrity in cytotoxic effector T lymphocyte activities.


Assuntos
Citoesqueleto de Actina/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Proteína 3 Relacionada a Actina/metabolismo , Movimento Celular/genética , Linfócitos T Citotóxicos/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/genética , Proteína 3 Relacionada a Actina/genética , Actinas/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células/genética , Sobrevivência Celular/genética , Regulação para Baixo , Técnicas de Silenciamento de Genes , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , RNA Interferente Pequeno , Análise de Célula Única , Linfócitos T Citotóxicos/citologia , Peixe-Zebra
11.
Nat Mater ; 18(7): 760-769, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30911119

RESUMO

Integrins are membrane receptors that mediate cell adhesion and mechanosensing. The structure-function relationship of integrins remains incompletely understood, despite the extensive studies carried out because of its importance to basic cell biology and translational medicine. Using a fluorescence dual biomembrane force probe, microfluidics and cone-and-plate rheometry, we applied precisely controlled mechanical stimulations to platelets and identified an intermediate state of integrin αIIbß3 that is characterized by an ectodomain conformation, ligand affinity and bond lifetimes that are all intermediate between the well-known inactive and active states. This intermediate state is induced by ligand engagement of glycoprotein (GP) Ibα via a mechanosignalling pathway and potentiates the outside-in mechanosignalling of αIIbß3 for further transition to the active state during integrin mechanical affinity maturation. Our work reveals distinct αIIbß3 state transitions in response to biomechanical and biochemical stimuli, and identifies a role for the αIIbß3 intermediate state in promoting biomechanical platelet aggregation.


Assuntos
Fenômenos Mecânicos , Agregação Plaquetária , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Fenômenos Biomecânicos , Humanos , Ligantes , Transdução de Sinais
12.
Proc Natl Acad Sci U S A ; 111(1): 379-84, 2014 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-24344262

RESUMO

Plexins and semaphorins comprise a large family of receptor-ligand pairs controlling cell guidance in nervous, immune, and vascular systems. How plexin regulation of neurite outgrowth, lymphoid trafficking, and vascular endothelial cell branching is linked to integrin function, central to most directed movement, remains unclear. Here we show that on developing thymocytes, plexinD1 controls surface topology of nanometer-scaled ß1 integrin adhesion domains in cis, whereas its ligation by sema3E in trans regulates individual ß1 integrin catch bonds. Loss of plexinD1 expression reduces ß1 integrin clustering, thereby diminishing avidity, whereas sema3E ligation shortens individual integrin bond lifetimes under force to reduce stability. Consequently, both decreased expression of plexinD1 during developmental progression and a thymic medulla-emanating sema3E gradient enhance thymocyte movement toward the medulla, thus enforcing the orchestrated lymphoid trafficking required for effective immune repertoire selection. Our results demonstrate plexin-tunable molecular features of integrin adhesion with broad implications for many cellular processes.


Assuntos
Moléculas de Adesão Celular Neuronais/metabolismo , Regulação da Expressão Gênica , Glicoproteínas/metabolismo , Integrina beta1/metabolismo , Proteínas de Membrana/metabolismo , Timócitos/citologia , Animais , Autoanticorpos/sangue , Adesão Celular , Quimiocinas/metabolismo , Quimiotaxia , Proteínas do Citoesqueleto , Integrinas/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Ligantes , Glicoproteínas de Membrana , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia Confocal , Proteínas do Tecido Nervoso , Semaforinas , Transdução de Sinais , Processos Estocásticos
13.
Biophys J ; 108(7): 1773-1784, 2015 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-25863068

RESUMO

The impact of flow disturbances on platelet adhesion is complex and incompletely understood. At the molecular scale, platelet glycoprotein Ibα (GPIbα) must associate with the von Willebrand factor A1 domain (VWF-A1) with a rapid on-rate under high hemodynamic forces, as occurs in arterial thrombosis, where various transport mechanisms are at work. Here, we theoretically modeled the coupled transport-reaction process of the two-dimensional (2D) receptor-ligand association kinetics in a biomembrane force probe to explicitly account for the effects of molecular length, confinement stiffness, medium viscosity, surface curvature, and separation distance. We experimentally verified the theoretical approach by visualizing association and dissociation of individual VWF-A1-GPIbα bonds in a real-time thermal fluctuation assay. The apparent on-rate, reciprocal of the average time intervals between sequential bonds, decreased with the increasing gap distance between A1- and GPIbα-bearing surfaces with an 80-nm threshold (beyond which bond formation became prohibitive) identified as the combined contour length of the receptor and ligand molecules. The biomembrane force probe spring constant and diffusivity of the protein-bearing beads also significantly influenced the apparent on-rate, in accordance with the proposed transport mechanisms. The global agreement between the experimental data and the model predictions supports the hypothesis that receptor-ligand association behaves distinctly in the transport- and reaction-limited scenarios. To our knowledge, our results represent the first detailed quantification of physical regulation of the 2D on-rate that allows platelets to sense and respond to local changes in their hemodynamic environment. In addition, they provide an approach for determining the intrinsic kinetic parameters that employs simultaneous experimental measurements and theoretical modeling of bond association in a single assay. The 2D intrinsic forward rate for VWF-A1-GPIbα association was determined from the measurements to be (3.5 ± 0.67) × 10(-4)µm(2) s(-1).


Assuntos
Complexo Glicoproteico GPIb-IX de Plaquetas/metabolismo , Fator de von Willebrand/metabolismo , Espaço Extracelular/química , Humanos , Cinética , Complexo Glicoproteico GPIb-IX de Plaquetas/química , Ligação Proteica , Viscosidade , Fator de von Willebrand/química
14.
Biophys J ; 109(9): 1781-4, 2015 Nov 03.
Artigo em Inglês | MEDLINE | ID: mdl-26536255

RESUMO

Leucine-rich repeat (LRR) is a versatile motif widely present in adhesive proteins and signal-transducing receptors. The concave structure formed by a group of LRRs is thought to facilitate binding to globular protein domains with increased affinities. However, little is known about the conformational dynamics of LRRs in such a structure, e.g., whether and how force induces conformational changes in LRRs to regulate protein binding and signal transduction. Here we investigated the platelet glycoprotein Ibα (GPIbα), a demonstrated mechanoreceptor with known crystal structures for the N-terminal domain (GPIbαN), as a model for LRR-containing proteins using a combined method of steered molecular dynamics simulations and single-molecule force spectroscopy with a biomembrane force probe. We found that force-induced unfolding of GPIbαN starts with LRR2-4 and propagates to other LRRs. Importantly, force-dependent lifetimes of individual VWF-A1 bonds with GPIbα are prolonged after LRR unfolding. Enhancement of protein-protein interactions by force-induced LRR unfolding may be a phenomenon of interest in biology.


Assuntos
Complexo Glicoproteico GPIb-IX de Plaquetas/metabolismo , Desdobramento de Proteína , Leucina/química , Simulação de Dinâmica Molecular , Ligação Proteica , Análise Espectral , Fator de von Willebrand/metabolismo
15.
Blood ; 122(10): 1695-706, 2013 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-23881916

RESUMO

Megakaryocytes generate platelets through extensive reorganization of the cytoskeleton and plasma membrane. Cdc42 interacting protein 4 (CIP4) is an F-BAR protein that localizes to membrane phospholipids through its BAR domain and interacts with Wiskott-Aldrich Syndrome Protein (WASP) via its SRC homology 3 domain. F-BAR proteins promote actin polymerization and membrane tubulation. To study its function, we generated CIP4-null mice that displayed thrombocytopenia similar to that of WAS(-) mice. The number of megakaryocytes and their progenitors was not affected. However, the number of proplatelet protrusions was reduced in CIP4-null, but not WAS(-), megakaryocytes. Electron micrographs of CIP4-null megakaryocytes showed an altered demarcation membrane system. Silencing of CIP4, not WASP, expression resulted in fewer proplatelet-like extensions. Fluorescence anisotropy studies showed that loss of CIP4 resulted in a more rigid membrane. Micropipette aspiration demonstrated decreased cortical actin tension in megakaryocytic cells with reduced CIP4 or WASP protein. These studies support a new biophysical mechanism for platelet biogenesis whereby CIP4 enhances the complex, dynamic reorganization of the plasma membrane (WASP independent) and actin cortex network (as known for WASP and cortical actin) to reduce the work required for generating proplatelets. CIP4 is a new component in the highly coordinated system of megakaryocytic membrane and cytoskeletal remodeling affecting platelet production.


Assuntos
Plaquetas/metabolismo , Membrana Celular/metabolismo , Citoesqueleto/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Citoesqueleto de Actina/metabolismo , Animais , Fenômenos Biomecânicos , Linhagem Celular , Ensaio de Unidades Formadoras de Colônias , Deleção de Genes , Técnicas de Silenciamento de Genes , Masculino , Megacariócitos/metabolismo , Megacariócitos/patologia , Megacariócitos/ultraestrutura , Fluidez de Membrana , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/deficiência , Antígenos de Histocompatibilidade Menor , Ploidias , Transporte Proteico , Células-Tronco/metabolismo , Células-Tronco/patologia , Trombocitopenia/metabolismo , Trombocitopenia/patologia , Proteína Neuronal da Síndrome de Wiskott-Aldrich/metabolismo
16.
J Biol Chem ; 288(45): 32289-32301, 2013 Nov 08.
Artigo em Inglês | MEDLINE | ID: mdl-24062306

RESUMO

Binding of platelet glycoprotein Ibα (GPIbα) to von Willebrand factor (VWF) initiates platelet adhesion to disrupted vascular surface under arterial blood flow. Flow exerts forces on the platelet that are transmitted to VWF-GPIbα bonds, which regulate their dissociation. Mutations in VWF and/or GPIbα may alter the mechanical regulation of platelet adhesion to cause hemostatic defects as found in patients with von Willebrand disease (VWD). Using a biomembrane force probe, we observed biphasic force-decelerated (catch) and force-accelerated (slip) dissociation of GPIbα from VWF. The VWF A1 domain that contains the N-terminal flanking sequence Gln(1238)-Glu(1260) (1238-A1) formed triphasic slip-catch-slip bonds with GPIbα. By comparison, using a short form of A1 that deletes this sequence (1261-A1) abolished the catch bond, destabilizing its binding to GPIbα at high forces. Importantly, shear-dependent platelet rolling velocities on these VWF ligands in a flow chamber system mirrored the force-dependent single-bond lifetimes. Adding the Gln(1238)-Glu(1260) peptide, which interacted with GPIbα and 1261-A1 but not 1238-A1, to whole blood decreased platelet attachment under shear stress. Soluble Gln(1238)-Glu(1260) reduced the lifetimes of GPIbα bonds with VWF and 1238-A1 but rescued the catch bond of GPIbα with 1261-A1. A type 2B VWD 1238-A1 mutation eliminated the catch bond by prolonging lifetimes at low forces, a type 2M VWD 1238-A1 mutation shifted the respective slip-catch and catch-slip transition points to higher forces, whereas a platelet type VWD GPIbα mutation enhanced the bond lifetime in the entire force regime. These data reveal the structural determinants of VWF activation by hemodynamic force of the circulation.


Assuntos
Plaquetas , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Adesividade Plaquetária , Fator de von Willebrand/química , Fator de von Willebrand/metabolismo , Substituição de Aminoácidos , Plaquetas/química , Plaquetas/metabolismo , Feminino , Humanos , Masculino , Glicoproteínas de Membrana/genética , Mutação de Sentido Incorreto , Complexo Glicoproteico GPIb-IX de Plaquetas , Ligação Proteica , Estrutura Terciária de Proteína , Fator de von Willebrand/genética
17.
Bioinformatics ; 29(12): 1511-8, 2013 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-23599504

RESUMO

MOTIVATION: Abrupt reduction/resumption of thermal fluctuations of a force probe has been used to identify association/dissociation events of protein-ligand bonds. We show that off-rate of molecular dissociation can be estimated by the analysis of the bond lifetime, while the on-rate of molecular association can be estimated by the analysis of the waiting time between two neighboring bond events. However, the analysis relies heavily on subjective judgments and is time-consuming. To automate the process of mapping out bond events from thermal fluctuation data, we develop a hidden Markov model (HMM)-based method. RESULTS: The HMM method represents the bond state by a hidden variable with two values: bound and unbound. The bond association/dissociation is visualized and pinpointed. We apply the method to analyze a key receptor-ligand interaction in the early stage of hemostasis and thrombosis: the von Willebrand factor (VWF) binding to platelet glycoprotein Ibα (GPIbα). The numbers of bond lifetime and waiting time events estimated by the HMM are much more than those estimated by a descriptive statistical method from the same set of raw data. The kinetic parameters estimated by the HMM are in excellent agreement with those by a descriptive statistical analysis, but have much smaller errors for both wild-type and two mutant VWF-A1 domains. Thus, the computerized analysis allows us to speed up the analysis and improve the quality of estimates of receptor-ligand binding kinetics.


Assuntos
Algoritmos , Complexo Glicoproteico GPIb-IX de Plaquetas/metabolismo , Fator de von Willebrand/metabolismo , Cinética , Ligantes , Cadeias de Markov , Complexo Glicoproteico GPIb-IX de Plaquetas/química , Ligação Proteica , Temperatura , Fator de von Willebrand/química
18.
ACS Nano ; 18(1): 299-313, 2024 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-38105535

RESUMO

Integrins are cell surface nanosized receptors crucial for cell motility and mechanosensing of the extracellular environment, which are often targeted for the development of biomaterials and nanomedicines. As a key feature of integrins, their activity, structure and behavior are highly mechanosensitive, which are regulated by mechanical forces down to pico-Newton scale. Using single-molecule biomechanical approaches, we compared the force-modulated ectodomain bending/unbending conformational changes of two integrin species, α5ß1 and αVß3. It was found that the conformation of integrin α5ß1 is determined by a threshold head-to-tail tension. By comparison, integrin αVß3 exhibits bistability even without force and can spontaneously transition between the bent and extended conformations with an apparent transition time under a wide range of forces. Molecular dynamics simulations observed almost concurrent disruption of ∼2 hydrogen bonds during integrin α5ß1 unbending, but consecutive disruption of ∼7 hydrogen bonds during integrin αVß3 unbending. Accordingly, we constructed a canonical energy landscape for integrin α5ß1 with a single energy well that traps the integrin in the bent state until sufficient force tilts the energy landscape to allow the conformational transition. In contrast, the energy landscape of integrin αVß3 conformational changes was constructed with hexa-stable intermediate states and intermediate energy barriers that segregate the conformational change process into multiple small steps. Our study elucidates the different biomechanical inner workings of integrins α5ß1 and αVß3 at the submolecular level, helps understand their mechanosignaling processes and how their respective functions are facilitated by their distinctive mechanosensitivities, and provides useful design principles for the engineering of protein-based biomechanical nanomachines.


Assuntos
Integrina alfa5beta1 , Integrinas , Integrina alfa5beta1/metabolismo , Integrinas/metabolismo , Simulação de Dinâmica Molecular , Integrina alfaVbeta3/metabolismo
19.
J Vis Exp ; (203)2024 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-38284529

RESUMO

Micropipette aspiration assays have long been a cornerstone for the investigation of live-cell mechanics, offering insights into cellular responses to mechanical stress. This paper details an innovative adaptation of the fluorescence-coupled micropipette aspiration (fMPA) assay. The fMPA assay introduces the capability to administer precise mechanical forces while concurrently monitoring the live-cell mechanotransduction processes mediated by ion channels. The sophisticated setup incorporates a precision-engineered borosilicate glass micropipette connected to a finely regulated water reservoir and pneumatic aspiration system, facilitating controlled pressure application with increments as refined as ± 1 mmHg. A significant enhancement is the integration of epi-fluorescence imaging, allowing for the simultaneous observation and quantification of cell morphological changes and intracellular calcium fluxes during aspiration. The fMPA assay, through its synergistic combination of epi-fluorescence imaging with micropipette aspiration, sets a new standard for the study of cell mechanosensing within mechanically challenging environments. This multifaceted approach is adaptable to various experimental setups, providing critical insights into the single-cell mechanosensing mechanisms.


Assuntos
Eritrócitos , Mecanotransdução Celular , Mecanotransdução Celular/fisiologia , Fluorescência , Estresse Mecânico , Pressão
20.
Adv Sci (Weinh) ; 11(30): e2401524, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-38757670

RESUMO

Use of extracorporeal membrane oxygenation (ECMO) for cardiorespiratory failure remains complicated by blood clot formation (thrombosis), triggered by biomaterial surfaces and flow conditions. Thrombosis may result in ECMO circuit changes, cause red blood cell hemolysis, and thromboembolic events. Medical device thrombosis is potentiated by the interplay between biomaterial properties, hemodynamic flow conditions and patient pathology, however, the contribution and importance of these factors are poorly understood because many in vitro models lack the capability to customize material and flow conditions to investigate thrombosis under clinically relevant medical device conditions. Therefore, an ECMO thrombosis-on-a-chip model is developed that enables highly customizable biomaterial and flow combinations to evaluate ECMO thrombosis in real-time with low blood volume. It is observed that low flow rates, decelerating conditions, and flow stasis significantly increased platelet adhesion, correlating with clinical thrombus formation. For the first time, it is found that tubing material, polyvinyl chloride, caused increased platelet P-selectin activation compared to connector material, polycarbonate. This ECMO thrombosis-on-a-chip model can be used to guide ECMO operation, inform medical device design, investigate embolism, occlusion and platelet activation mechanisms, and develop anti-thrombotic biomaterials to ultimately reduce medical device thrombosis, anti-thrombotic drug use and therefore bleeding complications, leading to safer blood-contacting medical devices.


Assuntos
Oxigenação por Membrana Extracorpórea , Dispositivos Lab-On-A-Chip , Ativação Plaquetária , Adesividade Plaquetária , Trombose , Oxigenação por Membrana Extracorpórea/métodos , Oxigenação por Membrana Extracorpórea/instrumentação , Trombose/etiologia , Humanos , Ativação Plaquetária/fisiologia , Plaquetas/metabolismo
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