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1.
Dev Cell ; 56(18): 2592-2606.e7, 2021 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-34508658

RESUMO

Membrane contact between intracellular organelles is important in mediating organelle communication. However, the assembly of molecular machinery at membrane contact site and its internal organization correlating with its functional activity remain unclear. Here, we demonstrate that a gel-like condensation of Cidec, a crucial protein for obesity development by facilitating lipid droplet (LD) fusion, occurs at the LD-LD contact site (LDCS) through phase separation. The homomeric interaction between the multivalent N terminus of Cidec is sufficient to promote its phase separation both in vivo and in vitro. Interestingly, Cidec condensation at LDCSs generates highly plastic and lipid-permeable fusion plates that are geometrically constrained by donor LDs. In addition, Cidec condensates are distributed unevenly in the fusion plate generating stochastic sub-compartments that may represent unique lipid passageways during LD fusion. We have thus uncovered the organization and functional significance of geometry-constrained Cidec phase separation in mediating LD fusion and lipid homeostasis.

2.
Biophys J ; 2021 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-34536388

RESUMO

Hepatic sinusoids present complex anatomical structures such as the endothelial sieve pores and the Disse space, which govern the microscopic blood flow in the sinusoids and are associated with structural variations in liver fibrosis and cirrhosis. However, the contributions of the permeability of endothelial and collagen layers and the roughness of hepatocyte microvilli to the features of this microflow remain largely unknown. Here, an immersed boundary method coupled with a lattice Boltzmann method was adopted in an in vitro hepatic sinusoidal model, and flow field and erythrocyte deformation analyses were conducted by introducing three new source terms including permeability of the endothelial layer, resistance of hepatocyte microvilli and collagen layers, and deformation of red blood cells (RBCs). Numerical calculations indicated that alterations in endothelial permeability could significantly affect the flow velocity and flow rate distributions in hepatic sinusoids. Interestingly, a biphasic regulating pattern of shear stress occurred simultaneously on the surface of hepatocytes and the lower side of endothelium, i.e., the shear stress increased with increased thickness of hepatocyte microvilli and collagen layer when the endothelial permeability was high but decreased with the increase of the thickness at low endothelial permeability. Additionally, this specified microflow manipulates typical RBC deformation inside the sinusoid, yielding one-third of the variation of deformable index with varied endothelial permeability. These simulations not only are consistent with experimental measurements using in vitro liver sinusoidal chip but also elaborate the contributions of endothelial and collagen layer permeability and wall roughness. Thus, our results provide a basis for further characterizing this microflow and understanding its effects on cellular migration and deformation in the hepatic sinusoids.

3.
FASEB J ; 35(5): e21521, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33811691

RESUMO

Transendothelial migration (TEM) of neutrophils under blood flow is critical in the inflammatory cascade. However, the role of endothelial plasticity in this process is not fully understood. Therefore, we used an in vitro model to test the dynamics of human polymorphonuclear neutrophil (PMN) TEM across lipopolysaccharide-treated human umbilical vein endothelial cell (HUVEC) monolayers. Interestingly, shRNA-E-selectin knockdown in HUVECs destabilized endothelial junctional integrity by reducing actin branching and increasing stress fiber at cell-cell junctions. This process is accomplished by downregulating the activation of cortactin and Arp2/3, which in turn alters the adhesive function of VE-cadherin, enhancing PMN transmigration. Meanwhile, redundant P-selectins possess overlapping functions in E-selectin-mediated neutrophil adhesion, and transmigration. These results demonstrate, to our knowledge, for the first time, that E-selectins negatively regulate neutrophil transmigration through alterations in endothelial plasticity. Furthermore, it improves our understanding of the mechanisms underlying actin remodeling, and junctional integrity, in endothelial cells mediating leukocyte TEM.


Assuntos
Movimento Celular , Selectina E/metabolismo , Endotélio Vascular/fisiologia , Junções Intercelulares/fisiologia , Neutrófilos/fisiologia , Migração Transendotelial e Transepitelial , Proteína 2 Relacionada a Actina/genética , Proteína 2 Relacionada a Actina/metabolismo , Proteína 3 Relacionada a Actina/genética , Proteína 3 Relacionada a Actina/metabolismo , Células Cultivadas , Selectina E/genética , Endotélio Vascular/citologia , Humanos , Neutrófilos/citologia , Pseudópodes
4.
Biomater Sci ; 9(10): 3776-3790, 2021 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-33876166

RESUMO

Mechanical or physical cues are associated with the growth and differentiation of embryonic stem cells (ESCs). While the substrate stiffness or topography independently affects the differentiation of ESCs, their cooperative regulation on lineage-specific differentiation remains largely unknown. Here, four topographical configurations on stiff or soft polyacrylamide hydrogel were combined to direct hepatic differentiation of human H1 cells via a four-stage protocol, and the coupled impacts of stiffness and topography were quantified at distinct stages. Data indicated that the substrate stiffness is dominant in stemness maintenance on stiff gel and hepatic differentiation on soft gel while substrate topography assists the differentiation of hepatocyte-like cells in positive correlation with the circularity of H1 clones initially formed on the substrate. The differentiated cells exhibited liver-specific functions such as maintaining the capacities of CYP450 metabolism, glycogen synthesis, ICG engulfment, and repairing liver injury in CCl4-treated mice. These results implied that the coupling of substrate stiffness and topography, combined with the biochemical signals, is favorable to improve the efficiency and functionality of hepatic differentiation of human ESCs.


Assuntos
Células-Tronco Embrionárias Humanas , Animais , Diferenciação Celular , Células Cultivadas , Células-Tronco Embrionárias , Fígado , Camundongos
5.
Mol Plant ; 14(6): 949-962, 2021 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-33722761

RESUMO

Leaf shape is highly variable within and among plant species, ranging from slender to oval shaped. This is largely determined by the proximodistal axis of growth. However, little is known about how proximal-distal growth is controlled to determine leaf shape. Here, we show that Arabidopsis leaf and sepal proximodistal growth is tuned by two phytohormones. Two class A AUXIN RESPONSE FACTORs (ARFs), ARF6 and ARF8, activate the transcription of DWARF4, which encodes a key brassinosteroid (BR) biosynthetic enzyme. At the cellular level, the phytohormones promote more directional cell expansion along the proximodistal axis, as well as final cell sizes. BRs promote the demethyl-esterification of cell wall pectins, leading to isotropic in-plane cell wall loosening. Notably, numerical simulation showed that isotropic cell wall loosening could lead to directional cell and organ growth along the proximodistal axis. Taken together, we show that auxin acts through biosynthesis of BRs to determine cell wall mechanics and directional cell growth to generate leaves of variable roundness.

6.
Nanoscale ; 13(12): 6053-6065, 2021 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-33683247

RESUMO

Conformational changes of proteins play a vital role in implementing their functions and revealing the underlying mechanisms in various biological processes. It is still challenging to monitor protein conformations with temporal fingerprints of current-resistance pulses in the nanopore technique. Here the low-resolution morphologies of different conformations of a typical integrin, αxß2, were estimated via relative blockade currents simulated from all-atom molecular dynamics (MD). Distinct conformational states of αxß2 were directly explained by the volume and shape identifiers. Protein modulation in ionic current was analyzed from the conductivity distribution inside the protein-blocked nanopore. Combining a discrete model with spheroidal approximation, a MD-based approach was developed to theoretically predict the volume and shape of the nanopore for sensing αxß2. This method was also applicable in specifying morphological identifiers of six other proteins, and the theoretical predictions are in good agreement with the experimental measurements. These results potentiated the validity of this method for the conformational identification of proteins in nanopores.


Assuntos
Nanoporos , Simulação de Dinâmica Molecular , Conformação Proteica , Transporte Proteico , Proteínas
7.
Biomech Model Mechanobiol ; 20(1): 205-222, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-32809130

RESUMO

Human embryonic stem cells (hESCs) can differentiate to three germ layers within biochemical and biomechanical niches. The complicated mechanical environments in vivo could have diverse effects on the fate decision and biological functions of hESCs. To globally screen mechanosensitive molecules, three typical types of mechanical stimuli, i.e., tensile stretch, shear flow, and mechanical compression, were applied in respective parameter sets of loading pattern, amplitude, frequency, and/or duration, and then, iTRAQ proteomics test was used for identifying and quantifying differentially expressed proteins in hESCs. Bioinformatics analysis identified 37, 41, and 23 proteins under stretch pattern, frequency, and duration, 13, 18, and 41 proteins under shear pattern, amplitude, and duration, and 4, 0, and 183 proteins under compression amplitude, frequency, and duration, respectively, where distinct parameters yielded the differentially weighted preferences under each stimulus. Ten mechanosensitive proteins were commonly shared between two of three mechanical stimuli, together with numerous proteins identified under single stimulus. More importantly, functional GSEA and WGCNA analyses elaborated the variations of the screened proteins with loading parameters. Common functions in protein synthesis and modification were identified among three stimuli, and specific functions were observed in skin development under stretch alone. In conclusion, mechanomics analysis is indispensable to map actual mechanosensitive proteins under physiologically mimicking mechanical environment, and sheds light on understanding the core hub proteins in mechanobiology.

8.
Artigo em Inglês | MEDLINE | ID: mdl-33296275

RESUMO

Extracellular matrix rigidity has important effects on cell behaviors and is increased sharply during liver fibrosis and cirrhosis. Hepatic blood flow is essential in maintaining hepatocytes (HC) functions. However, it is still unclear how matrix stiffness and shear stresses orchestrate HC phenotype in concert. A fibrotic 3D liver sinusoidal model is constructed using a porous membrane sandwiched between two PDMS layers with respective flow channels. The HC are cultured in collagen gels of various stiffness in the lower channel, while the upper channel is pre-seeded with liver sinusoidal endothelial cells (LSEC) and accessible to shear flow. The results reveal that HC cultured within stiffer matrices exhibit less albumin production and cytochrome P450 (CYP450) reductase expression. Low shear stresses enhance synthetic and metabolic functions of HC, while high shear stresses lead to the loss of HC phenotype. Furthermore, both two mechanical factors regulate HC functions in a cooperative way by complementing with each other. These observations are likely attributed to mechanically-induced mass transport or key signaling molecule of hepatocyte nuclear factor 4 alpha (HNF4α). Present results provide an insight in understanding the mechanisms of HC dysfunction in liver fibrosis and cirrhosis especially from viewpoint of matrix stiffness and blood flow.

9.
Curr Biol ; 30(20): 3972-3985.e6, 2020 10 19.
Artigo em Inglês | MEDLINE | ID: mdl-32916107

RESUMO

Plant organs can adopt a wide range of shapes, resulting from highly directional cell growth and divisions. We focus here on leaves and leaf-like organs in Arabidopsis and tomato, characterized by the formation of thin, flat laminae. Combining experimental approaches with 3D mechanical modeling, we provide evidence that leaf shape depends on cortical microtubule mediated cellulose deposition along the main predicted stress orientations, in particular, along the adaxial-abaxial axis in internal cell walls. This behavior can be explained by a mechanical feedback and has the potential to sustain and even amplify a preexisting degree of flatness, which in turn depends on genes involved in the control of organ polarity and leaf margin formation.

10.
Biophys J ; 119(5): 966-977, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32814058

RESUMO

Integrins are heterodimeric transmembrane proteins that mediate cellular adhesion and bidirectional mechanotransductions through their conformational allostery. The allosteric pathway of an I-domain-containing integrin remains unclear because of its complexity and lack of effective experiments. For a typical I-domain-containing integrin αXß2, molecular dynamics simulations were employed here to investigate the conformational dynamics in the first two steps of outside-in activation, the bindings of both the external and internal ligands. Results showed that the internal ligand binding is a prerequisite to the allosteric transmission from the α- to ß-subunits and the exertion of external force to integrin-ligand complex. The opening state of αI domain with downward movement and lower half unfolding of α7-helix ensures the stable intersubunit conformational transmission through external ligand binding first and internal ligand binding later. Reverse binding order induces a, to our knowledge, novel but unstable swingout of ß-subunit Hybrid domain with the retained close states of both αI and ßI domains. Prebinding of external ligand greatly facilitates the following internal ligand binding and vice versa. These simulations furthered the understanding in the outside-in activation of I-domain-containing integrins from the viewpoint of internal allosteric pathways.


Assuntos
Integrinas , Simulação de Dinâmica Molecular , Sítios de Ligação , Adesão Celular , Ligantes , Ligação Proteica
11.
Biochim Biophys Acta Gen Subj ; 1864(12): 129702, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-32814074

RESUMO

BACKGROUND: Liver sinusoidal endothelial cells (LSECs) display unique fenestrated morphology. Alterations in the size and number of fenestrae play a crucial role in the progression of various liver diseases. While their features have been visualized using atomic force microscopy (AFM), the in situ imaging methods and off-line analyses are further required for fenestra quantification. METHODS: Primary mouse LSECs were cultured on a collagen-I-coated culture dish, or a polydimethylsiloxane (PDMS) or polyacrylamide (PA) hydrogel substrate. An AFM contact mode was applied to visualize fenestrae on individual fixed LSECs. Collected images were analyzed using an in-house developed image recognition program based on fully convolutional networks (FCN). RESULTS: Key scanning parameters were first optimized for visualizing the fenestrae on LSECs on culture dish, which was also applicable for the LSECs cultured on various hydrogels. The intermediate-magnification morphology images of LSECs were used for developing the FCN-based, fenestra recognition program. This program enabled us to recognize the vast majority of fenestrae from AFM images after twice trainings at a typical accuracy of 81.6% on soft substrate and also quantify the statistics of porosity, number of fenestrae and distribution of fenestra diameter. CONCLUSIONS: Combining AFM imaging with FCN training is able to quantify the morphological distributions of LSEC fenestrae on various substrates. SIGNIFICANCE: AFM images acquired and analyzed here provided the global information of surface ultramicroscopic structures over an entire cell, which is fundamental in understanding their regulatory mechanisms and pathophysiological relevance in fenestra-like evolution of individual cells on stiffness-varied substrates.


Assuntos
Células Endoteliais/citologia , Fígado/citologia , Microscopia de Força Atômica , Animais , Células Cultivadas , Aprendizado Profundo , Células Endoteliais/ultraestrutura , Fígado/ultraestrutura , Masculino , Camundongos Endogâmicos C57BL , Microscopia de Força Atômica/métodos
12.
J Integr Plant Biol ; 62(12): 1853-1867, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32725947

RESUMO

The shape of comparable tissues and organs is consistent among individuals of a given species, but how this consistency or robustness is achieved remains an open question. The interaction between morphogenetic factors determines organ formation and subsequent shaping, which is ultimately a mechanical process. Using a computational approach, we show that the epidermal layer is essential for the robustness of organ geometry control. Specifically, proper epidermal restriction allows organ asymmetry maintenance, and the tensile epidermal layer is sufficient to suppress local variability in growth, leading to shape robustness. The model explains the enhanced organ shape variations in epidermal mutant plants. In addition, differences in the patterns of epidermal restriction may underlie the initial establishment of organ asymmetry. Our results show that epidermal restriction can answer the longstanding question of how cellular growth noise is averaged to produce precise organ shapes, and the findings also shed light on organ asymmetry establishment.


Assuntos
Arabidopsis/citologia , Arabidopsis/metabolismo , Epiderme Vegetal/citologia , Epiderme Vegetal/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo
13.
APL Bioeng ; 4(2): 026107, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32548541

RESUMO

Cyclic myometrial contractions of the non-pregnant uterus induce intra-uterine peristaltic flows, which have important roles in transport of sperm and embryos during early stages of reproduction. Hyperperistalsis in young females may lead to migration of endometrial cells and development of adenomyosis or endometriosis. We conducted an in vitro study of the biological response of a tissue engineered endometrial barrier exposed to peristaltic wall shear stresses (PWSSs). The endometrial barrier model was co-cultured of endometrial epithelial cells on top of myometrial smooth muscle cells (MSMCs) in custom-designed wells that can be disassembled for mechanobiology experiments. A new experimental setup was developed for exposing the uterine wall in vitro model to PWSSs that mimic the in vivo intra-uterine environment. Peristaltic flow was induced by moving a belt with bulges to deform the elastic cover of a fluid filled chamber that held the uterine wall model at the bottom. The in vitro biological model was exposed to peristaltic flows for 60 and 120 min and then stained for immunofluorescence studies of alternations in the cytoskeleton. Quantification of the F-actin mass in both layers revealed a significant increase with the length of exposure to PWSSs. Moreover, the inner layer of MSMCs that were not in direct contact with the fluid also responded with an increase in the F-actin mass. This new experimental approach can be expanded to in vitro studies of multiple structural changes and genetic expressions, while the tissue engineered uterine wall models are tested under conditions that mimic the in vivo physiological environment.

14.
Exp Cell Res ; 387(2): 111807, 2020 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-31891683

RESUMO

Lymphocyte function-associated antigen-1 (LFA-1) and macrophage-1 antigen (Mac-1) are key adhesion receptors to mediate neutrophil (PMN) recruitment and intracellular calcium (Ca2+) signaling. Binding of LFA-1 and Mac-1 to their ligands is essential in triggering Ca2+ transients and activating Ca2+-dependent kinases involved in cytoskeletal remodeling and migratory function. While mechanical forces are critical in regulating integrin-mediated Ca2+ transients, it is still unclear how the bond strength of ß2-integrin-ligand pair affects Ca2+ responses. Here three typical ligands with known mechanical features with LFA-1 and Mac-1 in our previous work were adopted to quantify their capabilities in inducing Ca2+ transients in adherent PMNs under shear flow. Data indicated that LFA-1 dominates Ca2+ transients in PMNs on intercellular adhesive molecule 1 (ICAM-1) and junctional adhesion molecule-A (JAM-A), while Mac-1 mediates Ca2+ transients induced by receptor for advanced glycation end products (RAGE), consistent with their corresponding bond strengths. These results link ß2 integrin-ligand bond strength with Ca2+ transients in PMNs, suggesting high bond strength gives rise to strong Ca2+ response especially under physiological-like shear flow. The outcomes provide a new insight in understanding the mechanical regulatory mechanisms of PMN recruitment.


Assuntos
Cálcio/metabolismo , Integrinas/metabolismo , Animais , Adesão Celular/fisiologia , Molécula 1 de Adesão Intercelular/metabolismo , Ligantes , Antígeno-1 Associado à Função Linfocitária/metabolismo , Antígeno de Macrófago 1/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neutrófilos/metabolismo , Receptor para Produtos Finais de Glicação Avançada/metabolismo , Transdução de Sinais/fisiologia
15.
EMBO J ; 39(3): e102374, 2020 02 03.
Artigo em Inglês | MEDLINE | ID: mdl-31830314

RESUMO

Renewal of integumentary organs occurs cyclically throughout an organism's lifetime, but the mechanism that initiates each cycle remains largely unknown. In a miniature pig model of tooth development that resembles tooth development in humans, the permanent tooth did not begin transitioning from the resting to the initiation stage until the deciduous tooth began to erupt. This eruption released the accumulated mechanical stress inside the mandible. Mechanical stress prevented permanent tooth development by regulating expression and activity of the integrin ß1-ERK1-RUNX2 axis in the surrounding mesenchyme. We observed similar molecular expression patterns in human tooth germs. Importantly, the release of biomechanical stress induced downregulation of RUNX2-wingless/integrated (Wnt) signaling in the mesenchyme between the deciduous and permanent tooth and upregulation of Wnt signaling in the epithelium of the permanent tooth, triggering initiation of its development. Consequently, our findings identified biomechanical stress-associated Wnt modulation as a critical initiator of organ renewal, possibly shedding light on the mechanisms of integumentary organ regeneration.


Assuntos
Regulação para Baixo , Odontogênese , Via de Sinalização Wnt , Animais , Fenômenos Biomecânicos , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Saco Dentário/citologia , Saco Dentário/metabolismo , Humanos , Integrina beta1/metabolismo , Modelos Biológicos , Cultura Primária de Células , Suínos , Porco Miniatura
16.
Stem Cell Res Ther ; 10(1): 349, 2019 11 27.
Artigo em Inglês | MEDLINE | ID: mdl-31775893

RESUMO

BACKGROUND: Distinct mechanical stimuli are known to manipulate the behaviors of embryonic stem cells (ESCs). Fundamental rationale of how ESCs respond to mechanical forces and the potential biological effects remain elusive. Here we conducted the mechanobiological study for hESCs upon mechanomics analysis to unravel typical mechanosensitive processes on hESC-specific fluid shear. METHODS: hESC line H1 was subjected to systematically varied shear flow, and mechanosensitive proteins were obtained by mass spectrometry (MS) analysis. Then, function enrichment analysis was performed to identify the enriched gene sets. Under a steady shear flow of 1.1 Pa for 24 h, protein expressions were further detected using western blotting (WB), quantitative real-time PCR (qPCR), and immunofluorescence (IF) staining. Meanwhile, the cells were treated with 200 nM trichostatin (TSA) for 1 h as positive control to test chromatin decondensation. Actin, DNA, and RNA were then visualized with TRITC-labeled phalloidin, Hoechst 33342, and SYTO® RNASelect™ green fluorescent cell stain (Life Technologies), respectively. In addition, cell stiffness was determined with atomic force microscopy (AFM) and annexin V-PE was used to determine the apoptosis with a flow cytometer (FCM). RESULTS: Typical mechanosensitive proteins were unraveled upon mechanomics analysis under fluid shear related to hESCs in vivo. Functional analyses revealed significant alterations in histone acetylation, nuclear size, and cytoskeleton for hESC under shear flow. Shear flow was able to induce H2B acetylation and nuclear spreading by CFL2/F-actin cytoskeletal reorganization. The resulting chromatin decondensation and a larger nucleus readily accommodate signaling molecules and transcription factors. CONCLUSIONS: Shear flow regulated chromatin dynamics in hESCs via cytoskeleton and nucleus alterations and consolidated their primed state.


Assuntos
Montagem e Desmontagem da Cromatina , Histonas/metabolismo , Células-Tronco Embrionárias Humanas/metabolismo , Mecanotransdução Celular , Processamento de Proteína Pós-Traducional , Resistência ao Cisalhamento , Acetilação , Linhagem Celular , Citoesqueleto/metabolismo , Células-Tronco Embrionárias Humanas/citologia , Humanos
17.
Biophys J ; 117(8): 1405-1418, 2019 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-31585706

RESUMO

Global cytoskeleton reorganization is well-recognized when cells are exposed to distinct mechanical stimuli, but the localized responses at a specified region of a cell are still unclear. In this work, we mapped the cell-surface mechanical property of single cells in situ before and after static point loading these cells using atomic force microscopy in PeakForce-Quantitative Nano Mechanics mode. Cell-surface stiffness was elevated at a maximum of 1.35-fold at the vicinity of loading site, indicating an enhanced structural protection of the cortex to the cell. Mechanical modeling also elucidated the structural protection from the stiffened cell cortex, in which 9-15% and 10-19% decrease of maximum stress and strain of the nucleus were obtained. Furthermore, the flat-ended atomic force microscopy probes were used to capture cytoskeleton reorganization after point loading quantitatively, revealing that the larger the applied force and the longer the loading time are, the more pronounced cytoskeleton reorganization is. Also, point loading using a microneedle combined with real-time confocal microscopy uncovered the fast dynamics of actin cytoskeleton reorganization for actin-stained live cells after point loading (<10 s). These results furthered the understandings in the transmission of localized mechanical forces into an adherent cell.


Assuntos
Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/ultraestrutura , Células HeLa , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Microscopia de Força Atômica , Estresse Mecânico
18.
Rev Sci Instrum ; 90(7): 075114, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31370504

RESUMO

Pathophysiological changes of astronauts under space microgravity involve complex factors and require an integrative perspective to fully understand the mechanisms. The readouts from space cell biology experiments strongly depend on the hardware and especially the cell bioreactor that is used in distinct spacecraft. Herein, a specialized cell culture bioreactor is designed for culturing mammalian cells on board the SJ-10 satellite. This hardware focuses mainly on satisfying the requirements of gas exchange, bubble separation, and flow control, as well as their functional and structural integration on cell culture within the technical and environmental constraints of the spacecraft platform under microgravity. A passive bubble separator is constructed and is connected in series to an individual cell culture chamber to remove the bubbles that were produced in orbit during cell growth. A moderate flow rate is preset to provide sufficient mass transfer and low shear stress in a well-designed flow circuit. Together with other modules of temperature control, in situ microscopic imaging, and online imaging acquisition, this novel space cell culture system is successfully used to culture human endothelial cells and rat bone marrow-derived mesenchymal stem cells in the SJ-10 mission. The advantages and shortcomings of the integration design are discussed for this type of the hardware.

19.
APL Bioeng ; 3(1): 016104, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31069337

RESUMO

Atherosclerosis is caused by chronic inflammation associated with the adhesion of neutrophils and endothelial cells (ECs) that is mediated by their respective cellular adhesive molecules to stiffened blood vessel walls. However, the stiffness dependence of calcium flux on neutrophils remains unclear yet. Here, the effect of substrate stiffness by ECs on neutrophils' calcium spike was quantified when the individual neutrophils that adhered to the human umbilical vascular endothelial cell (HUVEC) monolayer were pre-placed onto a stiffness-varied polyacrylamide substrate (5 or 34.88 kPa) or glass surface. Our data indicated that E-/P-selectins and intercellular adhesion molecule 1 (ICAM-1) on HUVECs and ß2-integrins, P-selectin glycoprotein ligand 1 (PSGL-1), and CD44s on neutrophils were all involved in mediating neutrophil calcium spike in a stiffness-dependent manner, in which the increase in substrate stiffness enhanced the calcium intensity and the oscillation frequency (spike number). Such stiffness-dependent calcium response is associated with the induced selectin related to ß2-integrin activation through the Syk/Src signaling pathway, and F-actin/myosin II are also involved in this. Moreover, tension-activated calcium ion channels displayed critical roles in initiating stiffness-dependent calcium spike. These results provide an insight into understanding how the stiffening of vascular walls could regulate the calcium flux of adhered neutrophils, and thus the immune responses in atherosclerosis.

20.
Am J Physiol Cell Physiol ; 316(5): C678-C689, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-30726114

RESUMO

L-selectin shedding induced by various cytokines is crucial in activating neutrophils (PMNs) in inflammatory cascade. While the real-time shedding in vivo lasts ~10 min after PMN activation, the impact of time-dependent shedding on binding kinetics of membrane-remaining L-selectins to its ligands is poorly understood at transient or steady state. Here, we developed an in vitro L-selectin shedding dynamics approach, together with competitive assays of cell adhesion, and proposed a theoretical model for quantifying the impact of real-time shedding on the binding kinetics of membrane-remaining L-selectins to P-selectin glycoprotein ligand-1 (PSGL-1). Our data indicated that the extent of L-selectin shedding on PMA activation is higher, but the terminating time is longer for Jurkat cells than those for human PMNs. Meanwhile, fMLF or IL-8 stimulation yields the longer terminating time than that on PMA stimulation but results in a similar shedding extent for PMNs. L-selectin shedding reduces L-selectin-PSGL-1-mediated cell adhesion in three ways: decreasing membrane-anchored L-selectins, increasing soluble L-selectins competitively binding to ligands, and presenting conformational alteration of membrane-remaining L-selectins themselves. Compared with those on intact cells, the binding affinities of membrane-remaining L-selectin-PSGL-1 pairs were all enhanced at initial and lowered at the late shedding phase for both PMN and Jurkat cells even with varied transition time points. The rolling velocities of both PMNs and Jurkat cells were increased following mechanically or biochemically induced shedding of L-selectin under shear flow. These findings help to further our understanding of the function of time-dependent L-selectin shedding during the inflammation cascade.


Assuntos
Membrana Celular/metabolismo , Micropartículas Derivadas de Células/metabolismo , Selectina L/metabolismo , Glicoproteínas de Membrana/metabolismo , Neutrófilos/metabolismo , Humanos , Células Jurkat , Cinética , Ligação Proteica/fisiologia
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