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1.
Nat Commun ; 11(1): 5995, 2020 11 25.
Artigo em Inglês | MEDLINE | ID: mdl-33239609

RESUMO

Infectious diseases are worldwide a major cause of morbidity and mortality. Fast and specific detection of pathogens such as bacteria is needed to combat these diseases. Optimal methods would be non-invasive and without extensive sample-taking/processing. Here, we developed a set of near infrared (NIR) fluorescent nanosensors and used them for remote fingerprinting of clinically important bacteria. The nanosensors are based on single-walled carbon nanotubes (SWCNTs) that fluoresce in the NIR optical tissue transparency window, which offers ultra-low background and high tissue penetration. They are chemically tailored to detect released metabolites as well as specific virulence factors (lipopolysaccharides, siderophores, DNases, proteases) and integrated into functional hydrogel arrays with 9 different sensors. These hydrogels are exposed to clinical isolates of 6 important bacteria (Staphylococcus aureus, Escherichia coli,…) and remote (≥25 cm) NIR imaging allows to identify and distinguish bacteria. Sensors are also spectrally encoded (900 nm, 1000 nm, 1250 nm) to differentiate the two major pathogens P. aeruginosa as well as S. aureus and penetrate tissue (>5 mm). This type of multiplexing with NIR fluorescent nanosensors enables remote detection and differentiation of important pathogens and the potential for smart surfaces.

2.
Chempluschem ; 85(7): 1465-1480, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32644301

RESUMO

Cells use biomolecules to convey information. For instance, neurons communicate by releasing chemicals called neurotransmitters, including several monoamines. The information transmitted by neurons is, in part, coded in the type and amount of neurotransmitter released, the spatial distribution of release sites, the frequency of release events, and the diffusion range of the neurotransmitter. Therefore, quantitative information about neurotransmitters at the (sub)cellular level with high spatiotemporal resolution is needed to understand how complex cellular networks function. So far, various analytical methods have been developed and used to detect neurotransmitter secretion from cells. However, each method has limitations with respect to chemical, temporal and spatial resolution. In this review, we focus on emerging methods for optical detection of neurotransmitter release and discuss fluorescent sensors/probes for monoamine neurotransmitters such as dopamine and serotonin. We focus on the latest advances in near infrared fluorescent carbon nanotube-based sensors and engineered fluorescent proteins for monoamine imaging, which provide high spatial and temporal resolution suitable for examining the release of monoamines from cells in cellular networks.

3.
Artigo em Inglês | MEDLINE | ID: mdl-32511874

RESUMO

Single-walled carbon nanotubes (SWCNTs) are a 1D nanomaterial that shows fluorescence in the near-infrared (NIR, >800 nm). In the past, covalent chemistry was less explored to functionalize SWCNTs as it impairs NIR emission. However, certain sp3 defects (quantum defects) in the carbon lattice have emerged that preserve NIR fluorescence and even introduce a new, red-shifted emission peak. Here, we report on quantum defects, introduced using light-driven diazonium chemistry, that serve as anchor points for peptides and proteins. We show that maleimide anchors allow conjugation of cysteine-containing proteins such as a GFP-binding nanobody. In addition, an Fmoc-protected phenylalanine defect serves as a starting point for conjugation of visible fluorophores to create multicolor SWCNTs and in situ peptide synthesis directly on the nanotube. Therefore, these quantum defects are a versatile platform to tailor both the nanotube's photophysical properties as well as their surface chemistry.

4.
Curr Biol ; 30(13): 2564-2573.e5, 2020 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-32470369

RESUMO

Many aspects in tissue morphogenesis are attributed to a collective behavior of the participating cells. Yet, the mechanism for emergence of dynamic tissue behavior is not well understood. Here, we report that the "yo-yo"-like nuclear movement in the Drosophila syncytial embryo displays emergent features indicative of collective behavior. Following mitosis, the array of nuclei moves away from the wave front by several nuclear diameters only to return to its starting position about 5 min later. Based on experimental manipulations and numerical simulations, we find that the ensemble of elongating and isotropically oriented spindles, rather than individual spindles, is the main driving force for anisotropic nuclear movement. ELMO-dependent F-actin restricts the time for the forward movement and ELMO- and dia-dependent F-actin is essential for the return movement. Our study provides insights into how the interactions among the cytoskeleton as individual elements lead to collective movement of the nuclear array on a macroscopic scale.

5.
Nanoscale ; 12(16): 9104-9115, 2020 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-32286598

RESUMO

Cells can take up nanoscale materials, which has important implications for understanding cellular functions, biocompatibility as well as biomedical applications. Controlled uptake, transport and triggered release of nanoscale cargo is one of the great challenges in biomedical applications of nanomaterials. Here, we study how human immune cells (neutrophilic granulocytes, neutrophils) take up nanomaterials and program them to release this cargo after a certain time period. For this purpose, we let neutrophils phagocytose DNA-functionalized single-walled carbon nanotubes (SWCNTs) in vitro that fluoresce in the near infrared (980 nm) and serve as sensors for small molecules. Cells still migrate, follow chemical gradients and respond to inflammatory signals after uptake of the cargo. To program release, we make use of neutrophil extracellular trap formation (NETosis), a novel cell death mechanism that leads to chromatin swelling, subsequent rupture of the cellular membrane and release of the cell's whole content. By using the process of NETosis, we can program the time point of cargo release via the initial concentration of stimuli such as phorbol 12-myristate-13-acetate (PMA) or lipopolysaccharide (LPS). At intermediate stimulation, cells continue to migrate, follow gradients and surface cues for around 30 minutes and up to several hundred micrometers until they stop and release the SWCNTs. The transported and released SWCNT sensors are still functional as shown by subsequent detection of the neurotransmitter dopamine and reactive oxygen species (H2O2). In summary, we hijack a biological process (NETosis) and demonstrate how neutrophils transport and release functional nanomaterials.

6.
J Cell Sci ; 133(5)2020 03 10.
Artigo em Inglês | MEDLINE | ID: mdl-32156720

RESUMO

Neutrophil extracellular traps (NETs) are one of the most intriguing discoveries in immunological research of the past few years. After their first description in 2004, the number of research articles on how NETs affect immunodefense, and also how they contribute to an ever-growing number of diseases, has skyrocketed. However, tempting as it may seem to plunge into pharmaceutical approaches to tamper with NET formation, our understanding of this complex process is still incomplete. Important concepts such as the context-dependent dual functions of NETs, in that they are both inflammatory and anti-inflammatory, or the major intra- and extracellular forces driving NET formation, are only emerging. In this Review, we summarize key aspects of our current understanding of NET formation (also termed NETosis), emphasize biophysical aspects and focus on three key principles - rearrangement and destabilization of the plasma membrane and the cytoskeleton, alterations and disassembly of the nuclear envelope, and chromatin decondensation as a driving force of intracellular reorganization.

8.
Nat Commun ; 11(1): 1495, 2020 03 20.
Artigo em Inglês | MEDLINE | ID: mdl-32198383

RESUMO

Imaging of complex (biological) samples in the near-infrared (NIR) is beneficial due to reduced light scattering, absorption, phototoxicity, and autofluorescence. However, there are few NIR fluorescent materials known and suitable for biomedical applications. Here we exfoliate the layered pigment CaCuSi4O10 (Egyptian Blue, EB) via ball milling and facile tip sonication into NIR fluorescent nanosheets (EB-NS). The size of EB-NS can be tailored to diameters <20 nm and heights down to 1 nm. EB-NS fluoresce at 910 nm and the fluorescence intensity correlates with the number of Cu2+ ions. Furthermore, EB-NS display no bleaching and high brightness compared with other NIR fluorophores. The versatility of EB-NS is demonstrated by in-vivo single-particle tracking and microrheology measurements in Drosophila melanogaster embryos. EB-NS can be uptaken by plants and remotely detected in a low-cost stand-off detection setup. In summary, EB-NS have the potential for a wide range of bioimaging applications.


Assuntos
Corantes Fluorescentes/efeitos da radiação , Raios Infravermelhos , Imagem Óptica/métodos , Óptica e Fotônica/métodos , Silicatos/efeitos da radiação , Animais , Cobre , Drosophila melanogaster/embriologia , Fluorescência , Íons , Modelos Teóricos , Nanopartículas
9.
Nano Lett ; 20(4): 2432-2442, 2020 04 08.
Artigo em Inglês | MEDLINE | ID: mdl-32097014

RESUMO

Near-infrared (nIR) fluorescent single-walled carbon nanotubes (SWCNTs) were designed and interfaced with leaves of Arabidopsis thaliana plants to report hydrogen peroxide (H2O2), a key signaling molecule associated with the onset of plant stress. The sensor nIR fluorescence response (>900 nm) is quenched by H2O2 with selectivity against other stress-associated signaling molecules and within the plant physiological range (10-100 H2O2 µM). In vivo remote nIR imaging of H2O2 sensors enabled optical monitoring of plant health in response to stresses including UV-B light (-11%), high light (-6%), and a pathogen-related peptide (flg22) (-10%), but not mechanical leaf wounding (<3%). The sensor's high biocompatibility was reflected on similar leaf cell death (<5%) and photosynthetic rates to controls without SWCNT. These optical nanosensors report early signs of stress and will improve our understanding of plant stress communication, provide novel tools for precision agriculture, and optimize the use of agrochemicals in the environment.

10.
J Biophotonics ; 13(1): e201960080, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31602799

RESUMO

Multispectral imaging combines the spectral resolution of spectroscopy with the spatial resolution of imaging and is therefore very useful for biomedical applications. Currently, histological diagnostics use mainly stainings with standard dyes (eg, hematoxylin + eosin) to identify tumors. This method is not applicable in vivo and provides low amounts of chemical information. Biomolecules absorb near infrared light (NIR, 800-1700 nm) at different wavelengths, which could be used to fingerprint tissue. Here, we built a NIR multispectral absorption imaging setup to study skin tissue samples. NIR light (900-1500 nm) was used for homogenous wide-field transmission illumination and detected by a cooled InGaAs camera. In this setup, images I(x, y, λ) from dermatological samples (melanoma, nodular basal-cell carcinoma, squamous-cell carcinoma) were acquired to distinguish healthy from diseased tissue regions. In summary, we show the potential of multispectral NIR imaging for cancer diagnostics.

11.
Front Immunol ; 10: 2428, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31708915

RESUMO

Neutrophil Extracellular Traps (NETs) are produced by neutrophilic granulocytes and consist of decondensed chromatin decorated with antimicrobial peptides. They defend the organism against intruders and are released upon various stimuli including pathogens, mediators of inflammation, or chemical triggers. NET formation is also involved in inflammatory, cardiovascular, malignant diseases, and autoimmune disorders like rheumatoid arthritis, psoriasis, or systemic lupus erythematosus (SLE). In many autoimmune diseases like SLE or dermatomyositis, light of the ultraviolet-visible (UV-VIS) spectrum is well-known to trigger and aggravate disease severity. However, the underlying connection between NET formation, light exposure, and disease exacerbation remains elusive. We studied the effect of UVA (375 nm), blue (470 nm) and green (565 nm) light on NETosis in human neutrophils ex vivo. Our results show a dose- and wavelength-dependent induction of NETosis. Light-induced NETosis depended on the generation of extracellular reactive oxygen species (ROS) induced by riboflavin excitation and its subsequent reaction with tryptophan. The light-induced NETosis required both neutrophil elastase (NE) as well as myeloperoxidase (MPO) activation and induced histone citrullination. These findings suggest that NET formation as a response to light could be the hitherto missing link between elevated susceptibility to NET formation in autoimmune patients and photosensitivity for example in SLE and dermatomyositis patients. This novel connection could provide a clue for a deeper understanding of light-sensitive diseases in general and for the development of new pharmacological strategies to avoid disease exacerbation upon light exposure.


Assuntos
Armadilhas Extracelulares/efeitos da radiação , Neutrófilos/efeitos da radiação , Raios Ultravioleta , Doenças Autoimunes/etiologia , Doenças Autoimunes/metabolismo , Relação Dose-Resposta à Radiação , Armadilhas Extracelulares/fisiologia , Humanos , Elastase de Leucócito/fisiologia , Neutrófilos/fisiologia , Peroxidase/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Riboflavina/química
12.
Front Immunol ; 10: 2320, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31632402

RESUMO

Neutrophils are the most abundant type of white blood cells. Upon stimulation, they are able to decondense and release their chromatin as neutrophil extracellular traps (NETs). This process (NETosis) is part of immune defense mechanisms but also plays an important role in many chronic and inflammatory diseases such as atherosclerosis, rheumatoid arthritis, diabetes, and cancer. For this reason, much effort has been invested into understanding biochemical signaling pathways in NETosis. However, the impact of the mechanical micro-environment and adhesion on NETosis is not well-understood. Here, we studied how adhesion and especially substrate elasticity affect NETosis. We employed polyacrylamide (PAA) gels with distinctly defined elasticities (Young's modulus E) within the physiologically relevant range from 1 to 128 kPa and coated the gels with integrin ligands (collagen I, fibrinogen). Neutrophils were cultured on these substrates and stimulated with potent inducers of NETosis: phorbol 12-myristate 13-acetate (PMA) and lipopolysaccharide (LPS). Interestingly, PMA-induced NETosis was neither affected by substrate elasticity nor by different integrin ligands. In contrast, for LPS stimulation, NETosis rates increased with increasing substrate elasticity (E > 20 kPa). LPS-induced NETosis increased with increasing cell contact area, while PMA-induced NETosis did not require adhesion at all. Furthermore, inhibition of phosphatidylinositide 3 kinase (PI3K), which is involved in adhesion signaling, completely abolished LPS-induced NETosis but only slightly decreased PMA-induced NETosis. In summary, we show that LPS-induced NETosis depends on adhesion and substrate elasticity while PMA-induced NETosis is completely independent of adhesion.


Assuntos
Armadilhas Extracelulares/imunologia , Imunidade Inata , Neutrófilos/imunologia , Neutrófilos/metabolismo , Biomarcadores , Adesão Celular/imunologia , Elasticidade , Armadilhas Extracelulares/efeitos dos fármacos , Humanos , Imunomodulação , Inflamação/etiologia , Inflamação/metabolismo , Inflamação/patologia , Lipopolissacarídeos/imunologia , Modelos Biológicos , Neutrófilos/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase/farmacologia
13.
Nano Lett ; 19(9): 6604-6611, 2019 09 11.
Artigo em Inglês | MEDLINE | ID: mdl-31418577

RESUMO

Serotonin is an important neurotransmitter involved in various functions of the nervous, blood, and immune system. In general, detection of small biomolecules such as serotonin in real time with high spatial and temporal resolution remains challenging with conventional sensors and methods. In this work, we designed a near-infrared (nIR) fluorescent nanosensor (NIRSer) based on fluorescent single-walled carbon nanotubes (SWCNTs) to image the release of serotonin from human blood platelets in real time. The nanosensor consists of a nonbleaching SWCNT backbone, which is fluorescent in the beneficial nIR tissue transparency window (800-1700 nm) and a serotonin binding DNA aptamer. The fluorescence of the NIRSer sensor (995 nm emission wavelength for (6,5)-SWCNTs) increases in response to serotonin by a factor up to 1.8. It detects serotonin reversibly with a dissociation constant of 301 nM ± 138 nM and a dynamic linear range in the physiologically relevant region from 100 nM to 1 µM. As a proof of principle, we detected serotonin release patterns from activated platelets on the single-cell level. Imaging of the nanosensors around and under the platelets enabled us to locate hot spots of serotonin release and quantify the time delay (≈ 21-30 s) between stimulation and release in a population of platelets, highlighting the spatiotemporal resolution of this nanosensor approach. In summary, we report a nIR fluorescent nanosensor for the neurotransmitter serotonin and show its potential for imaging of chemical communication between cells.


Assuntos
Técnicas Biossensoriais , Plaquetas/metabolismo , Corantes Fluorescentes/química , Nanotubos de Carbono/química , Serotonina/metabolismo , Plaquetas/ultraestrutura , Humanos
14.
Nat Nanotechnol ; 14(6): 541-553, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31168083

RESUMO

Nanobiotechnology has the potential to enable smart plant sensors that communicate with and actuate electronic devices for improving plant productivity, optimize and automate water and agrochemical allocation, and enable high-throughput plant chemical phenotyping. Reducing crop loss due to environmental and pathogen-related stresses, improving resource use efficiency and selecting optimal plant traits are major challenges in plant agriculture industries worldwide. New technologies are required to accurately monitor, in real time and with high spatial and temporal resolution, plant physiological and developmental responses to their microenvironment. Nanomaterials are allowing the translation of plant chemical signals into digital information that can be monitored by standoff electronic devices. Herein, we discuss the design and interfacing of smart nanobiotechnology-based sensors that report plant signalling molecules associated with health status to agricultural and phenotyping devices via optical, wireless or electrical signals. We describe how nanomaterial-mediated delivery of genetically encoded sensors can act as tools for research and development of smart plant sensors. We assess performance parameters of smart nanobiotechnology-based sensors in plants (for example, resolution, sensitivity, accuracy and durability) including in vivo optical nanosensors and wearable nanoelectronic sensors. To conclude, we present an integrated and prospective vision on how nanotechnology could enable smart plant sensors that communicate with and actuate electronic devices for monitoring and optimizing individual plant productivity and resource use.


Assuntos
Técnicas Biossensoriais/métodos , Biotecnologia/métodos , Produção Agrícola/métodos , Produtos Agrícolas , Nanotecnologia/métodos , Plantas Geneticamente Modificadas , Produtos Agrícolas/genética , Produtos Agrícolas/crescimento & desenvolvimento , Humanos , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/crescimento & desenvolvimento
15.
Nanoscale ; 11(23): 11159-11166, 2019 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-31149692

RESUMO

Single-walled carbon nanotubes (SWCNTs) have unique photophysical properties and serve as building blocks for biosensors, functional materials and devices. For many applications it is crucial to use chirality-pure SWCNTs, which requires sophisticated processes. Purification procedures such as wrapping by certain polymers, phase separation, density gradient centrifugation or gel chromatography have been developed and yield distinct SWCNT species wrapped by a specific polymer or surfactant. However, many applications require a different organic functionalization (corona) around the SWCNTs instead of the one used for the purification process. Here, we present a novel efficient and straightforward process to gain chirality pure SWCNTs with tunable functionalization. Our approach uses polyfluorene (PFO) polymers to enrich certain chiralities but the polymer is removed again and finally exchanged to any desired organic phase. We demonstrate this concept by dispersing SWCNTs in poly[(9,9-dioctylfluorenyl-2,7-diyl)-alt-co-(6,6'-{2,2'-bipyridine})] (PFO-BPy), which is known to preferentially solubilize (6,5)-SWCNTs. Then PFO-BPy is removed and recycled, while letting the SWCNTs adsorb/agglomerate on sodium chloride (NaCl) crystals, which act as a toluene-stable but water-soluble filler material. In the last step these purified SWCNTs are redispersed in different polymers, surfactants and ssDNA. This corona phase exchange purification (CPEP) approach was also extended to other PFO variants to enrich and functionalize (7,5)-SWCNTs. CPEP purified and functionalized SWCNTs display monodisperse nIR spectra, which are important for fundamental studies and applications that rely on spectral changes. We show this advantage for SWCNT-based nIR fluorescent sensors for the neurotransmitter dopamine and red-shifted sp3 defect peaks . In summary, CPEP makes use of PFO polymers for chirality enrichment but provides access to chirality enriched SWCNTs functionalized in any desired polymer, surfactant or biopolymer.

16.
Angew Chem Int Ed Engl ; 58(33): 11469-11473, 2019 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-31112007

RESUMO

Fluorescent nanomaterials such as single-walled carbon nanotubes (SWCNTs) have many advantages in terms of their photophysics, but it is difficult to target them to specific locations in living systems. In contrast, the green fluorescent protein (GFP) has been genetically fused to proteins in many cells and organisms. Therefore, GFP can be seen not only as a fluorophore but as a universal target/handle. Here, we report the conjugation of GFP-binding nanobodies to DNA-wrapped SWCNTs. This approach combines the targeting capabilities of GFP-binding nanobodies and the nonbleaching near-infrared fluorescence (850-1700 nm) of SWCNTs. These conjugates allow us to track single Kinesin-5-GFP motor proteins in developing embryos of Drosophila melanogaster. Additionally, they are sensitive to the neurotransmitter dopamine and can be used for targeted sensing of dopamine in the nm regime.


Assuntos
Técnicas Biossensoriais , Raios Infravermelhos , Nanotubos de Carbono/química , Animais , DNA/química , Dopamina/química , Dopamina/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Proteínas de Fluorescência Verde , Proteínas Associadas aos Microtúbulos/metabolismo , Transporte Proteico
17.
Front Immunol ; 10: 12, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30733715

RESUMO

The formation of neutrophil extracellular traps (NETs) is an immune defense mechanism of neutrophilic granulocytes. Moreover, it is also involved in the pathogenesis of autoimmune, inflammatory, and neoplastic diseases. For that reason, the process of NET formation (NETosis) is subject of intense ongoing research. In vitro approaches to quantify NET formation are commonly used and involve neutrophil stimulation with various activators such as phorbol 12-myristate 13-acetate (PMA), lipopolysaccharides (LPS), or calcium ionophores (CaI). However, the experimental conditions of these experiments, particularly the media and media supplements employed by different research groups, vary considerably, rendering comparisons of results difficult. Here, we present the first standardized investigation of the influence of different media supplements on NET formation in vitro. The addition of heat-inactivated (hi) fetal calf serum (FCS), 0.5% human serum albumin (HSA), or 0.5% bovine serum albumin (BSA) efficiently prevented NET formation of human neutrophils following stimulation with LPS and CaI, but not after stimulation with PMA. Thus, serum components such as HSA, BSA and hiFCS (at concentrations typically found in the literature) inhibit NET formation to different degrees, depending on the NETosis inducer used. In contrast, in murine neutrophils, NETosis was inhibited by FCS and BSA, regardless of the inducer employed. This shows that mouse and human neutrophils have different susceptibilities toward the inhibition of NETosis by albumin or serum components. Furthermore, we provide experimental evidence that albumin inhibits NETosis by scavenging activators such as LPS. We also put our results into the context of media supplements most commonly used in NET research. In experiments with human neutrophils, either FCS (0.5-10%), heat-inactivated (hiFCS, 0.1-10%) or human serum albumin (HSA, 0.05-2%) was commonly added to the medium. For murine neutrophils, serum-free medium was used in most cases for stimulation with LPS and CaI, reflecting the different sensitivities of human and murine neutrophils to media supplements. Thus, the choice of media supplements greatly determines the outcome of experiments on NET-formation, which must be taken into account in NETosis research.


Assuntos
Armadilhas Extracelulares/efeitos dos fármacos , Neutrófilos/efeitos dos fármacos , Albumina Sérica/farmacologia , Soro , Animais , Biomarcadores , Ionóforos de Cálcio/farmacologia , Bovinos , Armadilhas Extracelulares/imunologia , Armadilhas Extracelulares/metabolismo , Humanos , Imuno-Histoquímica , Lipopolissacarídeos/imunologia , Lipopolissacarídeos/farmacologia , Camundongos , Neutrófilos/imunologia , Neutrófilos/metabolismo , Ligação Proteica , Soro/metabolismo , Soroalbumina Bovina/metabolismo , Soroalbumina Bovina/farmacologia , Acetato de Tetradecanoilforbol/farmacologia
18.
J Invest Dermatol ; 139(3): 562-572, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30393081

RESUMO

Loss of E-cadherin and concomitant upregulation of N-cadherin is known as the cadherin switch, and has been implicated in melanoma progression. Mechanistically, homophilic ligation of N-cadherin-expressing melanoma cells with N-cadherin presented within the microenvironment is thought to facilitate invasion. However, the biophysical aspects governing molecular specificity and function of such interactions remain unclear. By using precisely defined nano-patterns of N- or E-cadherin (with densities tunable by more than one order of magnitude from 78 to 1,128 ligands/µm2), we analyzed adhesion and spreading of six different human melanoma cell lines with distinct constitutive cadherin expression patterns. Cadherin-mediated homophilic cell interactions (N/N and E/E) with cadherin-functionalized nano-matrices revealed an unexpected functional dichotomy inasmuch as melanoma cell adhesion was cadherin density-dependent, while spreading and lamellipodia formation were independent of cadherin density. Surprisingly, E-cadherin-expressing melanoma cells also interacted with N-cadherin-presenting nano-matrices, suggesting heterophilic (N/E) interactions. However, cellular spreading in these cases occurred only at high densities of N-cadherin (i.e., >285 ligands/µm2). Overall, our approach using nano-patterned biomimetic surfaces provides a platform to further refine the roles of cadherins in tumor cell behavior and it revealed an intriguing flexibility of mutually compensating N- and E-cadherin interactions relevant for melanoma progression.


Assuntos
Antígenos CD/genética , Caderinas/genética , Regulação Neoplásica da Expressão Gênica , Melanoma/patologia , Neoplasias Cutâneas/patologia , Microambiente Tumoral , Análise de Variância , Western Blotting , Comunicação Celular/genética , Linhagem Celular Tumoral/patologia , Progressão da Doença , Citometria de Fluxo , Humanos , Melanoma/metabolismo , Nanotecnologia , Neoplasias Cutâneas/metabolismo , Regulação para Cima
19.
Nat Commun ; 9(1): 3767, 2018 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-30218080

RESUMO

Neutrophilic granulocytes are able to release their own DNA as neutrophil extracellular traps (NETs) to capture and eliminate pathogens. DNA expulsion (NETosis) has also been documented for other cells and organisms, thus highlighting the evolutionary conservation of this process. Moreover, dysregulated NETosis has been implicated in many diseases, including cancer and inflammatory disorders. During NETosis, neutrophils undergo dynamic and dramatic alterations of their cellular as well as sub-cellular morphology whose biophysical basis is poorly understood. Here we investigate NETosis in real-time on the single-cell level using fluorescence and atomic force microscopy. Our results show that NETosis is highly organized into three distinct phases with a clear point of no return defined by chromatin status. Entropic chromatin swelling is the major physical driving force that causes cell morphology changes and the rupture of both nuclear envelope and plasma membrane. Through its material properties, chromatin thus directly orchestrates this complex biological process.


Assuntos
Cromatina/ultraestrutura , DNA/ultraestrutura , Armadilhas Extracelulares/metabolismo , Neutrófilos/ultraestrutura , Morte Celular , Membrana Celular , Forma Celular , Cromatina/metabolismo , DNA/metabolismo , Entropia , Humanos , Microscopia de Força Atômica , Microscopia de Fluorescência , Neutrófilos/metabolismo , Membrana Nuclear , Análise de Célula Única
20.
ACS Appl Mater Interfaces ; 10(21): 17693-17703, 2018 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-29708725

RESUMO

Integrins are transmembrane receptors that mediate cell-adhesion, signaling cascades and platelet-mediated blood clotting. Most integrins bind to the common short peptide Arg-Gly-Asp (RGD). The conformational freedom of the RGD motif determines how strong and to which integrins it binds. Here, we present a novel approach to tune binding constants by confining RGD peptide motifs via noncovalent adsorption of single-stranded DNA (ssDNA) anchors onto single-walled carbon nanotubes (SWCNTs). Semiconducting SWCNTs display fluorescence in the near-infrared (nIR) region and are versatile fluorescent building blocks for imaging and biosensing. The basic idea of this approach is that the DNA adsorbed on the SWCNT surface determines the conformational freedom of the RGD motif and affects binding affinities. The RGD motif was conjugated to different ssDNA sequences in both linear ssDNA-RGD and bridged ssDNA-RGD-ssDNA geometries. Molecular dynamics (MD) simulations show that the RGD motif in all the synthesized systems is mostly exposed to solvent and thus available for binding, but its flexibility depends on the exact geometry. The affinity for the human platelet integrin αIIbß3 could be modulated up to 15-fold by changing the ssDNA sequence. IC50 values varied from 309 nM for (C)20-RGD/SWCNT hybrids to 29 nM for (GT)15-RGD/SWCNT hybrids. When immobilized onto surface adhesion of epithelial cells increased 6-fold for (GT)15-RGD/SWCNTs. (GT)15-RGD/SWCNTs also increased the number of adhering human platelets by a factor of 4.8. Additionally, αIIbß3 integrins on human platelets were labeled in the nIR by incubating them with these ssDNA-peptide/SWCNT hybrids. In summary, we show that ssDNA-peptide hybrid structures noncovalently adsorb onto SWCNTs and serve as recognition units for cell surface receptors such as integrins. The DNA sequence affects the overall RGD affinity, which is a versatile and straightforward approach to tune binding affinities. In combination with the nIR fluorescence properties of SWCNTs, these new hybrid materials promise many applications in integrin targeting and bioimaging.


Assuntos
Nanotubos de Carbono , DNA de Cadeia Simples , Humanos , Integrinas , Oligopeptídeos
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