Mechanical interactions of cells with their immediately surrounding extracellular matrix (ECM) is now known to be critical in pathophysiology. For example, during cancer progression, while uncontrollable cell division leads to tumor formation, the subsequent metastatic migration of cells from the primary tumor site to distant parts of the body causes most cancer-related deaths. The metastatic journey requires cells to be able to adopt different shapes and move persistently through the highly fibrous native ECM, thereby requiring significant spatiotemporal reorganization of the cell cytoskeleton. While numerous studies performed on flat 2-dimensional culture platforms and physiological 3D gels have elucidated cytoskeletal reorganization, our understanding on how cells adapt to natural fibrous microenvironments and regulate their behavior in response to specific ECM biophysical cues including fiber size, spacing, alignment and stiffness remains in infancy. Here, we utilize the non -electrospinning Spinneret tunable engineered parameters (STEP) technique to manufacture ECM mimicking suspended fibrous matrices with precisely controlled fiber diameters, network architecture, inter-fiber spacing and structural stiffness to advance our fundamental understanding of how external cues affect cytoskeleton-based cellular forces in 3-distinct morphological processes of the cell cycle starting from division to spreading and migration. Mechanobiological insights from these studies are implemented to deliver intracellular cargo inside cells using electrical fields. Holistically, we conclude that fibrous environments elicit multiple new cell behaviors never before reported. Specifically, our new findings include (i) design of fiber networks regulates actin networks and cell forces to sculpt nuclei in varying shapes: compressed ovals, tear drop, and invaginations, and drive the nuclear translocation of transcription factors like YAP/TAZ. In all these shapes, nuclei remain rupture-free, thus demonstrating the unique adaptability of cells to fibers, (ii) dense crosshatch networks are fertile environments for persistent 1D migration in 3D shapes of rounded nuclei and low density of actin networks, while sparse fiber networks induce 2D random migration in flattened shapes and well-defined actin stress fibers, (iii) actin retraction fiber-based stability regulates mitotic errors. Cells undergoing mitosis on single fibers exhibit significant 3D movement, and those attached to two fibers can have rotated mitotic machinery, both conditions contributing to erroneous division, and (iv) a bi-phasic force response to electroporation that coincides with actin cytoskeleton remodeling. Cells on suspended fibers can withstand higher electric field abuse, which opens opportunities to deliver cargo of varying sizes inside the cell. Taken altogether, our findings provide new mechanobiological understanding of cell-fiber interactions at high spatiotemporal resolution impacting cell migration, division and nuclear mechanics-key behaviors in the study of pathophysiology. / Doctor of Philosophy / Cancer, one of the major pathophysiological conditions, progresses within the living body through spreading of malignant cells from the primary tumor to distant secondary sites, ultimately leading to life-ending outcomes. Such spreading of cancer also known as cancer metastasis requires mechanical interactions of cells with their immediately surrounding microenvironment or the extracellular matrix (ECM). Cells utilize their cytoskeleton, a dynamic internal network of filamentous proteins, to adopt various morphologies, exert mechanical forces and physically remodel their local environment as they navigate through the highly fibrous native ECM. While previous research has elucidated how biochemical factors and bulk matrix properties regulate such cytoskeletal organization and single cell behavior, our understanding of how cells adapt to fibrous environments and respond to local biophysical cues like fiber diameter, spacing, alignment and stiffness remains in infancy. Here we use the non -electrospinning Spinneret tunable engineered parameters (STEP) to generate suspended nanofiber networks of tunable geometric and mechanical properties to mimic the native cellular environment. We discover that cells elongated within these ECM-mimicking environments utilize a unique cytoskeletal caging structure to regulate the shape and response of their nuclei in a fiber -diameter and organization-dependent manner. Additionally, we demonstrate that these elongated cell morphologies often observed during metastatic cancer cell movements, is achievable not only in aligned fibers but can also be induced by dense networks of fibers in a crossing organization. Specifically, such dense crosshatch networks allow cells to migrate persistently at high speeds while cells on sparsely spaced networks demonstrate slower and random movements. As cells elongated during interphase rounded up to undergo division, we find that the underlying fiber-geometry modulates mitotic dynamics through differential levels of actin retraction fiber-mediated stability, leading to significant alterations in orientation of mitotic machinery and mitotic spindle defects. Finally, we utilize these mechanobiological insights on cytoskeletal organization and cell shape control to optimize intracellular delivery of cargo using high-voltage electric fields. We demonstrate suspended cells are capable of withstanding higher electric fields and identify multistage cell contractility recovery dynamics, which correlate with cytoskeletal disruption and reassembly. Taken altogether, our findings provide a comprehensive understanding of the fibrous ECM-mediated regulation of the cytoskeletal organization and its impact in cell migration, division and nuclear mechanics. Knowledge obtained from this study will improve our understanding of cancer metastasis and provide predictive data for in vivo cellular response, essential for cytoskeleton-targeting cancer therapies.

No / We report on the development and on the first use of the widefield surface plasmon (WSPR) microscope in the examination of the cell surface interface at submicron lateral resolutions. The microscope is Kohler illuminated and uses either a 1.45 numerical aperture (NA) oil immersion lens, or a 1.65 NA oil immersion lens to excite surface plasmons at the interface between a thin gold layer and a glass or sapphire cover slip. Like all surface plasmon microscope systems the WSPR has been proven in previous studies to also be capable of nanometric z-scale resolutions. In this study we used the system to image the interface between HaCaT cells and the gold layer. Imaging was performed in air using fixed samples and the 1.45 NA objective based system and also using live cells in culture media using the 1.65 NA based system. Imaging in air enabled the visualisation of high resolution and high-contrast submicron features identified by vinculin immunostaining as component of focal contacts and focal adhesions. In comparison, imaging in fluid enabled cell surface interfacial interactions to be tracked by time-lapse video WSPR microscopy. Our results indicate that the cell surface interface and thus cell signalling mechanisms may be readily interrogated in live cells without the use of labelling techniques.

Células de músculo liso vascular são capazes de perceber estímulos mecânicos do sistema cardiovascular, coordenando pressão sanguínea e perfusão tecidual por meio da modulação do tônus e do diâmetro vascular via resposta contrátil. O gatilho inicial à contração é um aumento na concentração intracelular de cálcio e diversas vias de sinalização têm sido descritas na sustentação deste sinal inicial. Evidências atuais indicam que adesões focais desempenham papel crucial na contração através da organização do citoesqueleto de actina e engajamento com o aparato contrátil. Nosso grupo demonstrou que a proteína rica em cisteína e glicina 3 (CRP3) interage com a quinase de adesão focal (FAK) em resposta a um aumento do estiramento mecânico e existem evidências de que CRP3 modula a dinâmica do citoesqueleto de actina. Neste trabalho testamos a hipótese de que a proteína CRP3 atua como uma proteína de adesão focal que regula a contração de células musculares lisas aórticas. Por meio de ensaios de imunoprecipitação e colocalização, verificou-se a presença de CRP3 nas adesões focais de células selvagens. Evidenciou-se que a ausência de CRP3 está associada a aumento no tamanho médio de adesões focais em células musculares lisas aórticas de forma independente do substrato. Entretanto, em resposta à angiotensina II, células nocaute para CRP3 apresentam incapacidade de maturação das adesões focais, um evento que está associado ao reduzido conteúdo proteico de FAK, paxilina e MLC2 (plataformas moleculares envolvidas na maturação de adesões focais) observada em células nocaute. Consistente com o maior tamanho médio das adesões focais, células nocaute são mais rígidas e, portanto, menos elásticas que células selvagens, sendo que a rigidez avaliada por citometria magnético-óptica de oscilação se reflete na reduzida capacidade contrátil, seja em condições basais, em resposta à angiotensina II ou ao inibidor de ROCK, como evidenciado no ensaio de contração em gel de colágeno. Em síntese, os dados deste trabalho mostram que CRP3 está presente nas adesões focais, regulando tamanho e sinalização, com reflexos na rigidez (viscoelasticidade) e capacidade contrátil, variáveis fundamentais ao correto funcionamento de células musculares lisas aórticas. Em conjunto, as evidências deste trabalho suportam a hipótese de que CRP3 é um modulador de contratilidade e mecanotransdução em células musculares lisas aórticas / Smooth muscle cells act also as mecanosensors of the cardiovascular system, coordinating blood pressure and tissue perfusion by means of vascular tone and diameter modulation via the contractile response. The trigger for contraction is a rise in the intracellular calcium concentration and several signaling pathways have been described to sustain the initial calcium signal. Recent evidences highlight the crucial role of focal adhesions to the contractile response, given its role in actin cytoskeleton assembly and engagement with the actomyosin contractile apparatus. We have demonstrated that the cysteine and glycine-rich protein-3 (CRP3) interacts with focal adhesion kinase (FAK) in response to increased hemodynamic stress. Additionally, it has also been shown that CRP3 modulates actin cytoskeleton dynamics. Here, we tested the hypothesis that CRP3 acts as a focal adhesion protein that regulates the contraction of aortic smooth muscle cells. Through colocalization and immunoprecipitation studies we found that CRP3 is a focal adhesion protein in aortic smooth muscle cells. Focal adhesion mean size evaluation showed that in the baseline, CRP3 KO smooth muscle cells display greater focal adhesion size. However, upon angiotensin II (a contraction-triggering molecule) stimulation, CRP3 KO cells fail to maturate focal adhesions, an event that might be related to the reduced protein levels of FAK, paxillin, and MLC2 (key signaling molecules involved in focal adhesion maturation) observed in KO cells. Consistent with the greater mean focal adhesion size, CRP3 KO cells exhibited increased stiffness and therefore, reduced viscoelasticity when compared to wild type cells. The reduced viscoelasticity of KO cells seems to influence cell contractility, as CRP3 KO cells also displayed reduced contractile response in the baseline and in response to angiotensin II. In summary, these data showed that CRP3 is present at focal adhesions, regulating their size and signaling. Thus, CRP3 at focal adhesions influences cell stiffness and contractile capacity, which are key features of smooth muscle cell physiology. Altogether, our findings support the idea that CRP3 is a key modifier of contractility and mechanotransduction in aortic smooth muscle cells

<p>Epithelial damage and shedding of the epithelium are common observations in many airway diseases such as asthma, Sjögren’s syndrome, chronic obstructive pulmonary disease and cystic fibrosis. The ability of the cells to attach to each other and/or to the matrix seems to be altered. In the present study, cultured normal human bronchial epithelial cells were used as a model system. The desmosomes and also the focal adhesions were investigated to see if changes in these structural components as well as metabolic alterations could explain the observed shedding of the epithelium.</p><p>Inflammatory mediators such as tumor necrosis factor alpha (TNF-α), interferon gamma (IFN-γ), interleukin-1 beta (IL-1β), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-13 (IL-13), hypochlorous acid (HOCl) and nitric oxide (NO) are present in increased amounts in inflammation. The Th-1 cytokines, IFN-γ and TNF-α, as well as HOCl and NO affected the number of desmosomes and their ability to attach to each other. Interestingly, the Th-2 cytokines IL-4, IL-5 and IL-13 did not affect the cell-cell adhesion. HOCl and NO also affected the focal adhesions of the cells. </p><p>Both morphological and functional studies indicated that TNF-α, IFN-γ, HOCl and NO affect the mitochondria. A decreased glucose oxidation rate could result in a decreased production of ATP, which in turn could lead to inhibition of many cellular activities including an impaired ability of the ciliary activity in bronchial epithelial cells and mucus transport. The antioxidant N-acetyl-L-cysteine and the nitric oxide synthase inhibitor N-propyl-L-arginine inhibited these effects of HOCl. This indicates that HOCl can induce damage both by induction of free radicals and also through an increased production of NO. TNF-α and IFN-γ also induced an increased production of NO. N^ω-monomethyl-L-arginine reduced the cytokine-induced production of NO. The NO donor DETA NONOate reduced the total protein biosynthesis as well as the DNA content. NO can react with superoxide anions generated by inflammatory cells in the airways to form peroxynitrite ions, which in turn could generate hydroxyl radicals. These toxic ions may contribute to damage of the airway epithelial cells. </p><p>In conclusion, pro-inflammatory cytokines such as TNF-α, IFN-γ and also the reactive oxygen species HOCl and NO could contribute to airway epithelial shedding by affecting the adhesion properties of the epithelial cells. More generalized morphological and metabolic changes could be other contributing factors, together with the increased production of NO.</p>

Epithelial damage and shedding of the epithelium are common observations in many airway diseases such as asthma, Sjögren’s syndrome, chronic obstructive pulmonary disease and cystic fibrosis. The ability of the cells to attach to each other and/or to the matrix seems to be altered. In the present study, cultured normal human bronchial epithelial cells were used as a model system. The desmosomes and also the focal adhesions were investigated to see if changes in these structural components as well as metabolic alterations could explain the observed shedding of the epithelium. Inflammatory mediators such as tumor necrosis factor alpha (TNF-α), interferon gamma (IFN-γ), interleukin-1 beta (IL-1β), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-13 (IL-13), hypochlorous acid (HOCl) and nitric oxide (NO) are present in increased amounts in inflammation. The Th-1 cytokines, IFN-γ and TNF-α, as well as HOCl and NO affected the number of desmosomes and their ability to attach to each other. Interestingly, the Th-2 cytokines IL-4, IL-5 and IL-13 did not affect the cell-cell adhesion. HOCl and NO also affected the focal adhesions of the cells. Both morphological and functional studies indicated that TNF-α, IFN-γ, HOCl and NO affect the mitochondria. A decreased glucose oxidation rate could result in a decreased production of ATP, which in turn could lead to inhibition of many cellular activities including an impaired ability of the ciliary activity in bronchial epithelial cells and mucus transport. The antioxidant N-acetyl-L-cysteine and the nitric oxide synthase inhibitor N-propyl-L-arginine inhibited these effects of HOCl. This indicates that HOCl can induce damage both by induction of free radicals and also through an increased production of NO. TNF-α and IFN-γ also induced an increased production of NO. Nω-monomethyl-L-arginine reduced the cytokine-induced production of NO. The NO donor DETA NONOate reduced the total protein biosynthesis as well as the DNA content. NO can react with superoxide anions generated by inflammatory cells in the airways to form peroxynitrite ions, which in turn could generate hydroxyl radicals. These toxic ions may contribute to damage of the airway epithelial cells. In conclusion, pro-inflammatory cytokines such as TNF-α, IFN-γ and also the reactive oxygen species HOCl and NO could contribute to airway epithelial shedding by affecting the adhesion properties of the epithelial cells. More generalized morphological and metabolic changes could be other contributing factors, together with the increased production of NO.

No description available.

A number of studies have demonstrated the pivotal role of collagen in modulating cell growth and differentiation. In bone, where the extracellular matrix is composed of approximately 85% type I collagen, cellular interaction with matrix components has been shown to be important in the regulation of the osteoblast phenotype. Preservation or enhancement of normal osteoblast function and appositional bone formation after implant placement represents a strategy that can be useful for the purpose of improving osseointegration. In order to further improve biocompatibility, we combined two known favorable compounds, namely the titanium alloy, Ti6A14V, with type I collagen. We assessed the in vitro behavior of primary osteoblasts grown on both fibrillar collagen-coated and tropocollagen-coated Ti6A14V in comparison with uncoated titanium alloy, using an improved adsorption procedure. As parameters of biocompatibility, a variety of processes, including cell attachment, spreading, cytoskeletal organization, focal contact formation, proliferation and expression of a differentiated phenotype, were investigated. Our results demonstrated for the first time that in comparison to uncoated titanium alloy, collagen-coated alloy enhanced spreading and resulted in a more rapid formation of focal adhesions and their associated stress fibers. Growing on collagen-coated Ti6A14V, osteoblasts had a higher proliferative capacity and the intracellular expression of osteopontin was upregulated compared to uncoated titanium alloy. Type I collagen-coated titanium alloy exhibits favorable effects on the initial adhesion and growth activities of osteoblasts, which is encouraging for its potential use as bone graft material. Moreover, collagen type I may serve as an excellent biocompatible carrier for osteotropic factors such as cell adhesion molecules (e.g. fibronectin) or bone-specific growth factors. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

Le T-DM1 est un immunoconjugué composé de l'anticorps trastuzumab qui cible HER2 lié au DM1, un agent anti-tubuline dérivé de la maytansine. Malgré son efficacité, la résistance acquise au T-DM1 a été démontré lors des tests précliniques et chez certains patients. Nous avons développé des lignées résistantes à partir de la lignée de cancer du sein MDA-MB-361 et de la lignée de cancer de l'œsophage OE-19, que nous avons exposées au T-DM1 à doses croissantes pendant une longue durée en absence ou en présence de ciclosporine A (CsA). A partir de ces conditions nous avons obtenus les lignées “TR” qui ont été exposées uniquement au T-DM1 et “TCR” qui ont été exposées au T-DM1 et CsA. Nous avons observé une augmentation de la vitesse de migration et une diminution de la force d'adhésion chez OE-19 TCR associées à une sensibilité accrue à un inhibiteur de RHOA. Aussi, la voie des prostaglandines était dérégulée chez OE-19 TR et TCR, avec une forte augmentation de l'expression de COX-2 et de prostaglandine E2 dans la lignée OE-19 TR. La sensibilité à l'aspirine, un inhibiteur des cyclooxygenases 1-2, était accrue chez les deux lignées OE-19 résistantes par rapport à la lignée parentale. En conclusion nous avons démontré que différentes voies de signalisation peuvent être impliquées dans la résistance au T-DM1. Nos résultats restent à être validés chez les patients. Nous suggérons que cibler la voie de régulation de la composition du cytosquelette ou la voie des prostaglandines pourrait permettre d'obtenir un effet thérapeutique dans le cas de cancers résistants au T-DM1 / T-DM1 is an antibody-drug conjugate composed of the monoclonal antibody trastuzumab linked to DM1, a potent tubulin binding agent. Despite its efficacy in the treatment of HER2-positive breast cancer patients, acquired resistance to T-DM1 was observed during clinical trials. In order to study resistance mechanisms to T-DM1, we developed resistance models using OE-19 (esophageal) and MDA-MB-361 (breast) cancer cell lines in the absence or presence of ciclosporin A (CsA), an inhibitor of MDR1 mediated efflux. Resistant cells selected with T-DM1 alone are named “TR” and cells selected in the presence of T-DM1 and CsA are called “TCR”. OE-19 TCR cells showed modifications in adhesion gene expression, migration and adhesion strength, combined with an increased sensitivity to a RHOA inhibitor. Also, OE-19 TR cells presented an overexpression of COX-2 associated with an increased amount of PGE2 in the supernatant. A deregulation of the genes involved in the prostaglandin pathways was found in OE-19 TR and TCR cells, associated with increased sensitivity to aspirin. In conclusion, we found two signaling pathways deregulated in cell lines resistant to T-DM1. These results need to be validated using samples from patients resistant to T-DM1. Targeting the adhesion or the prostaglandin pathway could be of benefit for patients with T-DM1 resistant cancers

A number of studies have demonstrated the pivotal role of collagen in modulating cell growth and differentiation. In bone, where the extracellular matrix is composed of approximately 85% type I collagen, cellular interaction with matrix components has been shown to be important in the regulation of the osteoblast phenotype. Preservation or enhancement of normal osteoblast function and appositional bone formation after implant placement represents a strategy that can be useful for the purpose of improving osseointegration. In order to further improve biocompatibility, we combined two known favorable compounds, namely the titanium alloy, Ti6A14V, with type I collagen. We assessed the in vitro behavior of primary osteoblasts grown on both fibrillar collagen-coated and tropocollagen-coated Ti6A14V in comparison with uncoated titanium alloy, using an improved adsorption procedure. As parameters of biocompatibility, a variety of processes, including cell attachment, spreading, cytoskeletal organization, focal contact formation, proliferation and expression of a differentiated phenotype, were investigated. Our results demonstrated for the first time that in comparison to uncoated titanium alloy, collagen-coated alloy enhanced spreading and resulted in a more rapid formation of focal adhesions and their associated stress fibers. Growing on collagen-coated Ti6A14V, osteoblasts had a higher proliferative capacity and the intracellular expression of osteopontin was upregulated compared to uncoated titanium alloy. Type I collagen-coated titanium alloy exhibits favorable effects on the initial adhesion and growth activities of osteoblasts, which is encouraging for its potential use as bone graft material. Moreover, collagen type I may serve as an excellent biocompatible carrier for osteotropic factors such as cell adhesion molecules (e.g. fibronectin) or bone-specific growth factors. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

Astrocytes are emerging as important regulators of neural circuit function and behavior in the healthy and diseased nervous system. In a screen for astrocyte molecules that modulate neuronal hyperexcitability we identified multiple components of focal adhesion complexes (FAs) as potent suppressors of genetically- or pharmacologically-induced seizure-like activity. Depletion of astrocytic Tensin, b-integrin, Talin, Focal adhesion kinase (FAK), or matrix metalloproteinase 1 (Mmp1), which degrades extracellular matrix to activate b-integrin receptors, resulted in enhanced recovery from, or resistance to seizure activity. Reciprocally, promoting FA signaling by overexpression of Mmp1 in astrocytes led to enhanced-seizure severity. Blockade of FA signaling in astrocytes led to reduced-astrocytic coverage of the synaptic neuropil and reduced expression of the excitatory amino acid transporter EAAT1. However, upon seizure induction, depletion of FA signaling components resulted in enhanced astrocyte coverage of the synaptic neuropil and a ~2-fold increase in EAAT1 levels compared to controls. Our data indicate that FAs promote astrocyte coverage in neuropil and EAAT1 expression under normal physiological conditions, but in the context of hyperexcitability, FAs negatively regulate the extent of astrocytic processes within neuropil and EAAT1 expression, thereby inhibiting a more rapid recovery from conditions of excessive neuronal activity.