Publications by authors named "Leyla Kocgozlu"

12 Publications

  • Page 1 of 1

Insensitivity of dental pulp stem cells migration to substrate stiffness.

Biomaterials 2021 Jun 15;275:120969. Epub 2021 Jun 15.

Inserm UMR-S1121, Centre de Recherche en Biomédecine de Strasbourg (CRBS), 1 rue Eugène Boeckel, 67084, Strasbourg, France; Université de Strasbourg, Faculté de Chirurgie Dentaire, 8 rue Sainte Elisabeth, 67000, Strasbourg, France; Fédération de Médecine Translationnelle, Strasbourg, France. Electronic address:

Dental pulp stem cells (DPSCs) are a promising cell source for regeneration of dental pulp. Migration is a key event but influence of the microenvironment rigidity (5 kPa at the center of dental pulp to 20 GPa for the dentin) is largely unknown. Mechanical signals are transmitted from the extracellular matrix to the cytoskeleton, to the nuclei, and to the chromatin, potentially regulating gene expression. To identify the microenvironmental influence on migration, we analyzed motility on PDMS substrates with stiffness increasing from 1.5 kPa up to 2.5 MPa. We found that migration speed slightly increases as substrate stiffness decreases in correlation with decreasing focal adhesion size. Motility is relatively insensitive to substrate stiffness, even on a bi-rigidity PDMS substrate where DPSCs migrate without preferential direction. Migration is independent of both myosin II activity and YAP translocation after myosin II inhibition. Additionally, inhibition of Arp2/3 complex leads to significant speed decrease for all rigidities, suggesting contribution of the lamellipodia in the migration. Interestingly, the chromatin architecture remains stable after a 7-days exposure on the PDMS substrates for all rigidity. To design scaffold mimicking dental pulp environment, similar DPSCs migration for all rigidity, leaves field open to choose this mechanical parameter.
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http://dx.doi.org/10.1016/j.biomaterials.2021.120969DOI Listing
June 2021

Enzyme assisted peptide self-assemblies trigger cell adhesion in high density oxime based host gels.

J Mater Chem B 2020 05 18;8(20):4419-4427. Epub 2020 Mar 18.

Université de Strasbourg, CNRS, Institut Charles Sadron UPR 22, 67034 Strasbourg, France.

Peptide supramolecular self-assemblies are recognized as important components in responsive hydrogel based materials with applications in tissue engineering and regenerative medicine. Studying the influence of hydrogel matrices on the self-assembly behavior of peptides and interaction with cells is essential to guide the future development of engineered biomaterials. In this contribution, we present a PEG based host hydrogel material generated by oxime click chemistry that shows cellular adhesion behavior in response to enzyme assisted peptide self-assembly (EASA) within the host gel. This hydrogel prepared from poly(dimethylacrylamide-co-diacetoneacrylamide), poly(DMA-DAAM) with high molar fractions (49%) of DAAM and dialkoxyamine PEG cross-linker, was studied in the presence of embedded enzyme alkaline phosphatase (AP) and a non-adhesive cell behavior towards NIH 3T3 fibroblasts was observed. When brought into contact with a Fmoc-FFpY peptide solution (pY: phosphorylated tyrosine), the gel forms intercalated Fmoc-FFY peptide self-assemblies upon diffusion of Fmoc-FFpY into the cross-linked hydrogel network as was confirmed by circular dichroism, fluorescence emission spectroscopy and confocal microscopy. Nevertheless, the mechanical properties do not change significantly after the peptide self-assembly in the host gel. This enzyme assisted peptide self-assembly promotes fibroblast cell adhesion that can be enhanced if Fmoc-F-RGD peptides are added to the pre-gelator Fmoc-FFpY peptide solution. Cell adhesion results mainly from interactions of cells with the non-covalent peptide self-assemblies present in the gel despite the fact that the mechanical properties are very close to those of the native host gel. This result is in contrast to numerous studies which showed that the mechanical properties of a substrate are key parameters of cell adhesion. It opens up the possibility to develop a diverse set of hybrid materials to control cell fate in culture due to tailored self-assemblies of peptides responding to the environment provided by the host guest gel.
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http://dx.doi.org/10.1039/d0tb00456aDOI Listing
May 2020

Phase Separation in Supramolecular Hydrogels Based on Peptide Self-Assembly from Enzyme-Coated Nanoparticles.

Langmuir 2019 08 6;35(33):10838-10845. Epub 2019 Aug 6.

Université de Strasbourg, CNRS, Institut Charles Sadron UPR 22 , 67034 Strasbourg , France.

Spatial localization of biocatalysts, such as enzymes, has recently proven to be an effective process to direct supramolecular self-assemblies in a spatiotemporal way. In this work, silica nanoparticles (NPs) functionalized covalently by alkaline phosphatase ([email protected]) induce the localized growth of self-assembled peptide nanofibers from NPs by dephosphorylation of Fmoc-FFY peptides (Fmoc: fluorenylmethyloxycarbonyl; F: phenylalanine; Y: tyrosine; : phosphate group). The fibrillary nanoarchitecture around [email protected] underpins a homogeneous hydrogel, which unexpectedly undergoes a macroscopic shape change over time. This macroscopic change is due to a phase separation leading to a dense phase (in NPs and nanofibers) in the center of the vial and surrounded by a dilute one, which still contains NPs and peptide self-assemblies. We thus hypothesize that the phase separation is not a syneresis process. Such a change is only observed when the enzymes are localized on the NPs. The dense phase contracts with time until reaching a constant volume after several days. For a given phosphorylated peptide concentration, the dense phase contracts faster when the [email protected] concentration is increased. For a given [email protected] concentration, it condenses faster when the peptide concentration increases. We hypothesize that the appearance of a dense phase is not only due to attractive interactions between [email protected] but also to the strong interactions of self-assembled peptide nanofibers with the enzymes, covalently fixed on the NPs.
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http://dx.doi.org/10.1021/acs.langmuir.9b01420DOI Listing
August 2019

Cell shape and substrate stiffness drive actin-based cell polarity.

Phys Rev E 2019 Jan;99(1-1):012412

Mechanobiology Institute, National University of Singapore, 117411 Singapore, Singapore.

A general trait of living cells is their ability to exert contractile stresses on their surroundings and thus respond to substrate rigidity. At the cellular scale, this response affects cell shape, polarity, and ultimately migration. The regulation of cell shape together with rigidity sensing remains largely unknown. In this article we show that both substrate rigidity and cell shape contribute to drive actin organization and cell polarity. Increasing substrate rigidity affects bulk properties of the actin cytoskeleton by favoring long-lived actin stress fibers with increased nematic interactions, whereas cell shape imposes a local alignment of actin fibers at the cell periphery.
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http://dx.doi.org/10.1103/PhysRevE.99.012412DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6464093PMC
January 2019

Chromatin de-condensation by switching substrate elasticity.

Sci Rep 2018 08 23;8(1):12655. Epub 2018 Aug 23.

Inserm UMR-S1121, 11 rue Humann, 67085, Strasbourg, France.

Mechanical properties of the cellular environment are known to influence cell fate. Chromatin de-condensation appears as an early event in cell reprogramming. Whereas the ratio of euchromatin versus heterochromatin can be increased chemically, we report herein for the first time that the ratio can also be increased by purely changing the mechanical properties of the microenvironment by successive 24 h-contact of the cells on a soft substrate alternated with relocation and growth for 7 days on a hard substrate. An initial contact with soft substrate caused massive SW480 cancer cell death by necrosis, whereas approximately 7% of the cells did survived exhibiting a high level of condensed chromatin (21% heterochromatin). However, four consecutive hard/soft cycles elicited a strong chromatin de-condensation (6% heterochromatin) correlating with an increase of cellular survival (approximately 90%). Furthermore, cell survival appeared to be reversible, indicative of an adaptive process rather than an irreversible gene mutation(s). This adaptation process is associated with modifications in gene expression patterns. A completely new approach for chromatin de-condensation, based only on mechanical properties of the microenvironment, without any drug mediation is presented.
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http://dx.doi.org/10.1038/s41598-018-31023-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6107547PMC
August 2018

Topological defects in epithelia govern cell death and extrusion.

Nature 2017 04;544(7649):212-216

Mechanobiology Institute, National University of Singapore, Singapore.

Epithelial tissues (epithelia) remove excess cells through extrusion, preventing the accumulation of unnecessary or pathological cells. The extrusion process can be triggered by apoptotic signalling, oncogenic transformation and overcrowding of cells. Despite the important linkage of cell extrusion to developmental, homeostatic and pathological processes such as cancer metastasis, its underlying mechanism and connections to the intrinsic mechanics of the epithelium are largely unexplored. We approach this problem by modelling the epithelium as an active nematic liquid crystal (that has a long range directional order), and comparing numerical simulations to strain rate and stress measurements within monolayers of MDCK (Madin Darby canine kidney) cells. Here we show that apoptotic cell extrusion is provoked by singularities in cell alignments in the form of comet-shaped topological defects. We find a universal correlation between extrusion sites and positions of nematic defects in the cell orientation field in different epithelium types. The results confirm the active nematic nature of epithelia, and demonstrate that defect-induced isotropic stresses are the primary precursors of mechanotransductive responses in cells, including YAP (Yes-associated protein) transcription factor activity, caspase-3-mediated cell death, and extrusions. Importantly, the defect-driven extrusion mechanism depends on intercellular junctions, because the weakening of cell-cell interactions in an α-catenin knockdown monolayer reduces the defect size and increases both the number of defects and extrusion rates, as is also predicted by our model. We further demonstrate the ability to control extrusion hotspots by geometrically inducing defects through microcontact printing of patterned monolayers. On the basis of these results, we propose a mechanism for apoptotic cell extrusion: spontaneously formed topological defects in epithelia govern cell fate. This will be important in predicting extrusion hotspots and dynamics in vivo, with potential applications to tissue regeneration and the suppression of metastasis. Moreover, we anticipate that the analogy between the epithelium and active nematic liquid crystals will trigger further investigations of the link between cellular processes and the material properties of epithelia.
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http://dx.doi.org/10.1038/nature21718DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5439518PMC
April 2017

Epithelial Cell Packing Induces Distinct Modes of Cell Extrusions.

Curr Biol 2016 11 13;26(21):2942-2950. Epub 2016 Oct 13.

Mechanobiology Institute, National University of Singapore, T-Lab, 5A Engineering Drive 1, 117411, Singapore.

The control of tissue growth, which is a key to maintain the protective barrier function of the epithelium, depends on the balance between cell division and cell extrusion rates [1, 2]. Cells within confluent epithelial layers undergo cell extrusion, which relies on cell-cell interactions [3] and actomyosin contractility [4, 5]. Although it has been reported that cell extrusion is also dependent on cell density [6, 7], the contribution of tissue mechanics, which is tightly regulated by cell density [8-12], to cell extrusion is still poorly understood. By measuring the multicellular dynamics and traction forces, we show that changes in epithelial packing density lead to the emergence of distinct modes of cell extrusion. In confluent epithelia with low cell density, cell extrusion is mainly driven by the lamellipodia-based crawling mechanism in the neighbor non-dying cells in connection with large-scale collective movements. As cell density increases, cell motion is shown to slow down, and the role of a supracellular actomyosin cable formation and its contraction in the neighboring cells becomes the preponderant mechanism to locally promote cell extrusion. We propose that these two distinct mechanisms complement each other to ensure proper cell extrusion depending on the cellular environment. Our study provides a quantitative and robust framework to explain how cell density can influence tissue mechanics and in turn regulate cell extrusion mechanisms.
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http://dx.doi.org/10.1016/j.cub.2016.08.057DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5423527PMC
November 2016

Micropillar substrates: a tool for studying cell mechanobiology.

Methods Cell Biol 2015 8;125:289-308. Epub 2015 Jan 8.

Mechanobiology Institute, National University of Singapore, Singapore; Institut Jacques Monod (IJM), CNRS UMR 7592 & Université Paris Diderot, Paris, France.

Increasing evidence has shown that mechanical cues from the environment play an important role in cell biology. Mechanotransduction or the study of how cells can sense these mechanical cues, and respond to them, is an active field of research. However, it is still not clear how cells sense and respond to mechanical cues. Thus, new tools are being rapidly developed to quantitatively study cell mechanobiology. Particularly, force measurement tools such as micropillar substrates have provided new insights into the underlying mechanisms of mechanosensing. In this chapter, we provide detailed protocol for fabrication, characterization, functionalization, and use of the micropillar substrates.
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http://dx.doi.org/10.1016/bs.mcb.2014.10.009DOI Listing
September 2015

Contribution of soft substrates to malignancy and tumor suppression during colon cancer cell division.

PLoS One 2013 22;8(10):e78468. Epub 2013 Oct 22.

Inserm UMR 1121, Strasbourg, France ; Université de Strasbourg, Faculté de Chirurgie Dentaire, Strasbourg, France ; Fédération de Médecine Translationnelle, Strasbourg, France.

In colon cancer, a highly aggressive disease, progression through the malignant sequence is accompanied by increasingly numerous chromosomal rearrangements. To colonize target organs, invasive cells cross several tissues of various elastic moduli. Whether soft tissue increases malignancy or in contrast limits invasive colon cell spreading remains an open question. Using polyelectrolyte multilayer films mimicking microenvironments of various elastic moduli, we revealed that human SW480 colon cancer cells displayed increasing frequency in chromosomal segregation abnormalities when cultured on substrates with decreasing stiffness. Our results show that, although decreasing stiffness correlates with increased cell lethality, a significant proportion of SW480 cancer cells did escape from the very soft substrates, even when bearing abnormal chromosome segregation, achieve mitosis and undergo a new cycle of replication in contrast to human colonic HCoEpiC cells which died on soft substrates. This observation opens the possibility that the ability of cancer cells to overcome defects in chromosome segregation on very soft substrates could contribute to increasing chromosomal rearrangements and tumor cell aggressiveness.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0078468PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3805547PMC
May 2014

Multiarray cell stretching platform for high-magnification real-time imaging.

Nanomedicine (Lond) 2013 Apr;8(4):543-53

Nanyang Technological University, 50 Nanyang Avenue, 639798 Singapore.

Aim: This article reports the development of a multiarray microchip with real-time imaging capability to apply mechanical strains onto monolayered cell cultures.

Materials & Methods: Cells were cultured on an 8-µm thick membrane that was positioned in the microscope focal plane throughout the stretching process. Each stretching unit was assembled from three elastomeric layers and a glass coverslip. A programmable pneumatic control system was developed to actuate this platform. Multiple stretching experiments were conducted with various cell lines.

Results: The platform provides a maximum uniform strain of 69%. Acute and long-term cell morphological changes were observed. The supreme imaging capability was verified by real-time imaging of transfected COS-7 stretching and poststretching imaging of immunofluorescence-stained PTK2.

Conclusion: The platform reported here is a powerful tool for studying mechanically induced physiological changes in cells. Such a device could be used in tissue regeneration for maintaining essential cell growth conditions.
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http://dx.doi.org/10.2217/nnm.13.45DOI Listing
April 2013

The control of chromosome segregation during mitosis in epithelial cells by substrate elasticity.

Biomaterials 2012 Jan 29;33(3):798-809. Epub 2011 Oct 29.

Institut National de la Santé et de la Recherche Médicale, INSERM Unité 977, 11 rue Humann, 67085 Strasbourg Cedex, France.

Materials of defined elasticity, including synthetic material scaffolds and tissue-derived matrices, can regulate biological responses of cells and in particular adhesion, migration, growth and differentiation which are essential parameters for tissue integration. These responses have been extensively investigated in interphase cells, but little is known whether and how material elasticity affects mitotic cells. We used polyelectrolyte multilayer films as model substrates with elastic modulus ranging from Eap = 0 up to Eap = 500 kPa and mitotic PtK2 epithelial cells to address these important questions. Soft substrates (Eap < 50 kPa) led to abnormal morphology in chromosome segregation, materialized by chromatin bridges and chromosome lagging. Frequency of these damages increased with decreasing substrate stiffness and was correlated with a pro-apoptotic phenotype. Mitotic spindle was not observed on soft substrates where formation of chromatin damages is due to low β1-integrin engagement and decrease of Rac1 activities. This work constitutes the first evidence that soft substrates hinder epithelial cell division. In perspective, our findings emphasize the prime incidence of the material elasticity on the fate of the phenotype, especially of stem cells in the mitotic phase.
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http://dx.doi.org/10.1016/j.biomaterials.2011.10.024DOI Listing
January 2012

Selective and uncoupled role of substrate elasticity in the regulation of replication and transcription in epithelial cells.

J Cell Sci 2010 Jan;123(Pt 1):29-39

Institut National de la Santé et de la Recherche Médicale, INSERM Unité 977, 67085 Strasbourg Cedex, France.

Actin cytoskeleton forms a physical connection between the extracellular matrix, adhesion complexes and nuclear architecture. Because tissue stiffness plays key roles in adhesion and cytoskeletal organization, an important open question concerns the influence of substrate elasticity on replication and transcription. To answer this major question, polyelectrolyte multilayer films were used as substrate models with apparent elastic moduli ranging from 0 to 500 kPa. The sequential relationship between Rac1, vinculin adhesion assembly, and replication becomes efficient at above 200 kPa because activation of Rac1 leads to vinculin assembly, actin fiber formation and, subsequently, to initiation of replication. An optimal window of elasticity (200 kPa) is required for activation of focal adhesion kinase through auto-phosphorylation of tyrosine 397. Transcription, including nuclear recruitment of heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1), occurred above 50 kPa. Actin fiber and focal adhesion signaling are not required for transcription. Above 50 kPa, transcription was correlated with alphav-integrin engagement together with histone H3 hyperacetylation and chromatin decondensation, allowing little cell spreading. By contrast, soft substrate (below 50 kPa) promoted morphological changes characteristic of apoptosis, including cell rounding, nucleus condensation, loss of focal adhesions and exposure of phosphatidylserine at the outer cell surface. On the basis of our data, we propose a selective and uncoupled contribution from the substrate elasticity to the regulation of replication and transcription activities for an epithelial cell model.
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http://dx.doi.org/10.1242/jcs.053520DOI Listing
January 2010