Publications by authors named "Kheya Sengupta"

36 Publications

On the control of dispersion interactions between biological membranes and protein coated biointerfaces.

J Colloid Interface Sci 2021 Sep 11;598:464-473. Epub 2021 Mar 11.

PULS Group, Department of Physics and Interdisciplinary Center for Nanostructured Films, Friedrich-Alexander-Universität Erlangen-Nürnberg, IZNF, Cauerstrasse 3, 91058 Erlangen, Germany; Division of Physical Chemistry, Ruđer Bošković Institute, Bijenička cesta 54, 10000 Zagreb, Croatia. Electronic address:

Hypothesis: Interaction of cellular membranes with biointerfaces is of vital importance for a number of medical devices and implants. Adhesiveness of these surfaces and cells is often regulated by depositing a layer of bovine serum albumin (BSA) or other protein coatings. However, anomalously large separations between phospholipid membranes and the biointerfaces in various conditions and buffers have been observed, which could not be understood using available theoretical arguments.

Methods: Using the Lifshitz theory, we here evaluate the distance-dependent Hamaker coefficient describing the dispersion interaction between a biointerface and a membrane to understand the relative positioning of two surfaces. Our theoretical modeling is supported by experiments where the biointerface is represented by a glass substrate with deposited BSA and protein layers. These biointerfaces are allowed to interact with giant unilamellar vesicles decorated with polyethylene glycol (PEG) using PEG lipids to mimic cellular membranes and their pericellular coat.

Results: We demonstrate that careful treatment of the van der Waals interactions is critical for explaining the lack of adhesiveness of the membranes with protein-decorated biointerfaces. We show that BSA alone indeed passivates the glass, but depositing an additional protein layer on the surface BSA, or producing multiple layers of proteins and BSA results in repulsive dispersion forces responsible for 100 nm large equilibrium separations between the two surfaces.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jcis.2021.02.078DOI Listing
September 2021

Biphasic mechanosensitivity of T cell receptor-mediated spreading of lymphocytes.

Proc Natl Acad Sci U S A 2019 03 8;116(13):5908-5913. Epub 2019 Mar 8.

Centre Interdisciplinaire de Nanoscience de Marseille (CINaM), CNRS, Aix Marseille University, 13009 Marseille, France;

Mechanosensing by T cells through the T cell receptor (TCR) is at the heart of immune recognition. While the mechanobiology of the TCR at the molecular level is increasingly well documented, its link to cell-scale response is poorly understood. Here we explore T cell spreading response as a function of substrate rigidity and show that remarkably, depending on the surface receptors stimulated, the cellular response may be either biphasic or monotonous. When adhering solely via the TCR complex, T cells respond to environmental stiffness in an unusual fashion, attaining maximal spreading on an optimal substrate stiffness comparable to that of professional antigen-presenting cells. However, in the presence of additional ligands for the integrin LFA-1, this biphasic response is abrogated and the cell spreading increases monotonously with stiffness up to a saturation value. This ligand-specific mechanosensing is effected through an actin-polymerization-dependent mechanism. We construct a mesoscale semianalytical model based on force-dependent bond rupture and show that cell-scale biphasic or monotonous behavior emerges from molecular parameters. As the substrate stiffness is increased, there is a competition between increasing effective stiffness of the bonds, which leads to increased cell spreading and increasing bond breakage, which leads to decreased spreading. We hypothesize that the link between actin and the receptors (TCR or LFA-1), rather than the ligand/receptor linkage, is the site of this mechanosensing.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1811516116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6442626PMC
March 2019

T Cells on Engineered Substrates: The Impact of TCR Clustering Is Enhanced by LFA-1 Engagement.

Front Immunol 2018 18;9:2085. Epub 2018 Sep 18.

CNRS, CINaM UMR 7325, Aix-Marseille Université, Marseille, France.

We created APC-mimetic synthetic substrates to study the impact of ligand clustering on T cell activation and spreading. The substrates exhibit antibodies directed against the TCR-complex in the form of a patterned array of sub micrometric dots surrounded by a fluid supported lipid bilayer (SLB) which may itself be functionalized with another bio-molecule. We show that for T cell adhesion mediated by T cell receptor (TCR) alone, in the patterned, but not in the corresponding homogeneous controls, the TCR, ZAP-70 and actin are present in the form of clusters or patches that co-localize with the ligand-dots. However, global cell scale parameters like cell area and actin distribution are only weakly impacted by ligand clustering. In presence of ICAM-1 - the ligand of the T cell integrin LFA-1 - on the SLB, the TCR is still clustered due to the patterning of its ligands, but now global parameters are also impacted. The actin organization changes to a peripheral ring, resembling the classical actin distribution seen on homogeneous substrates, the patterned membrane topography disappears and the membrane is flat, whereas the cell area increases significantly. These observations taken together point to a possible pivotal role for LFA-1 in amplifying the effect of TCR-clustering. No such effect is evident for co-engagement of CD28, affected its ligand B7.2. Unlike on ICAM-1, on B7.2 cell spreading and actin organization are similar for homogeneous and patterned substrates. However, TCR and ZAP-70 clusters are still formed in the patterned case. These results indicate complementary role for LFA-1 and CD28 in the regulation and putative coupling of TCR micro-clusters to actin. The engineered substrates presented here clearly have the potential to act as platform for fundamental research in immune cell biology, as well as translational analyses in immunotherapy, for example to screen molecules for their role in T cell adhesion/activation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fimmu.2018.02085DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6154019PMC
September 2019

Lamellipod Reconstruction by Three-Dimensional Reflection Interference Contrast Nanoscopy (3D-RICN).

Nano Lett 2018 10 6;18(10):6544-6550. Epub 2018 Sep 6.

Aix Marseille Univ , CNRS, INSERM, LAI , Marseille 13288 , France.

There are very few techniques to reconstruct the shape of a cell at nanometric resolution, and those that exist are almost exclusively based on fluorescence, implying limitations due to staining constraints and artifacts. Reflection interference contrast microscopy (RICM), a label-free technique, permits the measurement of nanometric distances between refractive objects. However, its quantitative application to cells has been largely limited due to the complex interferometric pattern caused by multiple reflections on internal or thin structures like lamellipodia. Here we introduce 3D reflection interference contrast nanoscopy, 3D-RICN, which combines information from multiple illumination wavelengths and aperture angles to characterize the lamellipodial region of an adherent cell in terms of its distance from the surface and its thickness. We validate this new method by comparing data obtained on fixed cells imaged with atomic force microscopy and quantitative phase imaging. We show that as expected, cells adhering to micropatterns exhibit a radial symmetry for the lamellipodial thickness. We demonstrate that the substrate-lamellipod distance may be as high as 100 nm. We also show how the method applies to living cells, opening the way for label-free dynamical study of cell structures with nanometric resolution.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.nanolett.8b03134DOI Listing
October 2018

Printing Functional Protein Nanodots on Soft Elastomers: From Transfer Mechanism to Cell Mechanosensing.

Nano Lett 2017 07 7;17(7):4284-4290. Epub 2017 Jun 7.

Aix Marseille University, CNRS, CINAM , Marseille, France.

Living cells sense the physical and chemical nature of their micro/nano environment with exquisite sensitivity. In this context, there is a growing need to functionalize soft materials with micro/nanoscale biochemical patterns for applications in mechanobiology. This, however, is still an engineering challenge. Here a new method is proposed, where submicronic protein-patterns are first formed on glass and are then printed on to an elastomer. The degree of transfer is shown to be governed mainly by hydrophobic interactions and to be influenced by grafting an appropriate fluorophore onto the core protein of interest. The transfer mechanism is probed by measuring the forces of adhesion/cohesion using atomic force microscopy. The transfer of functional arrays of dots with size down to about 400 nm, on elastomers with stiffness ranging from 3 kPa to 7 MPa, is demonstrated. Pilot studies on adhesion of T lymphocytes on such soft patterned substrates are reported.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.nanolett.7b01254DOI Listing
July 2017

Ligand Nano-cluster Arrays in a Supported Lipid Bilayer.

J Vis Exp 2017 04 23(122). Epub 2017 Apr 23.

Aix-Marseille Université, CNRS, UMR 7325, CINaM;

Currently there is considerable interest in creating ordered arrays of adhesive protein islands in a sea of passivated surface for cell biological studies. In the past years, it has become increasingly clear that living cells respond, not only to the biochemical nature of the molecules presented to them but also to the way these molecules are presented. Creating protein micro-patterns is therefore now standard in many biology laboratories; nano-patterns are also more accessible. However, in the context of cell-cell interactions, there is a need to pattern not only proteins but also lipid bilayers. Such dual proteo-lipidic patterning has so far not been easily accessible. We offer a facile technique to create protein nano-dots supported on glass and propose a method to backfill the inter-dot space with a supported lipid bilayer (SLB). From photo-bleaching of tracer fluorescent lipids included in the SLB, we demonstrate that the bilayer exhibits considerable in-plane fluidity. Functionalizing the protein dots with fluorescent groups allows us to image them and to show that they are ordered in a regular hexagonal lattice. The typical dot size is about 800 nm and the spacing demonstrated here is 2 microns. These substrates are expected to serve as useful platforms for cell adhesion, migration and mechano-sensing studies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3791/55060DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5565094PMC
April 2017

Nanometric thermal fluctuations of weakly confined biomembranes measured with microsecond time-resolution.

Soft Matter 2016 May;12(21):4755-68

Aix-Marseille Université, CNRS UMR 7325 (Centre Interdisciplinaire de Nanosciences de Marseille - CINaM), Marseille Cedex 9, France.

We probe the bending fluctuations of bio-membranes using highly deflated giant unilamellar vesicles (GUVs) bound to a substrate by a weak potential arising from generic interactions. The substrate is either homogeneous, with GUVs bound only by the weak potential, or is chemically functionalized with a micro-pattern of very strong specific binders. In both cases, the weakly adhered membrane is seen to be confined at a well-defined distance above the surface while it continues to fluctuate strongly. We quantify the fluctuations of the weakly confined membrane at the substrate proximal surface as well as of the free membrane at the distal surface of the same GUV. This strategy enables us to probe in detail the damping of fluctuations in the presence of the substrate, and to independently measure the membrane tension and the strength of the generic interaction potential. Measurements were done using two complementary techniques - dynamic optical displacement spectroscopy (DODS, resolution: 20 nm, 10 μs), and dual wavelength reflection interference contrast microscopy (DW-RICM, resolution: 4 nm, 50 ms). After accounting for the spatio-temporal resolution of the techniques, an excellent agreement between the two measurements was obtained. For both weakly confined systems we explore in detail the link between fluctuations on the one hand and membrane tension and the interaction potential on the other hand.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1039/c6sm00412aDOI Listing
May 2016

Nano-clustering of ligands on surrogate antigen presenting cells modulates T cell membrane adhesion and organization.

Integr Biol (Camb) 2016 Mar;8(3):287-301

Aix-Marseille Université, CNRS, CINaM-UMR 7325, Marseille, 13288, France.

We investigate the adhesion and molecular organization of the plasma membrane of T lymphocytes interacting with a surrogate antigen presenting cell comprising glass supported ordered arrays of antibody (α-CD3) nano-dots dispersed in a non-adhesive matrix of polyethylene glycol (PEG). The local membrane adhesion and topography, as well as the distribution of the T cell receptors (TCRs) and the kinase ZAP-70, are influenced by dot-geometry, whereas the cell spreading area is determined by the overall average density of the ligands rather than specific characteristics of the dots. TCR clusters are recruited preferentially to the nano-dots and the TCR cluster size distribution has a weak dot-size dependence. On the patterns, the clusters are larger, more numerous, and more enriched in TCRs, as compared to the homogeneously distributed ligands at comparable concentrations. These observations support the idea that non-ligated TCRs residing in the non-adhered parts of the proximal membrane are able to diffuse and enrich the existing clusters at the ligand dots. However, long distance transport is impaired and cluster centralization in the form of a central supramolecular cluster (cSMAC) is not observed. Time-lapse imaging of early cell-surface contacts indicates that the ZAP-70 microclusters are directly recruited to the site of the antibody dots and this process is concomitant with membrane adhesion. These results together point to a complex interplay of adhesion, molecular organization and activation in response to spatially modulated stimulation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1039/c5ib00293aDOI Listing
March 2016

Size-Tunable Organic Nanodot Arrays: A Versatile Platform for Manipulating and Imaging Cells.

Nano Lett 2015 Aug 15;15(8):5178-84. Epub 2015 Jul 15.

†Aix-Marseille Université, CNRS, CINaM-UMR 7325, Marseille 13288, France.

Arrays of protein nanodots with dot-size tuned independently of spacing (e.g., ∼100 to 600 nm diameter for 900 nm spacing) are fabricated. The mechanism of size control is demonstrated, by numerical simulations, to arise from shadow effects during deposition of a sacrificial metal mask. We functionalize the nanodots with antibodies and embed them in a polymer-cushion or in lipid-bilayers or transfer them to soft elastomers. Their ability to influence cell architecture and local membrane organization is demonstrated in T-lymphocytes, using reflection interference contrast and total internal reflection fluorescence microscopy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.nanolett.5b01400DOI Listing
August 2015

Crowding of receptors induces ring-like adhesions in model membranes.

Biochim Biophys Acta 2015 Nov 28;1853(11 Pt B):2984-91. Epub 2015 May 28.

Institut für Theoretische Physik and Cluster of Excellence: Engineering of Advanced Materials, Friedrich Alexander Universität Erlangen-Nürnberg, 91052 Erlangen, Germany; Insitut Ruđer Bošković, 10000 Zagreb, Croatia. Electronic address:

The dynamics of formation of macromolecular structures in adherent membranes is a key to a number of cellular processes. However, the interplay between protein reaction kinetics, diffusion and the morphology of the growing domains, governed by membrane mediated interactions, is still poorly understood. Here we show, experimentally and in simulations, that a rich phase diagram emerges from the competition between binding, cooperativity, molecular crowding and membrane spreading. In the cellular context, the spontaneously-occurring organization of adhesion domains in ring-like morphologies is particularly interesting. These are stabilized by the crowding of bulky proteins, and the membrane-transmitted correlations between bonds. Depending on the density of the receptors, this phase may be circumvented, and instead, the adhesions may grow homogeneously in the contact zone between two membranes. If the development of adhesion occurs simultaneously with membrane spreading, much higher accumulation of binders can be achieved depending on the velocity of spreading. The mechanisms identified here, in the context of our mimetic model, may shed light on the structuring of adhesions in the contact zones between two living cells. This article is part of a Special Issue entitled: Mechanobiology.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bbamcr.2015.05.025DOI Listing
November 2015

Association rates of membrane-coupled cell adhesion molecules.

Biophys J 2014 Dec 2;107(11):L33-6. Epub 2014 Dec 2.

Institut für Theoretische Physik and Cluster of Excellence Engineering of Advanced Materials, Friedrich-Alexander-Universität, Erlangen, Germany; Institute Ruđer Bošković, Division of Physical Chemistry, Zagreb, Croatia. Electronic address:

Thus far, understanding how the confined cellular environment affects the lifetime of bonds, as well as the extraction of complexation rates, has been a major challenge in studies of cell adhesion. Based on a theoretical description of the growth curves of adhesion domains, we present a new (to our knowledge) method to measure the association rate k(on) of ligand-receptor pairs incorporated into lipid membranes. As a proof of principle, we apply this method to several systems. We find that the k(on) for the interaction of biotin with neutravidin is larger than that for integrin binding to RGD or sialyl Lewis(x) to E-selectin. Furthermore, we find k(on) to be enhanced by membrane fluctuations that increase the probability for encounters between the binders. The opposite effect on k(on) could be attributed to the presence of repulsive polymers that mimic the glycocalyx, which points to two potential mechanisms for controlling the speed of protein complexation during the cell recognition process.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4255260PMC
http://dx.doi.org/10.1016/j.bpj.2014.10.033DOI Listing
December 2014

Ligand-mediated friction determines morphodynamics of spreading T cells.

Biophys J 2014 Dec 2;107(11):2629-38. Epub 2014 Dec 2.

Adhesion & Inflammation, Aix-Marseille University, Inserm UMR 1067, CNRS UMR 7333, Marseille, France. Electronic address:

Spreading of T cells on antigen presenting cells is a crucial initial step in immune response. Spreading occurs through rapid morphological changes concomitant with the reorganization of surface receptors and of the cytoskeleton. Ligand mobility and frictional coupling of receptors to the cytoskeleton were separately recognized as important factors but a systematic study to explore their biophysical role in spreading was hitherto missing. To explore the impact of ligand mobility, we prepared chemically identical substrates on which molecules of anti-CD3 (capable of binding and activating the T cell receptor complex), were either immobilized or able to diffuse. We quantified the T cell spreading area and cell edge dynamics using quantitative reflection interference contrast microscopy, and imaged the actin distribution. On mobile ligands, as compared to fixed ligands, the cells spread much less, the actin is centrally, rather than peripherally distributed and the edge dynamics is largely altered. Blocking myosin-II or adding molecules of ICAM1 on the substrate largely abrogates these differences. We explain these observations by building a model based on the balance of forces between activation-dependent actin polymerization and actomyosin-generated tension on one hand, and on the frictional coupling of the ligand-receptor complexes with the actin cytoskeleton, the membrane and the substrate, on the other hand. Introducing the measured edge velocities in the model, we estimate the coefficient of frictional coupling between T Cell receptors or LFA-1 and the actin cytoskeleton. Our results provide for the first time, to our knowledge, a quantitative framework bridging T cell-specific biology with concepts developed for integrin-based mechanisms of spreading.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bpj.2014.10.044DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4255222PMC
December 2014

Adaptive amphiphilic dendrimer-based nanoassemblies as robust and versatile siRNA delivery systems.

Angew Chem Int Ed Engl 2014 Oct 12;53(44):11822-7. Epub 2014 Sep 12.

Aix-Marseille Université, CNRS, Centre Interdisciplinaire de Nanoscience de Marseille, UMR 7325 (France); Centre de Recherche en Cancérologie de Marseille, INSERM, UMR1068 (France); Institut Paoli-Calmettes, Marseille (France); Aix-Marseille Université (France); CNRS, UMR7258, Marseille (France).

siRNA delivery remains a major challenge in RNAi-based therapy. Here, we report for the first time that an amphiphilic dendrimer is able to self-assemble into adaptive supramolecular assemblies upon interaction with siRNA, and effectively delivers siRNAs to various cell lines, including human primary and stem cells, thereby outperforming the currently available nonviral vectors. In addition, this amphiphilic dendrimer is able to harness the advantageous features of both polymer and lipid vectors and hence promotes effective siRNA delivery. Our study demonstrates for the first time that dendrimer-based adaptive supramolecular assemblies represent novel and versatile means for functional siRNA delivery, heralding a new age of dendrimer-based self-assembled drug delivery in biomedical applications.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/anie.201406764DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4485617PMC
October 2014

Nanometric protein-patch arrays on glass and polydimethylsiloxane for cell adhesion studies.

Nano Lett 2013 Jul 28;13(7):3372-8. Epub 2013 Jun 28.

Aix-Marseille Université , CNRS, CINaM UMR 7325, 13288 Marseille, France.

We present a simple cost-effective benchtop protocol to functionalize glass and polydimethylsiloxane (PDMS) with nanometric protein patches for cell adhesion studies. Evaporation masks, covering macroscopic areas on glass, were made using improved strategies for self-assembly of colloidal microbeads which then served as templates for creating the protein patch arrays via the intermediate steps of organo-aminosilane deposition and polyethylene-glycol grafting. The diameter of the patches could be varied down to about 80 nm. The glass substrates were used for advanced optical imaging of T-lymphocytes to explore adhesion by reflection interference contrast microscopy and the possible colocalization of T-cell receptor microclusters and the activating protein patches by total internal reflection fluorescence microscopy. The selectively functionalized glass could also serve as template for transferring the protein nanopatches to the surface of a soft elastomer. We demonstrated successful reverse contact printing onto the surface of thin layers of PDMS with stiffness ranging from 30 KPa to 3 MPa.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/nl401696mDOI Listing
July 2013

A bola-phospholipid bearing tetrafluorophenylazido chromophore as a promising lipid probe for biomembrane photolabeling studies.

Org Biomol Chem 2013 Aug 20;11(30):5000-5. Epub 2013 Jun 20.

Aix-Marseille University, Centre Interdisciplinaire de Nanoscience de Marseille, CNRS UMR 7325, 163, avenue de Luminy, 13288 Marseille, France.

A bola-phospholipid probe, carrying a tetrafluorophenylazido chromophore in the middle of the transmembrane diacyl chain, was synthesized and characterized with a view to studying biomembranes by a photolabeling approach. This probe shows the advantageous stability of bola-lipids in giant vesicle formation alongside excellent photochemical properties conferred by the tetrafluorophenylazido chromophore, and thus constitutes a promising probe for biomembrane photolabeling studies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1039/c3ob40659hDOI Listing
August 2013

Giant vesicles as cell models.

Integr Biol (Camb) 2012 Sep 25;4(9):982-95. Epub 2012 Jul 25.

Leiden Institute of Physics: Physics of Life Processes, Leiden University, The Netherlands.

Tremendous progress has been made in recent years in understanding the working of the living cell, including its micro-anatomy, signalling networks, and regulation of genes. However, an understanding of cellular phenomena using fundamental laws starting from first principles is still very far away. Part of the reason is that a cell is an active and exquisitely complex system where every part is linked to the other. Thus, it is difficult or even impossible to design experiments that selectively and exclusively probe a chosen aspect of the cell. Various kinds of idealised systems and cell models have been used to circumvent this problem. An important example is a giant unilamellar vesicle (GUV, also called giant liposome), which provides a cell-sized confined volume to study biochemical reactions as well as self-assembly processes that occur on the membrane. The GUV membrane can be designed suitably to present selected, correctly-oriented cell-membrane proteins, whose mobility is confined to two dimensions. Here, we present recent advances in GUV design and the use of GUVs as cell models that enable quantitative testing leading to insight into the working of real cells. We briefly recapitulate important classical concepts in membrane biophysics emphasising the advantages and limitations of GUVs. We then present results obtained over the last decades using GUVs, choosing the formation of membrane domains and cell adhesion as examples for in-depth treatment. Insight into cell adhesion obtained using micro-interferometry is treated in detail. We conclude by summarising the open questions and possible future directions.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1039/c2ib00188hDOI Listing
September 2012

Photoactivatable phospholipids bearing tetrafluorophenylazido chromophores exhibit unprecedented protonation-state-dependent 19F NMR signals.

Org Lett 2011 Aug 18;13(16):4248-51. Epub 2011 Jul 18.

Centre Interdisciplinaire de Nanoscience de Marseille, CNRS UPR 3118, 13288 Marseille, France.

Phospholipids bearing tetrafluorophenylazido chromophores were synthesized with perfectly conserved amphiphilicity and photochemical activity. Interestingly however, those phospholipids harboring the amine-linked chromophores exhibited unusual (19)F NMR signals which depended on the protonation state of the lipid headgroup. These probes may serve as powerful tools for studying various pH-dependent events in biomembranes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/ol201593kDOI Listing
August 2011

Switching from ultraweak to strong adhesion.

Adv Mater 2011 Jun 15;23(22-23):2622-6. Epub 2011 Apr 15.

Institute of Complex Systems 7: Biomechanics Forschungszentrum Jülich GmbH, 52425 Jülich, Germany.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/adma.201004097DOI Listing
June 2011

Blebbing dynamics during endothelial cell spreading.

Eur J Cell Biol 2011 Jan 18;90(1):37-48. Epub 2010 Nov 18.

Fischell Department of Bioengineering, University of Maryland, College Park, MD 20742, United States.

Cell spreading is a critical component of numerous physiological phenomena including cancer metastasis, embryonic development, and mitosis. We have previously illustrated that cellular blebs appear after abrupt cell-substrate detachment and play a critical role in regulating membrane tension; however, the dynamics of bleb-substrate interactions during spreading remains unclear. Here we explore the role of blebs during endothelial cell spreading using chemical and osmotic modifications to either induce or inhibit bleb formation. We track cell-substrate dynamics as well as individual blebs using surface sensitive microscopic techniques. Blebbing cells (both control and chemically induced) exhibit increased lag times prior to fast growth. Interestingly, lamellae appear later for blebbing compared to non-blebbing cells, and in all cases, lamellae signal the start of fast spreading. Our results indicate that cellular blebs play a key role in the early stage of cell spreading, first by controling the initial cell adhesion and then by presenting a dynamic inhibition of cell spreading until a lamella appears and fast spreading ensues.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ejcb.2010.09.013DOI Listing
January 2011

Cell blebbing and membrane area homeostasis in spreading and retracting cells.

Biophys J 2010 Sep;99(6):1726-33

Fischell Department of Bioengineering, University of Maryland, College Park, Maryland, USA.

Cells remodel their plasma membrane and cytoskeleton during numerous physiological processes, including spreading and motility. Morphological changes require the cell to adjust its membrane tension on different timescales. While it is known that endo- and exocytosis regulate the cell membrane area in a timescale of 1 h, faster processes, such as abrupt cell detachment, require faster regulation of the plasma membrane tension. In this article, we demonstrate that cell blebbing plays a critical role in the global mechanical homeostasis of the cell through regulation of membrane tension. Abrupt cell detachment leads to pronounced blebbing (which slow detachment does not), and blebbing decreases with time in a dynamin-dependent fashion. Cells only start spreading after a lag period whose duration depends on the cell's blebbing activity. Our model quantitatively reproduces the monotonic decay of the blebbing activity and accounts for the lag phase in the spreading of blebbing cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bpj.2010.07.031DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2944031PMC
September 2010

Adhesion of soft membranes controlled by tension and interfacial polymers.

Phys Rev Lett 2010 Feb 25;104(8):088101. Epub 2010 Feb 25.

CNRS, Aix-Marseille University, CINaM-UPR3118, Campus Luminy, Case 913 F-13288, Marseille, France.

We examine experimental and theoretical aspects of nonspecific adhesion of giant vesicles on modified surfaces as model systems for cell spreading. Using dual-wave interference microscopy and new analysis, membrane undulations as well as large scale vesicle shape are monitored. Measurements and modelling show that the nucleation of adhesion depends critically on the interfacial polymer and membrane tension. Patch growth is governed by local membrane geometry, adhesion energy, and local viscosity. Finally, spreading stops when tension induced by adhesion unfolds excess membrane area.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1103/PhysRevLett.104.088101DOI Listing
February 2010

Probing biomembrane dynamics by dual-wavelength reflection interference contrast microscopy.

Chemphyschem 2009 Nov;10(16):2828-38

Institute of Bio- and Nanosystems 4 (IBN 4), Research Centre Jülich, Wilhelm-Johnen-Strasse, 52425 Jülich, Germany.

We present an improved analysis of reflection interference contrast microscopy (RICM) images, recorded to investigate model membrane systems that mimic cell adhesion. The model systems were giant unilamellar vesicles (GUV) adhering via specific ligand-receptor interactions to supported lipid bilayers (SLB) or to patterns of receptors. Conventional RICM and dual-wavelength RICM (DW-RICM) were applied to measure absolute optical distances between the biomembranes and planar substrates. We developed algorithms for a straightforward implementation of an automated, time-resolved reconstruction of the membrane conformations from RICM/DW-RICM images, taking into account all the interfaces in the system and blurring of the data due to camera noise. Finally, we demonstrate the validity and usefulness of this new approach by analyzing the topography and fluctuations of a bound membrane in the steady state and its dynamic adaptation to osmotic pressure changes. These measurements clearly show that macroscopic membrane flow through tightly adhered area is possible in our system.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/cphc.200900645DOI Listing
November 2009

Quantitative reflection interference contrast microscopy (RICM) in soft matter and cell adhesion.

Chemphyschem 2009 Nov;10(16):2752-68

Adhesion and Inflammation, CNRS UMR 6212, Inserm U600, Aix-Marseille University, Luminy, Marseille, France.

Adhesion can be quantified by measuring the distance between the interacting surfaces. Reflection interference contrast microscopy (RICM), with its ability to measure inter-surface distances under water with nanometric precision and milliseconds time resolution, is ideally suited to studying the dynamics of adhesion in soft systems. Recent technical developments, which include innovative image analysis and the use of multi-coloured illumination, have led to renewed interest in this technique. Unambiguous quantitative measurements have been achieved for colloidal beads and model membranes, thus revealing new insights and applications. Quantification of data from cells shows exciting prospects. Herein, we review the basic principles and recent developments of RICM applied to studies of dynamical adhesion processes in soft matter and cell biology and provide practical hints to potential users.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/cphc.200900601DOI Listing
November 2009

Large-scale ordered plastic nanopillars for quantitative live-cell imaging.

Small 2009 Apr;5(4):449-53

Centre Interdisciplinaire de Nanosciences de Marseille CINaM UPR CNRS 3118, Aix-Marseille Université Campus de Luminy 13288 Marseille cedex 9, France.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/smll.200800836DOI Listing
April 2009

Dynamics of specific vesicle-substrate adhesion: from local events to global dynamics.

Phys Rev Lett 2008 Nov 13;101(20):208103. Epub 2008 Nov 13.

II. Institut für Theoretische Physik, Universität Stuttgart, D-70550 Stuttgart, Germany.

We present a synergistic combination of simulations and experimental data on the dynamics of membrane adhesion. We show that a change in either the density or the strength of the bonds results in very different dynamics. Such behavior is explained by introducing an effective binding affinity that emerges as a result of the competition between the strength of the chemical bonds and the environment defined by the fluctuating membrane.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1103/PhysRevLett.101.208103DOI Listing
November 2008

Diffusion and intermembrane distance: case study of avidin and E-cadherin mediated adhesion.

Langmuir 2009 Jan;25(2):1074-85

Institute of Bio- and Nanosystems (IBN), Research Centre Julich, 52425 Julich, Germany.

We present a biomimetic model system for cell-cell adhesion consisting of a giant unilamellar vesicle (GUV) adhering via specific ligand-receptor interactions to a supported lipid bilayer (SLB). The modification of in-plane diffusion of tracer lipids and receptors in the SLB membrane due to adhesion to the GUV is reported. Adhesion was mediated by either biotin-neutravidin (an avidin analogue) or the extracellular domains of the cell adhesion molecule E-cadherin (Ecad). In the strong interaction (biotin-avidin) case, binding of soluble receptors to the SLB alone led to reduced diffusion of tracer lipids. From theoretical considerations, this could be attributed partially to introduction of obstacles and partially to viscous effects. Further specific binding of a GUV membrane caused additional slowing down of tracers (up to 15%) and immobilization of receptors, and led to accumulation of receptors in the adhesion zone until full coverage was achieved. The intermembrane distance was measured to be 7 nm from microinterferometry (RICM). We show that a crowding effect due to the accumulated receptors alone is not sufficient to account for the slowing downan additional friction from the membrane also plays a role. In the weak binding case (Ecad), the intermembrane distance was about 50 nm, corresponding to partial overlap of the Ecad domains. No significant change in diffusion of tracer lipids was observed upon either protein binding or subsequent vesicle binding. The former was probably due to very small effective size of the obstacles introduced into the bilayer by Ecad binding, whereas the latter was due to the fact that, with such high intermembrane distance, the resulting friction is negligible. We conclude that the effect of intermembrane adhesion on diffusion depends strongly on the choice of the receptors.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/la803227sDOI Listing
January 2009

Tuning the formation and rupture of single ligand-receptor bonds by hyaluronan-induced repulsion.

Biophys J 2008 Oct 3;95(8):3999-4012. Epub 2008 Jul 3.

INSERM UMR 600, Centre National de la Recherche Scientifique, Université de la Méditerranée, Marseille, France.

We used a combination of laminar flow chamber and reflection interference microscopy to study the formation and rupture of single bonds formed between Fc-ICAM-1 attached to a substrate and anti-ICAM-1 carried by micrometric beads in the presence of a repulsive hyaluronan (HA) layer adsorbed onto the substrate. The absolute distance between the colloids and the surface was measured under flow with an accuracy of a few nanometers. We could verify the long-term prediction of classical lubrication theory for the movement of a sphere near a wall in a shear flow. The HA polymer layer exerted long-range repulsive steric force on the beads and the hydrodynamics at the boundary remained more or less unchanged. By incubating HA at various concentrations, the thickness of the layer, as estimated by beads most probable height, was tuned in the range 20-200 nm. Frequency of bond formation was decreased by more than one order of magnitude by increasing the thickness of the repulsive layer, while the lifetime of individual bonds was not affected. This study opens the way for further quantitative studies of the effect of molecular environment and separation distance on ligand-receptor association and dissociation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1529/biophysj.108.135947DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2553109PMC
October 2008

Force-induced growth of adhesion domains is controlled by receptor mobility.

Proc Natl Acad Sci U S A 2008 May 7;105(19):6906-11. Epub 2008 May 7.

II. Institut für Theoretische Physik II, Universität Stuttgart, Pfaffenwaldring 57/III, D-70550 Stuttgart, Germany.

In living cells, adhesion structures have the astonishing ability to grow and strengthen under force. Despite the rising evidence of the importance of this phenomenon, little is known about the underlying mechanism. Here, we show that force-induced adhesion-strengthening can occur purely because of the thermodynamic response to the elastic deformation of the membrane, even in the absence of the actively regulated cytoskeleton of the cell, which was hitherto deemed necessary. We impose pN-forces on two fluid membranes, locally pre-adhered by RGD-integrin binding. One of the binding partners is always mobile whereas the mobility of the other can be switched on or off. Immediate passive strengthening of adhesion structures occurs in both cases. When both binding partners are mobile, strengthening is aided by lateral movement of intact bonds as a transient response to force-induced membrane-deformation. By extending our microinterferometric technique to the suboptical regime, we show that the adhesion, as well as the resistance to force-induced de-adhesion, is greatly enhanced when both, rather than only one, of the binding partners are mobile. We formulate a theory that explains our observations by linking the macroscopic shape deformation with the microscopic formation of bonds, which further elucidates the importance of receptor mobility. We propose this fast passive response to be the first-recognition that triggers signaling events leading to mechanosensing in living cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.0801706105DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2383988PMC
May 2008

Fibroblast adaptation and stiffness matching to soft elastic substrates.

Biophys J 2007 Dec;93(12):4453-61

Institute for Medicine and Engineering, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.

Many cell types alter their morphology and gene expression profile when grown on chemically equivalent surfaces with different rigidities. One expectation of this change in morphology and composition is that the cell's internal stiffness, governed by cytoskeletal assembly and production of internal stresses, will change as a function of substrate stiffness. Atomic force microscopy was used to measure the stiffness of fibroblasts grown on fibronectin-coated polyacrylamide gels of shear moduli varying between 500 and 40,000 Pa. Indentation measurements show that the cells' elastic moduli were equal to, or slightly lower than, those of their substrates for a range of soft gels and reached a saturating value at a substrate rigidity of 20 kPa. The amount of cross-linked F-actin sedimenting at low centrifugal force also increased with substrate stiffness. Together with enhanced actin polymerization and cross-linking, active contraction of the cytoskeleton can also modulate stiffness by exploiting the nonlinear elasticity of semiflexible biopolymer networks. These results suggest that within a range of stiffness spanning that of soft tissues, fibroblasts tune their internal stiffness to match that of their substrate, and modulation of cellular stiffness by the rigidity of the environment may be a mechanism used to direct cell migration and wound repair.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1529/biophysj.106.101386DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2098710PMC
December 2007

Modulation of vesicle adhesion and spreading kinetics by hyaluronan cushions.

Biophys J 2007 Nov 13;93(9):3300-13. Epub 2007 Jul 13.

Adhésion Cellulaire, Centre National de Recherche Scientifique UMR 6212, INSERM U600, Université de Mediterranée, Luminy, Marseille, France.

The adhesion of giant unilamellar phospholipid vesicles to planar substrates coated with extracellular matrix mimetic cushions of hyaluronan is studied using quantitative reflection interference contrast microscopy. The absolute height of the vesicle membrane at the vicinity of the substrate is measured by considering, for the first time, the refractive indices of the reflecting media. The thickness of the cushion is varied in the range of approximately 50-100 nm, by designing various coupling strategies. On bare protein-coated substrates, the vesicles spread fast (0.5 s) and form a uniform adhesion disk, with the average membrane height approximately 4 nm. On thick hyaluronan cushions (>80 nm), the membrane height is approximately the same as the thickness of the cushion, implying that the vesicle lies on top of the cushion. On a thin and inhomogeneous hyaluronan cushion, the adhesion is modified but not prevented. The spreading is slow ( approximately 20 s) compared to the no-cushion case. The average membrane height is approximately 10 nm and the adhesion disk is studded with blisterlike structures. Observations with fluorescent hyaluronan indicate that the polymer is compressed under, rather than expelled from, the adhesion disk. The adhesion energy density is approximately threefold higher in the no-cushion case (1.2 microJ/m(2)) as compared to the thin-cushion case (0.54 microJ/m(2)). In the thin-cushion case, the presence of short ( approximately 4 nm) glyco-polymers on the vesicles results in a hitherto unreported stable partial adhesion state--the membrane height ranges from zero to approximately 250 nm. The minimal model system presented here mimics in vitro the hyaluronan-modulated early stages of cell adhesion, and demonstrates that the presence of a polymer cushion influences both the final equilibrium adhesion-state and the spreading kinetics.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1529/biophysj.107.105544DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2025668PMC
November 2007