AFM investigations of cellular response to environmental and local chemo-mechanical stimulus. Fernando Suárez Sánchez - PDF

AFM investigations of cellular response to environmental and local chemo-mechanical stimulus. By Fernando Suárez Sánchez Centre for the Physics of Materials Department of Physics McGill University, Montréal

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AFM investigations of cellular response to environmental and local chemo-mechanical stimulus. By Fernando Suárez Sánchez Centre for the Physics of Materials Department of Physics McGill University, Montréal June, A thesis submitted to McGill University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Fernando Suarez Sanchez, 2 Abstract Several cell types are found in organisms. Each type displays specific characteristics such as morphology, proliferation rate, genetic expression, mechanical properties, etc. Many investigations have been performed to study the effect of chemical cues on these cell properties. This is in contrast to the present work, which investigates the effect of mechanical properties of the global or local surroundings. Here, we have studied the effect of the matrix stiffness on the mechanical properties of airway smooth muscle cells using Atomic Force Microscopy (AFM). Our results show that the elastic modulus (G ) of these cells increases to 820 ± 360 Pa when cultured on stiff gels when compared to the elastic modulus of 340 ± 160 Pa for cells cultured on soft polyacrylamide gels. We notice no significant difference in elastic modulus for cells plated on a glass substrate when compared to the stiffer gels. There is no evident effect of substrate stiffness on the loss modulus. The variability of the measured elastic modulus is attributed to cellular variability. This variability is smaller for cells cultured on the soft gel. When the cell cultures were labeled with Red-phalloidin, we observed an increase in the organization of the actin fibers at the cell cortex for stiffer substrates. We thus hypothesize that the increase in cellular stiffness is the consequence of the actin organization beneath the cell membrane. Proliferation rate was significantly diminished when the cells were cultured on the softer polyacrylamide gels. Matrix stiffness also had an effect on genetic expression as demonstrated by gene arrays. We observed a significant difference of genetic expression when cells were cultured on a glass substrate. All these results indicate that smooth muscle cells respond structurally and genetrically to the mechanical properties of the environment. Neurons are mechanically much more fragile and responsive than smooth muscle cells. We locally changed the mechano-chemical environment of axons and observed that this was sufficient to induce major structural changes such as synapse formation and even the extraction of proteins containing membrane strings. We developed a new approach to induce and study the creation of presynaptic site formation in axons through a combination of local modification to A the mechano-chemical environment using a combination of AFM and fluorescence microscopy. First, we use a poly-d-lysine coated bead attached to an AFM tip to induce a synapse. We used transfection techniques and fluorescence microscopy to study the recruitment of two synaptic proteins, bassoon and synaptophysin, and measure their absolute arrival times to the presynaptic site. We find that bassoon arrives after 23 ± 10 minutes and that synaptophysin arrives after 43 ± 9 minutes. Finally, we observed the formation of long (several 10s of μm) membrane strings as the AFM tip was withdrawn from the axon. These membrane strings seemed functionally intact. It is conceivable that these strings might be a mechanism by which new neurites and branch points along existing neurites can be generated in situ. B Résumé Plusieurs types de cellules se retrouvent dans les organismes. Chaque type présente des caractéristiques spécifiques telles que la morphologie, le taux de prolifération, l expression génétique, les propriétés mécaniques, etc. De nombreuses enquêtes ont été réalisées afin d étudier l effet des signaux chimiques dans la cellule. Ceci contraste avec les travaux actuels qui étudie l effet des propriétés mécaniques sur l environnement global et local. Ici, nous avons étudié l influence de la rigidité du substrat sur les propriétés mécaniques et l expression génétique des cellules musculaires lisses des voies aériennes. Nos résultats démontrent que le module d élasticité (G ) des cellules augmente à 820 ± 360 Pa lorsqu elles sont cultivées sur les gels rigides par rapport à 340 ± 160 Pa aux cellules cultivées sur les gels polyacrylamides doux. Nous n avons pas remarqué de différence significative dans le module d élasticité pour les cellules étalées sur un substrat de verre en comparaison aux gels rigides. L effet de rigidité sur le module de perte n est donc pas observé. La variabilité du module d élasticité mesuré est attribuée à la variabilité cellulaire. Cette variabilité cellulaire est moins effective pour les cellules cultivées sur les gels doux. Lorsque les cultures de cellules ont été marquées avec Red-phalloïdine, nous observons une augmentation dans l organisation des fibres d actine au niveau du cortex cellulaire. Ainsi, nous émettons l hypothèse que l augmentation de la rigidité cellulaire est une conséquence de l organisation d actine sous la membrane cellulaire. Le taux de prolifération a significativement diminué lorsque les cellules ont été cultivées dans les gels de polyacrylamide plus doux. La rigidité du substrat a également une influence sur l expression génétique comme démontrée dans les réseaux de gènes. D un autre côté, une variation significative sur l expression génétique a été observée dans les cellules cultivées sur du verre. Tous ces résultats suggèrent que les cellules musculaires lisses répondent aux propriétés fournis par l environment. Les neurones sont mécaniquement beaucoup plus fragiles et sensibles que les cellules musculaires lisses. Nous avons localement changé l environment C mécanochimique des axones et avons observé que cela suffisait pour induire des changements structurels significatifs tels que la formation des synapses et même l extraction de protéines contenant des chaînes membranaires. Nous avons développé une nouvelle approche pour inciter et étudier la formation des sites présynaptiques dans les axones par une combinaison de modifications locales de l environnement mécano-chimique en utilisant une combinaison de l'afm et de la microscopie à fluorescence. Tout d abord, nous utilisons une bille enrobée de poly-d-lysine pour attacher sur une pointe d AFM dans le but d induire une synapse. Nous avons utilisé des techniques de transfection et la microscopie à fluorescence pour étudier le recrutement de deux protéines synaptiques, basson et synaptophysine, et de mesurer leur temps d'arrivée absolue aux sites présynaptiques. Nous constatons que le basson arrive après 23 ± 10 minutes et que la synaptophysine arrive après 43 ± 9 minutes. Finalement, nous avons observé la formation de longues chaînes membranaires contenant des protéines de l ordre de 10µm quand la pointe d AFM a été retirée de l axone. Ces chaînes membranaires semblent être fonctionnellement intactes. Il est concevable que ces chaînes pourraient être un mécanisme rénovateur par lequel les nouvelles neurites et les nouveaux points de branchement au long des neurites existants peuvent être générés in situ.. D Acknowledgments I want to thanks the institutions and people that make possible the development of the present study. First, I thank the the Natural Science and Engineering Research Council of Canada and the Canadian Institute for Health Research and the Consejo Nacional de Ciencia y Tecnologia for their financial assistance, both through scholarship awards and the research funding provided. I am deeply grateful to my supervisor, Dr. Peter Grütter, who has been very supportive through the development of this study. His insight and advices have contributed importantly to improve my research skills. I thank Dr. Barbara Tolloczko and Dr. James Martin. Both have helped me to increase my biological knowledge and have guided me through many of the the biological methods employed in this thesis. Dr. David Colman has been a supportive figure in the neuro-physics study described in this thesis. He facilitated the access to neuronal cultures by providing us access to the installations in his laboratory. My colleges Peter Thostrup and Jeff LeDue at the physics department, Paul Andre, Taisuke Jo and technician Jamilah Saeed at the Meakins-Christie Laboratories as well as Anna Lisa Lucido at the Montreal Neurological Institute deserve my appreciation and credit. Talks with them were very helpful to organize the ideas and discuss the results. Especial thanks to Helene Bourque at the physics department for her friendship and encourage during the first days of my time Canada. She taught me much of the AFM methodologies employed in this thesis. I thank my lab-mates for their partnership and to all my friends that helped me in different manners and circumstances. I want to thank my parents for their love and support during all this years and thanks to Lizbel, who has been supportive and has been with me since the first days of my doctoral studies. E Table of Content Abstract Résumé Acknowledgments Table of Contents Statement of Originality A C E F K 1 Introduction Why study mechanics in biological systems? Synaptic formation in Neurons. 9 2 Material and Methods (Atomic Force Microscope) AFM and inverted optical microscope setup AFM basics and optimization Operation modes Contact mode Tapping mode Metrology and time resolution in contact and dynamic modes The force sensor Spring constant Attachment of polystyrene beads to cantilever tips Force spectroscopy Force Volume Imaging Hertz model 34 F 2.5.3 Indentation Modulation 40 3 Cellular mechanics Methods to measure mechanical properties of cells Mechanical properties of cells 55 4 Material and Methods (Airway Smooth Muscle Cells) Experimental Setup (Temperature and ph control) Cell isolation and cultures for the AFM experiments Cell localization and methodology to identify the area to indent Polyacrylamide gel preparation Gel functionalization Gel characterization Proliferation assay RNA extraction protocol Immunofluorescence Analysis of the data 68 5 Application of force spectroscopy to Airway Smooth Muscle Cells (Results) Gel functionalization and its mechanical properties Sources of uncertainty and applicability of the Hertz model to determine the Young s modulus of substrates Cell morphology on gels and actin network. 77 G 5.4 Dependence of the elastic properties of ASMC on the substrate stiffness Cell Proliferation Genetic expression Summary, Conclusions and Discussion Mechanical properties of the polyacrylamide gel and fibronectin functionalization The use of the AFM to investigate the mechanics of the cells Correlation between the substrate stiffness, the viscoelasticity of ASMC and the actin content Cell proliferation rate and genetic expression is influenced by the substrate stiffness Outlook Protein recruitment at presynaptic sites Material and methods (Neurons) Primary Cultures of Rat Hippocampal Neurons Time-lapse Protein Recruitment and Adhesion Experiments Image analysis and vesicle speed calculation Applications of the AFM to the study of presynaptic formation (Results) Procedure for successful axon/bead contacts Synaptic constituents are transported in vesicles Bassoon is recruited before synaptophysin 118 H 8.4 Adhesion occurs before protein recruitment Membrane-bound strings can be pulled out of an axon Properties of axon strings Discussion Conclusion General Conclusion Appendix Appendix Bibliography 146 I J Statement of Originality The author claims the following aspects of this thesis as original contributions to the field of biophysics. 1. Cellular response to matrix stiffness study. The first study where the viscoelastic properties of airway smooth muscle cells cultured on different matrix stiffnesses were measured. The study sheds light on the influence of the substrate mechanics in the proliferative and genetic expression of airway smooth muscle cells. Description of a methodology to determine the the Hertzian best fit on force distance curves adquired with the AFM The first study where the mechanical properties, the proliferation, morphology and the genetic expression of ASMC are determined as a function of the matrix stiffness. 2. Time-lapse presynaptic formation study. The first study where the formation of presynaptic buttons is induced in a controlled fashion. The process is initiated by the contact of a poly-lysin coated bead and the axon membrane. The formation of long strings (potentially functional neurites) was formed after bead pulling. Movement of vesicles in the strings was observed. Determination of the presence of important structural molecules for protein and vesicle transport (tubulin) as well as actin in the strings. Synaptophysin and bassoon proteins normally found moving along the axon shaft or stationed on the presynaptic sites were also observed. following. Essential procedures in this thesis not performed by the author are the K Gene arrays were performed by Genome Quebec Innovation Center, McGill University with total RNA provided by our laboratory. Neuron culture preparation. Procedure explained in brief in material and methods. Prepared by Dr. Peter Thostrup, Physics department, McGill University. L 1 Introduction In his book Imagined Worlds Freeman Dayson, a notable US theoretical physicist and mathematician wrote: The effect of concept-driven revolution is to explain old things in new ways. The effect of tool-driven revolution is to discover new things that have yet to be explained. Freeman Dyson, Imagined Worlds This is to say that the use of tools, especially when new ones are used in science, have the potential to generate numerous new findings that can lead to a search of its basics principles, understanding of previous unsolved questions or originate new ones that have to be explored. In this logic, the aim of the use of the Atomic Force Microscope (AFM) tool in this thesis is to explore two biological questions and make new discoveries that then stimulate a more systematic examination in more detail by biologists. This will lead to a deeper understanding of cell behavior. Here, we are motivated in how mechanical cues affect cell response and properties. We will explore the effect of the global substrate stiffness on cell mechanical properties. We will also use a local mechano-chemical cue to investigate the formation of synapses in neurites. We will demonstrate that this technique can be used to understand the dynamics of proteins known to be involved in the formation of synapses. In particular, we determined the arrival time of two different proteins to the AFM induced newly formed presynaptic sites. However, before going deeper in the uses of the AFM in biology, it is worth to give some examples of its use to explore questions in physics and the type of information that can be obtained. Above all, after its introduction by Binnig et al., (1986), the AFM was first employed by physicists. For instance, the AFM has been used to detect atomic interactions, something that requires very sensitive techniques due to the small forces involved. To achieve that, two approaches that have been used include the utilization of a cantilever oscillating at its resonance frequency above the sample while keeping track of small shifts 1 in the resonance frequency. Any change in the resonance frequency is associated to the interactions that occur between the cantilever tip and the closest atom on the sample. In the other case, atomic force spectroscopy at low temperature was exploited (Lantz et al., 2001) for a similar purpose. In this case, the AFM was used to detect and directly measure the formation of a chemical bond. A silicon tip was approached to a silicon (111) 7 7 surface and quantitative and atomic-scale information about the interaction forces was acquired by the frequency shift methodology. The short-range forces measured and depicted in the force-distance plot shown in Figure 1 were compared to the chemical bond interaction calculated by basic principles (Perez et al., 1998). This study showed that there is a very good agreement between the two values and thus the sensitivity of the AFM to measure atomic interactions was evidenced. Atomic manipulation is another area where AFM has shown is value and flexibility. Sugimoto et al., (2007) demonstrated that vacancy-mediated lateral manipulations of the Si atoms in the same type of surface used in the previous work is possible. It was achieved as a result of structural relaxations that weaken the adatom 1 surface bonds when the tip is in close proximity to the surface. This, in turn, enables the hopping of atoms to new locations by lowering the energetic barriers that keep the atoms still. Similarly, atomic manipulation was achieved by Oyabu et al., (2003) that demonstrated that repulsive short-range interaction forces during soft nanoindentation allows the removal of a silicon atom. Atom deposition was also achieved and both spatial manipulation processes were purely mechanical. Another study where the ability of the AFM to precisely control the spatial movement of the probe has been exploited is in the deposition of molecules on surfaces. For instance, Moldovan et al., (2006) designed and fabricated a nanoprobe system that consist of an ultrasharp volcano-like tip with microfluidic capabilities. The tip is connected to an on-chip reservoir through a channel inside 1 Contraction of adsorbed atom. An adatom is an atom that lies on a crystal surface, and can be thought of as the opposite of a surface vacancy. 2 Figure 1: Force-distance curve above the Si surface and a fit to the data using a sphere-plane model for the Van der Waals interaction force (upper left image). Total force (red line) and short-range force (yellow line) determined above an atom (lower left image, Lantz et al., 2001) In the inset, a comparison between the measured short range force and the calculated bonding interaction by first-principles is shown. Calculated diffusion barriers for adatoms on a lattice when the tip is at different height above the surface (upper right image). Topographic images from a series of vacancy-mediated manipulations mediated by the AFM tip (lower right images, Sugimoto et al., 2007) the cantilever. This system is an upgrade of the dip-pen nanolithography where the ink is only deposited on the surface of the cantilever tip (Piner et al., 1999) and used for writing. The potential uses of the volcano-like tip system in biology include localized drug delivery at specific sites of the cells or close to membrane receptors and biofluid sampling applications. This potentially allows the detection of cells that are different of the rest by sampling the secreted molecules for individual cells at specific locations. 3 Combination of AFM with volcano-like tips and fluorescence microscopy could be very valuable. Detection of spatial distribution of single molecules can be performed with fluorescence imaging (Eckel et al., 2006). Force spectroscopy by atomic force microscopy allows addressing, manipulation and quantitative probing of the nanomechanical properties of individual macromolecules thus allow the investigation of optical and mechanical properties at the single molecule level. In Eckel et al., (2006) study, they report the distance-controlled quenching of semiconductor quantum dot clusters with an AFM tip. In biology, the AFM as been used more often in recent years. A traditional applications is the collection of topographic data to reveal cellular structures. Prior to the AFM introduction, such information was to the best scarce or difficult to obtain. However, nowadays, contact and dynamic modes have been employed with success. It is evidenced by AFM images of cells (Le Grimellec et al., 1998, Espen
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