MOLECULAR MECHANISMS OF MECHANOSENSING AT CELL-CELL AND CELL-MATRIX ADHESIONS YAO MINGXI NATIONAL UNIVERSITY OF SINGAPORE 2014 MOLECULAR MECHANISMS OF MECHANOSENSING AT CELL-CELL AND CELL-MATRIX ADHESIONS YAO MINGXI B. Sci. (Hons.), NUS, 2009 A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY MECHANOBIOLOGY INSTITUTE NATIONAL UNIVERSITY OF SINGAPORE 2014 DECLEARATION I hereby declare that this thesis is my original work and it has been written by me in its entirety. I have duly acknowledged all the sources of information which have been used in the thesis. This thesis has not been submitted for any degree in any university previously. Yao Mingxi August 18, 2014 Acknowledgment It is truly a rewarding experience in the past five years as an PhD student in mechanobiology institute. It is such a vibrant institute where exciting research takes place. I am grateful for the opportunity to work in this dynamic environment surrounded by excellent colleagues. I would like to thank my supervisor Dr Yan Jie for his guidance and support over the years. He has been instrumental for creating a warm and stimulating atmosphere in the lab. I keep being amazed by his work altitude and passion for science. It is a great pleasure working with him and I learned a lot from him both academically and in life. I also want to thank my collaborators - Dr Rene-Marc Mege, Dr Benoit Ladoux, Dr Benjamin T Goult, Dr Mike Sheetz and Qiu Wu for their insightful suggestions and great work. Two excellent undergraduate students, Guo Yingjian and Kasper Graves Hvid, have helped me in many aspect of experiments. I am grateful for their contributions. I would like to express my gratitude to friends and colleagues in Yan Jie’s lab - Chen Hu, Fu Hongxia, Peiwen Cong, Yuan Xin, Lim Ciji, Zhang Xinghua, Le Shimin, Qu Yuanyuan, Chen Jin, Artem, Rickson, Lee Xinyi, Wong Weijuan, Zhao Xiaodan, Li You, Li Yanan, Rangit, Dugarao. They make the lab a warm and fun place to be. I am very grateful for their friendship and support along the way. December 15, 2014 iv Contents Introduction 1.1 Mechanosensitivity of cells . . . . . . . . . . . . . . . . . . . 1.2 Review of cell adhesions . . . . . . . . . . . . . . . . . . . . 1.2.1 Cadherin based adherens junctions . . . . . . . . . . 1.2.2 integrin based cell-matrix adhesions . . . . . . . . . . Literature survey on mechanosensing related proteins at celladhesions . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.3 1.4 1.3.1 vinculin . . . . . . . . . . . . . . . . . . . . . . . . . 10 1.3.2 talin . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 1.3.3 α-catenin . . . . . . . . . . . . . . . . . . . . . . . . . 14 1.3.4 other mechanosensing proteins at cell adhesions . . . 16 Key question: Mechanosensing mechanisms of talin and αcatenin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 Strategies and Methods 2.1 2.2 2.3 19 Theory of force induced structural transitions of protein . . . 20 2.1.1 Structural states during two state protein unfoding and refolding transitions . . . . . . . . . . . . . . . . 20 2.1.2 Force-extension curves of the structural states . . . . 22 2.1.3 Force dependent free energy differences between states 23 2.1.4 Free energy landscape along the transition coordinate 28 Magnetic tweezers . . . . . . . . . . . . . . . . . . . . . . . . 31 2.2.1 Magnetic tweezers setup . . . . . . . . . . . . . . . . 32 2.2.2 Force determination for magnetic tweezers . . . . . . 33 Other Methods . . . . . . . . . . . . . . . . . . . . . . . . . 37 2.3.1 Protein Expression . . . . . . . . . . . . . . . . . . . 37 2.3.2 Force calibration . . . . . . . . . . . . . . . . . . . . 38 2.3.3 Data-analysis . . . . . . . . . . . . . . . . . . . . . . 39 2.3.4 Hidden Markov models . . . . . . . . . . . . . . . . . 39 v 2.3.5 Bioconjugation and surface chemistries . . . . . . . . 40 Force response of talin rod and α-catenin 3.1 Introduction . . . . . . . . . . . . . . . . . . . 3.2 Results and Discussion . . . . . . . . . . . . . 3.2.1 The force response of talin rod domain 3.2.2 The force response of αE-catenin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vinculin binding to talin and α-catenin fine-tuned by mechanical forces 4.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2 Results and discussion . . . . . . . . . . . . . . . . . . . . . 4.2.1 The effects of VD1 domain binding on the effect of talin rod . . . . . . . . . . . . . . . . . . . . . . . . 4.2.2 Force dependent interaction of vinculin head to αCM 4.2.3 The effect of full length vinculin on the samples of talin and α-catenin . . . . . . . . . . . . . . . . . . . Discussion and Conclusions 43 43 44 44 52 61 61 62 62 70 77 81 vi Summary Over the past decade, mechanical forces have been identified to take part in many important biological processes ranging from embryo development to tissue maintenance and cancer. A novel class of proteins, termed mechanosensing proteins, is found to be able to convert mechanical forces into biochemical signals that direct cellular responses. These proteins are particularly enriched at cell adhesion sites where cells’ cytoskeleton connects with their micro-environment and mechanical forces are transmitted and sensed making cell adhesion sites signaling hubs for detecting mechanical cues. Established mechanosensing mechanisms of these proteins include force dependent channel opening, phosporylation and catch bond formation. Talin and α-catenin, two mechanosensing proteins located at focal adhesions and adherens junctions respectively, are critical for the force dependent initialization and growth cell adhesions.It has been suggested by cell and structural studies that mechanical force applied to the two proteins will increase their binding affinity to vinculin, a protein that promotes cytoskeleton linkage leading to growth and maturation of cell adhesions. Unlike many mechanosensors, accumulating data suggests that talin and α-catenin respond to applied force by expose their cryptic vinculin binding sites. However, molecular level mechanisms of this process have not been quantitatively understood with direct experimental evidence. In this thesis work, I used state-of-art magnetic-tweezers technology to study the mechanosensing mechanism of talin and α-catenin. The hypothesis is that the two proteins change their conformations upon application of force and modulate their binding affinity to vinculin. In Chapter 1, I review the biological background on mechanosensing, focusing on the role talin and α-catenin plays during initiation of cell adhesions. In Chapter 2, I describe the methods used for my thesis, introducing magnetic tweezers and theoretic background of force induced protein unfolding. In chapter I study the mechanical stability of the rod domains of talin and central domain of α-catenin using both wild type and mutant constructs. Both talin vii rod domain and α-catenin central domain undergo well-defined conformation changes at forces greater than pN, suggesting physiological relevant forces could expose cryptic vinculin binding sites in the two proteins. In Chapter 4, I compare the mechanical responses of talin rod domain and αcatenin central domain to show that vinculin binding to talin and α-catenin only upon application of force and vinculin binding inhibits the refolding of these proteins. In addition, at forces larger than 30 pN, bound vinculin can be displaced from these proteins, implying the binding of vinculin is biphasic with force. Finally in Chapter I discuss the biological implications of the findings. The work in this thesis establishes a molecular mechanism of mechanosensing at early adhesion formations where the force dependent conformational changes of talin and α-catenin play key role in the initiation of adhesioncytoskeleton linkage. Besides providing novel mechanistic insights into the function mechano-sensitive proteins,the single molecule manipulation methods developed in this work opens up possibility to study other forcedependent protein-protein interactions such as ligand receptor interaction, which has important implication in many biological and pathological processes. viii List of Figures 1.1 Anchoring junctions . . . . . . . . . . . . . . . . . . . . . . . 1.2 Types of cell-matrix adhesions . . . . . . . . . . . . . . . . . 1.3 Vinculin domain map . . . . . . . . . . . . . . . . . . . . . . 11 1.4 The domain map of talin . . . . . . . . . . . . . . . . . . . . 12 1.5 α-catenin domain map . . . . . . . . . . . . . . . . . . . . . 14 2.1 The conformational states of protein under force . . . . . . . 21 2.2 Energy landscape of two state model . . . . . . . . . . . . . 21 2.3 Calculated force-extension curve of folded and unfolded i27 . 23 2.4 Calculated force-dependent Gibbs free energy folded and unfolded I27 . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 2.5 Calculated force-dependent unfolding and refolding rate of I27 27 2.6 Calculated force-dependent free energy landscape of I27 as a function of extension . . . . . . . . . . . . . . . . . . . . . 30 2.7 Force geometry affect the free energy of transition states . . 31 2.8 Photo of the vertical magnetic tweezers used in this study . 32 2.9 Illustration of magnetic tweezers setup . . . . . . . . . . . . 34 2.10 Calibration of permanent magnets using λ-DNA . . . . . . . 36 2.11 Calibration of the strength of individual magnetic beads . . 38 3.1 Sketch of the conformation changes of talin R1-R3 domain under mechanical force . . . . . . . . . . . . . . . . . . . . . 44 3.2 The schematic figure of experimental setup for talin stretching experiment . . . . . . . . . . . . . . . . . . . . . . . . . 45 3.3 Force cycle experiments of the talin R1-R3 domain. . . . . . 46 3.4 2-D histogram of the unfolding force and unfolding size of WT talin R1-R3 domain . . . . . . . . . . . . . . . . . . . . 47 3.5 Unfolding force histograms of two R1-R3 talin domains . . . 47 3.6 Two state fluctuations of talin R3 domain . . . . . . . . . . 49 ix 3.7 3.8 3.9 3.10 3.11 3.12 3.13 3.14 Unfolding force histogram of wildtype and IVVI mutant talin R1-R3 domains of talin at pN/s constant loading rate. . Unfolding force responses of the R9-R12 region of talin rod Unfolding force responses of the R7-R9 region of talin rod Experimental setup of αCM stretching. . . . . . . . . . . . Force responses of wild type αCM . . . . . . . . . . . . . . Repeated unfolding-refolding force cycle experiments on a single αCM tether . . . . . . . . . . . . . . . . . . . . . . . Unfurling of αCM and ∼ pN forces . . . . . . . . . . . . The force responses of L344P mutant of αCM . . . . . . . 4.1 4.2 Mechanosensitivity of talin R1-R3 . . . . . . . . . . . . . Concentration dependence of VD1 binding to talin R1-R3 domain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3 VD1 dissociate from talin rod at high forces . . . . . . . . . 4.4 Observation of five VD1 dissociation steps . . . . . . . . . . 4.5 Detecting the binding of VD1 to the peptide chain of unfolded vinculin binding α-helices at high force in 100 nM VD1 . . 4.6 Effect of VD1 on the unfolding/refolding of αCM . . . . . . 4.7 Correlation between vinculin dissociation and αCM folding 4.8 The mechanosensitivity of αCM folding on vinculin binding 4.9 High force displaces the bound VD1 from the vinculin binding site in αCM . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.10 Detecting the binding of full length vinculin to talin R1-R3 and αCM . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.11 Dissociation of full length vinculin from the αCM at high force . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1 5.2 . . . . . 50 51 52 53 55 . 56 . 57 . 59 . 64 . 65 . 66 . 68 . . . . 69 71 73 75 . 76 . 78 . 79 Model of fore-dependent talin-vinculin interaction . . . . . . 87 Model of fore-dependent α-catenin-vinculin interaction . . . 88 x cell-ECM and cell-cell transduced mechanical load, an important factor of tissue reshaping, tumor progression, and collective cell migration. Figure 5.2: Schematics of biphasic dependence of VD1 binding to α-catenin on force. At low force (< pN), α-catenin exists under an auto-inhibited helix bundle conformation that prevents VD1 binding. At high force (> 30 pN), VD1 binding is also inhibited because the α-helix conformation of the vinculin binding site bound with VD1 is unstable. In the intermediate force range (5-30 pN), the auto-inhibited conformation of α-catenin is released, and the vinculin binding site is exposed for VD1 binding. The force-dependent process centered on α-catenin and talin we unraveled here may further cooperate with the binding of other α-catenin partners to regulate the strength of the cytoskeleton linkage to cell-cell junctions. A ∼ pN force deforms the modulation domain of α-catenin and initializes binding of VD1 . The resulting lockage of α -catenin in a partially unfolded conformation may have a downstream impact on interactions between αcatenin and other junctional and cytoskeleton proteins, such as α-catenin itself, afadin, ZO-1, α-actinin and F-actin. However, further understanding of these processes will require precise structural and biochemical data on these complexes that are lacking so far. Up to date, a complete structural description of αE-catenin under both its closed and open conformations is still lacking. It is postulated that the robust functions of cells are carried out by modular subsets consisted with finite group of molecules termed as ”functional modules”. Individual functional modules represent evolutionarily constraint design principles that can be reused in different circumstances in cells [147]. The tissue and cellular level mechanosensing could be con88 sidered as emergent properties of related functional modules responding to mechanical forces in individual cells. For example, cells seems to have delicate sarcomere like apparatus that could deform their substrates and sense the mechanical rigidity of the environment [12, 13]. These functional modules could be reused by cells in different circumstances for distinct purposes. Thus it is very important to understand the underline molecular mechanism and regulation of these functional modules, as they may provide general principles of performing defined molecular functions. The mechanosensing mechanism revealed in this work could be a common feature of other mechanosensitive adhesion proteins that couple to actin. There are many proteins that bind to cytoskeletons upon stretch [47], and it is likely that many rely upon exposure of buried binding sites. It is possible that the ∼5 pN low force threshold for talin and α-catenin is a common feature of other mechanosensitive adhesion proteins that couple to actin. 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Nature 402, C47–52. 103 [...]... related proteins at cell- adhesions As cell adhesions play a pivotal role in the proper function of cells, misregulation of cell adhesions often leads to severe pathological consequences Altered cell- cell and cell- matrix adhesion function is one of the hall marks of cancer [49, 50] The proper functions of cell adhesions, both in terms of adhesion strength and underlying signaling pathways are critical... form the optically dense matrix adhesion plaques [41] Integrin based cell- matrix adhesions can generate a highly diverse set of adhesive structures that have distinct morphology and behaviors depending on the micro-environment and phases of adhesion maturation In 2D culture system, in the initialization phase of cell spreading, when a cell is just in contact with its substrate, cells form nascent dynamic... for anchoring intermediate filaments to the cell- cell contact sites and are present in cells that experiencing high shear stress [20 , 22 ] The most well-studied cellcell adhesions that play critical role in maintaining mechanical integrity of tissues and regulate cell fates are cadherin based adherens junctions The cadherin family is the most diverse class of CAMs for cell- cell adhesions It contains over... Figure 1 .2: Different cell- matrix adhesions have distinct morphologies and functions depending on cell type and spatial-temporal phases of the cell Used by permission from MBInfo: www.mechanobio.info; Mechanobiology Institute, National University of Singapore Cell- matrix adhesions start to form upon stimulation, either external when cells gets in contact with ECM, or internal, by the activation of signaling... architecture facilitated by specific interactions between adhesion proteins [44] Mechanical force is a critical factor that couples tightly with the formation and dynamics of cell- matrix adhesions Most cell- matrix adhesions, such as focal complex and focal adhesions, are contractile that actively exert forces to their substrate [45] Stable focal adhesions are lost when actomyosin contraction of cells is inhibited... inhibited by blebbistatin In addition, the size of focal adhesion is proportional to the forces they exerted to the substrate [46] and many focal adhesion proteins show force dependent localization to focal adhesions [47] Cell- matrix adhesions are also responsible for sensing the rigidity of the substrate, directing the behaviors of cell spreading (Reviewed in [ 12] ) As cell- matrix adhesions are force... the formation and functioning of tissues Cell- cell adhesion is one of the corner stones in the arising of multicellular organisms Depending on the context, cell- cell adhesion could serve different roles such as supporting mechanical integrity of tissues, signal transduction across cells, cellular recognition and triggering of immune responses Tissue organizations do not only depend on cell- cell adhesions... due to heart and brain defects [ 52] Vinculin does not possess enzymatic activity - all its biological functions are carried out through highly regulated molecular interactions that can be fine-tuned by conformation changes At subcellular level, vinculin plays important roles in regulating cell- matrix and cell- cell adhesions Cells lacking vinculin develop less stable focal adhesions and migrate faster... epithelium and muscles, cells are surrounded by a fibrous protein network called extra-cellular matrix (ECM) Main components of ECM include collagen, proteoglycans and multiadhesive matrix proteins such as fibronectin ECM acts as an organization scaffold for tissues and is responsible for the signaling and regulation of variety of cellular processes such as cell growth, migration and gene-expression [20 ] To... α-catenin, demonstrating the mechanosensitivity of adherens junctions [37] Adherens junction mechanosensing has been shown to be involved in im6 portant biological processes such as development, tissue repair and diseases Great efforts have been made to elucidate the key players and underlying mechanosensing mechanisms (Reviewed in [38]) 1 .2. 2 integrin based cell- matrix adhesions Integrin based cell- matrix . MOLECULAR MECHANISMS OF MECHANOSENSING AT CELL- CELL AND CELL- MATRIX ADHESIONS YAO MINGXI NATIONAL UNIVERSITY OF SINGAPORE 20 14 MOLECULAR MECHANISMS OF MECHANOSENSING AT CELL- CELL AND CELL- MATRIX. force . . . . . . . 21 2. 2 Energy landscape of two state model . . . . . . . . . . . . . 21 2. 3 Calculated force-extension curve of folded and unfolded i27 . 23 2. 4 Calculated force-dependent Gibbs. and regulation of variety of cellular processes such as cell growth, migration and gene-expression [20 ]. To fulfill such a set of functions, eukaryotic cells have evolved delicate and robust molecular