Intra cellular endoplasmic reticulum dynamics, distribution and function in response to cell engineered surface adhesion

INTRA-CELLULAR ENDOPLASMIC RETICULUM DYNAMICS, DISTRIBUTION AND FUNCTION IN RESPONSE TO CELL-ENGINEERED SURFACE ADHESION ZHANG XIN (B.Sci (Hons.), NUS) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY GRADUATE PROGRAMME IN BIOENGINEERING−SCHOOL OF MEDICINE (GPBE−SOM) NATIONAL UNIVERSITY OF SINGAPORE 2009 i ACKNOWLEDGEMENTS First and foremost, I would like to thank my supervisor, Prof Hanry Yu and A/P Zhang Yong-Wei for their guidance and being very encouraging throughout my graduate studies. I wish to express my gratitude to my husband Deng Da and my parents for their support and understanding in my graduate studies. I would like to thank Miss Tee Yee-Han and Mr Heng Kiang, Justin for their moral support and help; it has been a great pleasure to have worked with them. I would also like to thank Miss Hu Xian and Dr Felix Margadant as well as Mr Zhang Jie for their advice in my project. My colleagues at the Cell and Tissue Engineering Lab in both NUS and IBN have all made my research experience memorable. Last, but not least, I would like to thank NUS, A*STAR, BMRC, ARC and NMRC for their financial support. ii LIST OF PUBLISHED WORK z Xin Zhang, Annette S Vincent, Berry Halliwell and Kim Ping Wong. (2004) A mechanism of sulfite neurotoxicity: direct inhibition of glutamate dehydrogenase. J. Biol Chem. 279: 14035-14045. z Lee-lee Ong, Pao-chun Lin, Xin Zhang, Ser-mien Chia and Hanry Yu. (2006) Kinectin-dependent assembly of translation elongation factor-1 complex on endoplasmic reticulum regulates protein synthesis. J. Biol Chem. 281: 3362133634. z Yi-Chin Toh, Susanne Ng, Yuet Mei Khong, Xin Zhang, Yajuan Zhu, Pao Chun Lin, Chee Min Ten, Wanxin Sun and Hanry Yu. (2006) Cellular responses to a nanofibrous environment. Nano Today. 1(3): 34-43 z Ping Liu, Yong W. Zhang, Hanry Yu, Xin Zhang, Qian H. Cheng, William Bonfield. (2008) Spreading of an anchorage-dependent cell on a selectively ligand-coated substrate mediated by receptor-ligand binding. J. Biomed Mater Res A. (Epub ahead of print). z Xin Zhang, Yee-Han Tee, Justin K. Heng, Yajuan Zhu, Xian Hu, Felix Margadant, Christoph Ballestrem, Alexander Bershadsky, Gareth Griffiths, Hanry Yu. (2009) Kinectin-mediated endoplasmic reticulum dynamics supports focal adhesion growth in cellular lamella. J. Cell Sci. (in revision). LIST OF CONFERENCE PAPER z Xin Zhang, Justin K. Heng, Hanry Yu. (2007) Endoplasmic reticulum at cell leading edge is essential to regulate cell motility. The 47th Annual Meeting of the American Society for Cell Biology in Washington, DC, December 1-5, 2007. iii TABLE OF CONTENTS ACKNOWLEDGEMENTS …………………………………………………… ii LIST OF PUBLISHED WORK ………………………………………………. iii SUMMARY vi LIST OF TABLES …………………………………………………………… . viii LIST OF FIGURES ……………………………………………………………. ix LIST OF ABBREVIATIONS ………………………………………………… xii CHAPTER I Aims and Approaches … .……………………… ……. CHAPTER II Background and Significance 2.1 2.2 2.3 2.4 2.5 Interaction of cells with ECM --- cell adhesion ……………………… 2.1.1 Integrin induced focal complex assembly ……………………… . 2.1.2 Novel components at focal complexes …………………………… 12 Interaction of cells with ECM --- cytoskeleton and cell morphology 14 2.2.1 Actin filaments in cell adhesion …………………………………. 14 2.2.2 Microtubules in cell adhesion …………………………………… 18 2.2.3 Cell morphology in cell adhesion ……………………………… 19 Cytoskeleton dependent intra-cellular ER dynamics ……………… 21 2.3.1 Microtubule dependent ER dynamics …………………………… 21 2.3.2 Actin filaments and ER dynamics 24 Kinectin regulated ER dynamics …………………………………… . 26 2.4.1 Introduction of kinectin …………………………………………. 26 2.4.2 Kinectin regulated ER dynamics ……………………………… . 28 Significance and rationale of thesis research ………………………… 31 CHAPTER III Materials and Methods ……………………………… . CHAPTER IV ER accumulation and dynamics in cellular lamella upon cell adhesion ……………………………………………. 51 CHAPTER V Kinectin mediated ER tubule dynamics in cellular lamella…………………………………………………… 71 34 iv CHAPTER VI Effects on cell behaviors via disruption of kinectin-kinesin interaction and inhibition of ER dynamics………… . 96 CHAPTER VII Discussion ……………………………………………… 120 CHAPTER VIII Reference ………………………………………………. 127 APPENDIX A Fabrication and characterization of the bead………… AI v SUMMARY In tissue engineering research, the regulation of cell behaviors in extra-cellular environments with chemical and mechanical signals is crucial for the development of cells into desired tissues that can perform physiological functions. The current understanding of the cell behavior regulation is limited to signals that can induce cytoskeleton rearrangements and cellular deformations. In fact, the cell is a complicated and integrated system with numerous intra-cellular proteins, vesicles and organelles. The dynamics, distribution and functions of these intra-cellular components can greatly influence cell phenotypes and cellular functions. However, there is a lack of understanding in these intra-cellular component responses upon cell adhering onto a substrate surface. This thesis reports an intra-cellular organelle response upon cell membrane adhering to an extra-cellular surface coated with fibronectin. I have discovered that intra-cellular endoplasmic reticulum (ER) tubule dynamics in cellular lamella is towards the cell periphery upon cell membrane adhesion and spreading. The ER tubules in the lamella can interact with nascent adhesions --- focal complexes and are required in the process of focal complex growth (increase of the size of the focal complex). ER tubule dynamics in the lamella, the sheet-like region proximal with membrane protrusions, was observed upon cell membrane adhesion onto both the fibronectin coated bead’s surface and flat surfaces. The dynamics was mediated by the interaction of the microtubule motor protein kinesin with its receptor kinectin on vi ER membrane. Over-expression of the minimal kinectin-kinesin interaction domain (vd4) of kinectin disrupted the interaction and hence inhibited ER tubule dynamics. This method was then used to study the consequences of inhibited ER dynamics to the cell behaviors such as cell migration and spreading. The interaction of ER tubules to focal complexes was required in the growth of focal complexes, whereas the lack of ER dynamics resulted in unstable focal complexes in the lamella. In addition, as a consequence of the inhibited ER dynamics upon cell membrane adhesion, both the cell migration and cell spreading rate were significantly reduced. In conclusion, this thesis describes the spatial and functional co-relations of ER tubule dynamics with focal complexes in the lamella and with cell behaviors such as cell migration and spreading upon the cell membrane adhesion. The finding of ER’s participation in the growth of focal complexes, cell migration and cell spreading implies the possibility that other intra-cellular components would also be involved in processes of cell adhesion. With more understanding of intra-cellular component regulated cell behaviors, better design of extra-cellular environments in tissue engineering with chemical and mechanical signals which can induce controllable intra-cellular organelle changes, protein expression or vesicle transportation may be applied to achieve the desired cell/tissue performance. vii LIST OF TABLES Table Summary of ER-microtubule linkers. Table Fibronectin concentration and density on the coated bead’s surface. viii LIST OF FIGURES Figure Illustration of the series of intra-cellular events triggered upon cellsubstrate surface interactions. Figure Two strategies applied to study ER’s response in cell adhesion. Figure Integrin-fibronectin interaction induced integrin clustering and assembly of focal complexes. Figure Novel components found at focal complexes. Focal complexes are induced by the interaction of cells with fibronectin coated beads. Figure Actin filaments arrangement in response to cell adhesion. Figure Cell morphologies in response to cell adhesion on different scaffolds. Figure Illustration of protein primary structure of kinectin. Figure The interaction of kinectin and kinesin drives ER extension along microtubule. Figure The feedback loop of inter-related extra-cellular and intra-cellular signals and events regulates cell behaviors. Figure 10 Surface activated magnetic beads with tosyl groups that can be replaced by amine groups in proteins (Dynabead® Invitrogen). Figure 11 XPS wide scanning spectrums of the un-coated bead’s surface and the protein coated bead’s surface. Figure 12 Scanning Electron Microscopy (SEM) photos of the surface of the uncoated bead and the fibronectin coated bead. Figure 13 Fibronectin coating efficiency on the bead’s surface. Figure 14 Cell membrane around the coated bead’s surface. Figure 15 ER accumulation around the bead coated with different ligands. Figure 16 ER accumulation around fibronectin coated bead’s surface. Figure 17 Quantification of ER accumulation. Figure 18 Transmission electron microscopy (TEM) image of ER near cell-bead interface. Figure 19 Time lapse images of ER tubule dynamics on the coated bead’s surface. Figure 20 The dynamics of ER tubules towards cell leading edge. ix Figure 21 The zoomed in image (x4) of the dynamics of ER tubules towards the cell edge. Figure 22 Interaction of ER tubules with focal complexes. Figure 23 ER tubule dynamics towards focal complexes. Figure 24 The distribution of ER tubules and microtubules. Figure 25 The zoomed in images of the distribution of ER tubules and microtubules. Figure 26 Dynamics of ER tubules along the direction of microtubule polymerization. Figure 27 Co-localization of ER with immuno-stained cellular kinectin in DsRed2-ER HeLa cells at cell periphery. Figure 28 Co-localization of ER and kinectin around the coated bead. Figure 29 ER accumulation around fibronectin coated beads. Figure 30 Kinectin accumulation around fibronectin coated beads. Figure 31 The inhibition of ER extension into the cell edge with the overexpression of vd4 domain versus control vector. Figure 32 Over-expression of kinectin vd4 domain inhibited the ER recovery after nocodazole treatment. Figure 33 Over-expression of kinectin vd4 domain affected the ER dynamics in cell leading edge. Figure 34 Over-expression of kinectin vd4 domain affected the cell membrane extension. Figure 35 Time lapse images of ER tubule dynamics during cell membrane extension in DsRed2-ER HeLa cells. 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Paramagnetic materials not retain any magnetization in the absence of an externally applied magnetic field, whereas magnetic materials always have magnetization. C D A B Figure A1. The schematic cross-section of the paramagnetic polymer bead (Wang, C. H. et al 1992). (A) represents a polymer core, (B) is a metal oxide/polymer coating, (C) is the protect polymer coating and (D) is a functionalized polymer coating. The polymer core can be obtained by emulsion polymerization or suspension polymerization (Wang, C. H. et al 1992). Both of them are commonly used to fabricate polymers. Emulsion polymerization is a type of radical polymerization that usually starts with an emulsion with water, monomers, and surfactant. Suspension polymerization is a polymerization process that uses mechanical agitation to mix the monomer or the mixture of monomers in a liquid phase such as water; and the monomers can be polymerized when they are dispersed by continuous agitation. I There are different types of monomers can be used to prepare the polymer core, such as styrene, methyl methacrylate and vinyltoluene. The mixture of monomers can also be applied (Chen, K. S. et al 2005). For example, the Dynabead® polystyrene bead has a polymer core with a copolymer of 80% styrene and 20% divinylbenzene (Ugelstad, J. et al 1987). The monomer used for magnetic metal oxide coating and protective coating may or may not be the same as the polymer core. The metal oxide is prepared from a mixture of ferrous and ferric salt. It is produced by heating and precipitating the mixture of divalent and trivalent ferrous and ferric sulfate or chloride with sodium hydroxide solution (Wang, C. H. et al 1992). The molar ratio of divalent to trivalent metal salt can be controlled to obtain desirable magnetic characteristics of the metal oxide (Wang, C. H. et al 1992). The prepared metal oxide is mixed with monomers and coated onto the polymer core to form the metal oxide/polymer layer. In addition, the thickness of this layer can be controlled by varying the weight ratio of the monomers in polymer core and monomers in metal oxide/polymer layer (Wang, C. H. et al 1992). In order to prevent the falling off of the magnetic oxide, a polystyrene protective layer can be used to coat the magnetic particles (Wang, C. H. et al 1992). Covalent conjugation with biological materials on beads’ surface can be achieved by coating the beads with a functionalized polymer layer with different functional groups such as carboxyl, amino, tosyl or hydroxyl groups. These functional groups can be coupled to the polymer particle surfaces. For example, II post-polymerization nitration can be used to introduce nitro group on the polymeric particle surfaces. (Fonnum, G. et al 2006). Alternatively, they can be resulted from the use of functionalized co-mononers. For instance, monomers can be mixed and polymerize with epoxides such as glycidol or allylgllycidyl ether to form a functionalized layer on the polymeric particle surfaces (Fonnum, G. et al 2006). As the size of the bead ranges from µm to more than 10 µm, the bead can be used as three-dimensional external environments with various types of signals or with different signal densities to interact with cells (Trans, H. et al 2002). For example, after the conjugation with different types of ECM ligands, such as fibronectin, the cellular responses upon the cell-bead interaction can be systematically analyzed. Moreover, because DNAs and proteins such as antigen or antibody can be conjugated to beads’ surfaces, the bead can be used to separate cells with specific receptors or to purify cells in biomedical applications (Ugelstad, J. et al 1987; Wang, C. H. et al 1992; Fonnum, G. et al 2006). The paramagnetism of the bead provides the ease to separate bio-molecule conjugated beads from non-conjugated beads. III A2 Characterization A2.1 Structural characterization The size, shape and surface features of the beads can be characterized by different types of electron microscope. Different from light microscope, electron microscope uses electrons to generate an image with high magnification. Scanning electron microscope (SEM) and transmission electron microscope (TEM) have been heavily used in both material science and biological science. SEM can be used to provide high resolution image of a sample surface with a three-dimensional appearance. Its resolution can range from nm to 20 nm. For example, the structure of porous polymer scaffolds, nanofibers (Li, W. et al 2001; Huang, Z. et al 2003; Zhang, S. et al 2005), nanopillars (Kuo, C. W. et al 2003; Mao, P. and Han, J. 2005), as well as nanochannels (Lee, C. et al 2003; Mao, P. and Han, J. 2005) have all been characterized by SEM. The size and the shape of beads I used in this thesis were characterized by SEM as shown in Figure A2. However, the functional groups and conjugated ligands on beads’ surface are hardly to be seen using SEM. A B Figure A2. Paramagnetic beads with diameter of 4.5µm viewed by SEM. (A) Lower magnification (4,000×) was used to examine the size, shape and uniformity of the bead. (B) Higher magnification (15,000×) was used to examine surface topography. IV A2.2 Chemical characterization Chemical instruments can be used to characterize the chemical components on the ligand conjugated bead’s surface. For examples, X-ray photoelectron spectroscopy (XPS) spectra are obtained by irradiating a material with a beam of X-ray; it can be used to quantitatively analyze the chemical elements of a surface with a depth of 1-10 nm as well as the chemical or electronic state of each element of the surface. Therefore, the use of XPS helps the detection of a successful surface modification (Kang, E. T. and Tan, K. L. 1996; Barry, J. J. A. et al 2006). The XPS graphs of Figure in Chapter III showed the introduction of N1s on the bead surface by coating fibronectin. Besides XPS, another potential instrument can be used for surface chemical characterization is energy dispersive X-ray spectroscopy (EDX), in which an electron or photon beam is used to excite the samples with ground state electrons in inner shell of an element to result in the electron hole in an atom’s electronic structure; and the filling in of this hole by the electrons from the outer shell can emit excess energy in the form of X-ray that can be detected. Each specific element will have highly specific spectral lines. Similar as XPS, EDX has also been used for chemical characterization of many types of three-dimensional scaffold for surface modifications. For deeper surface characterization down to µm of a surface, fourier transform infrared spectroscopy (FTIR) can be used. FTIR is particularly useful to analyze functional groups such as C=O, C-N or –NO2 on surfaces because each functional group has characteristic infrared absorptions. The surface compositions and properties of various three-dimensional scaffolds like fibers (Nazarov, R. et al 2004), particles V and surface coatings can be applied for FTIR analysis. A2.3 Mechanical characterization The mechanical properties such as the stiffness and topography of the bead’s surface can be characterized by atomic force microscopy (AFM). Comparing with SEM, AFM can provide three-dimensional surface profile and higher resolution images. The samples viewed by AFM not require special treatments, such as coating of a metal, to avoid any damage to the sample. In addition to its topographic measurement, AFM can sense piconewton forces and subnanometer displacement and hence can measure the stiffness of the bead or any engineered scaffold (Dufrene, Y. F. 2002). Moreover, AFM can measure the binding force between integrins and ligands on the bead’s surface to evaluate the cell-bead interactions (Moy, V. T. et al 1994; Lehenkari, P. P. and Horton, M. A. 1999). Another instrument that can be used to characterize the cell-bead interaction is optical tweezers, which use light to manipulate microscopic objects at low temperature (Ashkin, A. 1970; Chu. S.1991). Its principle is based on the radiation pressure exerted by a light beam on dielectric objects. Besides manipulation of small objects, optical tweezers can be used to measure intermolecular forces in the range of piconewtons (Kuo. S. C and Sheetz, M. P. 1993), as well as the elasticity of scaffold such as nanofibers (Smith, S. B. et al 1996). VI A3 References Ashkin, A. (1970) Acceleration and trapping of particles by radiation pressure. Physical Review Letters 24: 156-159 Barry, J. J. A., Howard, D., Shakesheff, K. M., Howdle, S. M., and Alexander, M. R. (2006) Using a core-sheath distribution of surface chemistry through 3D tissue engineering scaffolds to control cell ingress. Advanced Materials 18 (11): 1406-1410 Chen, K. S., Lin, I. K., and Ko, F. H. 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Composites Science and Technology 63. 2223-2253 Kang, E. T. and Tan, K. L. (1996) Surface modification and functionalization of polytetrafluoroethylene films. Macromolecules 29 (21): 6872-6879 Kuo, C. W., Shiu, J. Y., and Chen, P. (2003) Size- and shape-controlled fabrication of large-area periodic nanopillar arrays. Chemistry of Materials 15: 1917-1920 Kuo. S. C and Sheetz, M. P. (1993) Force of single kinesin molecules measured with optical tweezers. Science 260 (5105): 232-234 Lee, C., Yang E. H., Myung, N. V., and George, T. A (2003) nanochannel fabrication technique using chemical-mechanical polishing (CMP) and thermal oxidation. VII Nanotechnology IEEE 2: 553-556 Lehenkari, P. P. and Horton, M. A. (1999) Single integrin molecule adhesion forces in intact cells measured by atomic force microscopy. Biochemical and Biophysical Research Communications 259 (3): 645-650 Li, W., Laurencin, C. T., Caterson, E. J., Tuan, R. S., and Ko F. K. (2001) Electrospun nanofibrous structure: a novel scaffold for tissue engineering. Journal of Biomedical Materials Research 60. 613-621 Mao, P. and Han, J. (2005) Fabrication and characterization of 20nm planar nanofluidic channels by glass-glass and glass-silicon bonding. Lab on a Chip 5: 837-844 Moy, V. T., Florin, E. L., and Gaub, H. E. (1994) Intermolecular forces and energies between ligands and receptors. Science 266 (5138): 257-159 Nazarov, R., Jin, H. J., and Kaplan, D. L. (2004) Porous 3-D scaffolds from regenerated silk fibroin. Biomacromolecules (3): 718-726 Smith, S. B., Cui, Y., and Bustamante, C. (1996) Overstretching B-DNA: The elastic response of individual double-stranded and single-stranded DNA molecules. Science 271 (5250): 795-799 Trans, H., Pankov, R., Tran, S. D., Hampton, B., Burgess, W. H., and Yamada, K. M. (2002) Integrin clustering induces kinectin accumulation. Journal of Cell Science 115: 2031-2040 Wang, C. H. and Shah, D. O. 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Seminars in Cancer Biology 15: 413-420 VIII [...]... adaptor proteins talin, α-actinin and vinculin Red lines indicate the direct binding of talin and α-actinin with integrin; green lines indicate the binding of talin, α-actinin and vinculin with actin filaments; and blue lines indicate the binding of vinculin with talin and α-actinin The indirect interacts provide connection between actin filaments and ECM, and hence the forces generated from actin filament... ligands and integrins leads to the clustering of integrin heteodimers and the establishment of physical connection of integrins to actin cytoskeleton via the recruitment of interacting cytoskeletal proteins including talin, vinculin and α-actinin (Ziegler et al., 2008) Integrin β subunit binds to talin and α-actinin that interact with vinculin All these three proteins can bind to actin cytoskeleton... and β subunits vinculin actin Intra- cellular α-actinin talin FAK α- paxillin a cti actinin vinculin PLC, Rac1, RhoA… Figure 3 Integrin-fibronectin interaction induced integrin clustering and assembly of focal complexes (A) The interation of integrins on cell membrane with RGD domains on fibronectins in ECM triggers integrin clustering and the assembly of intra- cellular proteins to form focal complex... described in eukaryotic cells, but research has also found prokaryotic cytoskeleton (Carballido-Lopez and Errington, 2003; Shih and Rothfield, 2006) The cytoskeleton network provides intracellular scaffolding on which motor proteins such as kinesin, myosin and dynein can translocate to move organelles or generate internal force (Janmey, 1998) Extracellular stimuli can induce cytoskeleton rearrangement and intra- cellular. .. transform into focal adhesions containing integrin β3 or β1, vinculin and paxillin at cell periphery In contrast, at more central positions, fibrillar adhesions are found in association with fibronectin fibrils, containing integrin β1 and tensin Focal complexes are early adhesion, some of which will recruit various structural and signaling proteins, and develop into focal adhesions The recruiting process... fibronectin, laminin and collagen consists of three amino acids: arginine-glycine-aspartic acid and is the recognition domain for integrins (Engel et al., 1981) Once the integrins on cell membrane interacts with fibronectins in an extra -cellular environment, integrin clutering occurs with the immobilization of fibronectins (Fig 3A) The interaction of cells with ECM also involves the spreading and extension... motor protein kinesin interacts with its receptor kinectin on ER and is important for ER tubule dynamics in the cellular lamella The microtubule and ER connection can be disrupted via the over-expression of the minimal kinectin-kinesin interaction domain vd4 on kinectin The resulted ER extension and microtubule structure in the leading lamella is examined using confocal microscopy • Cells with ER and. .. dynamics and distribution may play important roles in regulating cell behaviors in an external environment Figure 1 Illustration of the series of intra- cellular events triggered upon cell- substrate surface interactions The initial interaction occurs in sub-second to second timescale; early cell responses take seconds to minutes, involving cytoskeleton rearrangement, reinforcement of the linkages between cell- substrate... cytoskeleton rearrangement (Alt-Holland et al., 2008; Jaasma et al., 2007; Stella et al., 2008) and intra- cellular organelle dynamics and distribution (Nakanishi et al., 2007) The collected data and information of intracellular changes in response to seeding cells in extra -cellular environments with chemical and mechanical signals (1) can be used as the guidelines in scaffold development (Koegler and. .. complexes localized in the newly adhered cell membrane via integrin-fibronectin interaction, suggesting the possible ER’s dynamics, distribution and function which is described and explained in this project 2.1 Interaction of cells with ECM - cell adhesion 2.1.1 Integrin induced focal complex assembly The first step of cell- ECM interaction is the interaction of ECM ligands in an extra -cellular environment . i INTRA-CELLULAR ENDOPLASMIC RETICULUM DYNAMICS, DISTRIBUTION AND FUNCTION IN RESPONSE TO CELL-ENGINEERED SURFACE ADHESION ZHANG XIN (B.Sci (Hons.), NUS) . binding protein EB1 (the moving of this protein indiates the polymerizing of microtubule) using confocal microscopy. • Microtubule motor protein kinesin interacts with its receptor kinectin. cell- substrate surface interactions. Figure 2 Two strategies applied to study ER’s response in cell adhesion. Figure 3 Integrin-fibronectin interaction induced integrin clustering and assembly