Versatile microsphere attachment of GFP-labeled motors and other tagged proteins with preserved functionality
Main Article Content
Keywords
force spectroscopy, microsphere coupling, motor proteins, optical tweezers, PEGylation
Abstract
Microspheres are often used as handles for protein purification or force spectroscopy. For example, optical tweezers apply forces on trapped particles to which motor proteins are attached. However, even though many attachment strategies exist, procedures are often limited to a particular biomolecule and prone to non-specific protein or surface attachment. Such interactions may lead to loss of protein functionality or microsphere clustering. Here, we describe a versatile coupling procedure for GFP-tagged proteins via a polyethylene glycol linker preserving the functionality of the coupled proteins. The procedure combines well-established protocols, is highly reproducible, reliable, and can be used for a large variety of proteins. The coupling is efficient and can be tuned to the desired microsphere-to-protein ratio. Moreover, microspheres hardly cluster or adhere to surfaces. Furthermore, the procedure can be adapted to different tags providing flexibility and a promising attachment strategy for any tagged protein.
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References
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6. Oguchi Y, Uchimura S, Ohki T, Mikhailenko SV, Ishiwata S (2011) The bidirectional depolymerizer MCAK generates force by disassembling both microtubule ends. Nat Cell Biol 13: 846-852.
7. Spudich JA, Rice SE, Rock SR, Purcell TJ, Warrick HM (2011) Attachment of Anti-GFP Antibodies to Microspheres for Optical Trapping Experiments. Cold Spring Harb Protoc 11: 1370-1.
8. Guydosh NR, Block SM (2009) Direct observation of the binding state of the kinesin head to the microtubule. Nature 461: 125-128.
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PEGylation of microspheres for therapeutic embolization: Preparation, characterization and biological performance evaluation. Biomaterials 28(6):1198-208.
14. Byeon HJ, Kim I, Choi JS, Lee ES, Shin BS, Youn YS (2015) PEGylated apoptotic protein-loaded PLGA microspheres for cancer therapy. Int J Nanomedicine 10: 739-748.
15. Caldwell KD (1997) Surface modifications with adsorbed poly(ethylene oxide)-based block copolymers. Physical characteristics and biological use. ACS Symposium Series 680: 400-419.
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28. Tolic-Nørrelykke SF, Schäffer E, Howard J, Pavone FS, Jülicher F, Flyvbjerg H (2006) Calibration of optical tweezers with positional detection in the back focal plane. Rev Sci Instrum 77: 103101.
29. Varga V, Helenius J, Tanaka K, Hyman AA, Tanaka TU, Howard J (2006) Yeast kinesin-8 depolymerizes microtubules in a length-dependent manner. Nat Cell Biol 8: 957-962.
30. Trushko A, Schäffer E, Howard J (2013) The growth of microtubules with XMAP215-coated beads coupled to their ends is increased by tensile force. Proc Natl Acad Sci USA 110: 14670-14675.
31. Svoboda K, Block SM (1994) Force and velocity measured for single kinesin molecules. Cell 77: 773-784.
32. Mayr MI, Storch M, Howard J, Mayer TU (2011) A Non-Motor Microtubule Binding Site Is Essential for the High Processivity and Mitotic Function of Kinesin-8 Kif18A. PLOS ONE 6(11): e27471.
33. Gittes F, Schmidt CF (1998) Signals and noise in micromechanical measurements. Methods Cell Biol 55: 129-56.
34. Bugiel M, Böhl E, Schäffer E (2015) The kinesin-8, Kip3, switches microtubule protofilaments in a sideward random walk asymmetrically biased by force. Biophys J 108: 2019-2027.
35. Bohren CF, Huffmann DR (1983) Absorption and scattering of light by small particles. Wiley-Interscience. pp. 317-318.
36. Bormuth V, Varga V, Howard J, Schäffer E (2009) Protein Friction Limits Diffusive and Directed movements of Kinesin Motors on Microtubules. Science 325: 870-873.
37. Svoboda K, Schmidt CF, Schnapp BJ, Block SM (1993) Direct observation of kinesin stepping by optical trapping interferometry. Nature 365: 721-727.
38. Hermsdorf G (2013) Single-molecule fluorescence combined with optical tweezers to study membrane mechanics. Diploma thesis, Technische Universität Dresden.
39. Fantana HA (2011) Mechanics of spindle centering in Caenorhabditis elegans. PhD thesis, Technische Universität Dresden.
2. Block SM, Goldstein LS, Schnapp BJ (1990) Bead movement by single kinesin molecules studied with optical tweezers. Nature: 348:348-352.
3. Kerssemakers, JWJ, Howard J, Hess H, Diez S (2006) The distance that kinesin-1 holds its cargo from the microtubule surface measured by fluorescence interference contrast microscopy. Proc Natl Acad Sci USA 103: 15812-15817.
4. Asbury CL, Fehr AN, Block SM (2003) Kinesin moves by an asymmetric hand-over-hand mechanism. Science 302: 2130-2134.
5. Brunnbauer M, Dombi R, Ho TH, Schliwa M, Rief M, Ökten Z (2012) Torque Generation of Kinesin Motors Is Governed by the Stability of the Neck Domain. Mol Cell 46: 147-158.
6. Oguchi Y, Uchimura S, Ohki T, Mikhailenko SV, Ishiwata S (2011) The bidirectional depolymerizer MCAK generates force by disassembling both microtubule ends. Nat Cell Biol 13: 846-852.
7. Spudich JA, Rice SE, Rock SR, Purcell TJ, Warrick HM (2011) Attachment of Anti-GFP Antibodies to Microspheres for Optical Trapping Experiments. Cold Spring Harb Protoc 11: 1370-1.
8. Guydosh NR, Block SM (2009) Direct observation of the binding state of the kinesin head to the microtubule. Nature 461: 125-128.
9. Jamison D, Driver J, Rogers A, Constantinou P, Diehl M (2010) Two kinesins transport cargo primarily via the action of one motor: implications for intracellular transport. Biophys J. 99: 2967-2977.
10. Bormuth V, Zörgibel F, Schäffer E, Howard J (2011) Functional Surface Attachment in a Sandwich Geometry of GFP-Labeled Motor Proteins. In: Mashanov GI, Batters C, editors. Single Molecule Enzymology. Vol. 778 of Methods in Molecular Biology. Humana Press. pp. 11-18.
11. Gbadamosi JK, Hunter AC, Moghimi SM (2002) PEGylation of microspheres generates a heterogeneous population of particles with differential surface characteristics and biological performance. FEBS letters 532(3): 338-344.
12. Sinha V, Aggarwal A, Trehan A (2004) Biodegradable PEGylated microspheres and nanospheres. Am j drug deliv 2(3): 157-171.
13. Madani F, Bessodes M, Lakrouf A, Vauthier C, Scherman D, Chaumeil J (2007)
PEGylation of microspheres for therapeutic embolization: Preparation, characterization and biological performance evaluation. Biomaterials 28(6):1198-208.
14. Byeon HJ, Kim I, Choi JS, Lee ES, Shin BS, Youn YS (2015) PEGylated apoptotic protein-loaded PLGA microspheres for cancer therapy. Int J Nanomedicine 10: 739-748.
15. Caldwell KD (1997) Surface modifications with adsorbed poly(ethylene oxide)-based block copolymers. Physical characteristics and biological use. ACS Symposium Series 680: 400-419.
16. Valentine MT, Perlman ZE, Gardel ML, Shin JH, Matsudaira P, Mitchison TJ (2004) Colloid Surface Chemistry Critically Affects Multiple Particle Tracking Measurements of Biomaterials. Biophys J 86: 4004-4014.
17. Upadhyayula S, Quinata T, Bishop S, Gupta S, Johnson NR, Bahmani B (2012) Coatings of Polyethylene Glycol for Suppressing Adhesion between Solid Microspheres and Flat Surfaces. Langmuir 28(11): 5059-5069.
18. Lu HB, Campbell CT, Castner DG (2000) Attachment of Functionalized Poly(ethylene glycol) Films to Gold Surfaces. Langmuir 16(4): 1711-1718.
19. Prime K, Whitesides G (1997) Self-assembled organic monolayers: model systems for studying adsorption of proteins at surfaces. Science 252: 1164-1167.
20. Kanaras AG, Kamounah FS, Schaumburg K, Kiely CJ, Brust M (2002) Thioalkylated tetraethylene glycol: a new ligand for water soluble monolayer protected gold clusters. Chem Commun 20: 2294-2295.
21. Uyeda HT, Medintz IL, Jaiswal JK, Simon SM, Mattoussi H (2005) Synthesis of Compact Multidentate Ligands to Prepare Stable Hydrophilic Quantum Dot Fluorophores. J Am Chem Soc 127(11): 3870-3878.
22. Müller M, Vörös J, Csúcs G, Walter E, Danuser G, Merkle HP (2003) Surface modification of PLGA microspheres. J Biomed Mater Res A 66(1): 55-61.
23. Breed DR, Thibault R, Xie F, Wang Q, Hawker CJ, Pine DJ (2009) Functionalization of Polymer Microspheres Using Click Chemistry. Langmuir 25(8): 4370-4376.
24. Hermanson GT (2008) Bioconjugate techniques. Academic Press. pp. 215-223.
25. Jannasch A, Bormuth V, Storch M, Howard J, Schäffer E (2013) Kinesin-8 Is a Low-Force Motor Protein with a Weakly Bound Slip State. Biophys J 104: 2456-2464.
26. Bormuth V, Howard J, Schäffer E (2007) LED illumination for video-enhanced DIC imaging of single microtubules. J Microsc 226: 1-5.
27. Schäffer E, Nørrelykke S, Howard J (2007) Surface forces and drag coefficients of microspheres near a plane surface measured with optical tweezers. Langmuir 23: 3654-3665.
28. Tolic-Nørrelykke SF, Schäffer E, Howard J, Pavone FS, Jülicher F, Flyvbjerg H (2006) Calibration of optical tweezers with positional detection in the back focal plane. Rev Sci Instrum 77: 103101.
29. Varga V, Helenius J, Tanaka K, Hyman AA, Tanaka TU, Howard J (2006) Yeast kinesin-8 depolymerizes microtubules in a length-dependent manner. Nat Cell Biol 8: 957-962.
30. Trushko A, Schäffer E, Howard J (2013) The growth of microtubules with XMAP215-coated beads coupled to their ends is increased by tensile force. Proc Natl Acad Sci USA 110: 14670-14675.
31. Svoboda K, Block SM (1994) Force and velocity measured for single kinesin molecules. Cell 77: 773-784.
32. Mayr MI, Storch M, Howard J, Mayer TU (2011) A Non-Motor Microtubule Binding Site Is Essential for the High Processivity and Mitotic Function of Kinesin-8 Kif18A. PLOS ONE 6(11): e27471.
33. Gittes F, Schmidt CF (1998) Signals and noise in micromechanical measurements. Methods Cell Biol 55: 129-56.
34. Bugiel M, Böhl E, Schäffer E (2015) The kinesin-8, Kip3, switches microtubule protofilaments in a sideward random walk asymmetrically biased by force. Biophys J 108: 2019-2027.
35. Bohren CF, Huffmann DR (1983) Absorption and scattering of light by small particles. Wiley-Interscience. pp. 317-318.
36. Bormuth V, Varga V, Howard J, Schäffer E (2009) Protein Friction Limits Diffusive and Directed movements of Kinesin Motors on Microtubules. Science 325: 870-873.
37. Svoboda K, Schmidt CF, Schnapp BJ, Block SM (1993) Direct observation of kinesin stepping by optical trapping interferometry. Nature 365: 721-727.
38. Hermsdorf G (2013) Single-molecule fluorescence combined with optical tweezers to study membrane mechanics. Diploma thesis, Technische Universität Dresden.
39. Fantana HA (2011) Mechanics of spindle centering in Caenorhabditis elegans. PhD thesis, Technische Universität Dresden.
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Published
Nov 17, 2015
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Bugiel M, Fantana H, Bormuth V, Trushko A, Schiemann F, Howard J, Schäffer E, Jannasch A. Versatile microsphere attachment of GFP-labeled motors and other tagged proteins with preserved functionality. J Biol Methods [Internet]. 2015 Nov. 17 [cited 2024 Apr. 26];2(4):e30. Available from: https://jbmethods.org/index.php/jbm/article/view/79
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