CHAPTER
Micropatterning Microtubules
3 Didier Portran
Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA 94305, USA
CHAPTER OUTLINE Introduction .............................................................................................................. 40 3.1 Micropatterned Substrate Fabrication...................................................................41 3.1.1 Equipment.......................................................................................41 3.1.2 Materials .........................................................................................41 3.1.3 Methods ..........................................................................................41 3.1.3.1 Coverslip Passivation ................................................................. 41 3.1.3.2 Micropatterning ......................................................................... 42 3.2 Micropattern Functionalization for MTs Polymerization .........................................43 3.2.1 Materials .........................................................................................44 3.2.2 Methods ..........................................................................................45 3.2.2.1 MT Seeds Polymerization........................................................... 45 3.2.2.2 Flow Chamber Assembly............................................................ 45 3.2.2.3 MT Seeds Elongation ................................................................. 45 3.2.2.4 Observation in TIRF Microscopy................................................. 46 3.3 Discussion..........................................................................................................48 3.3.1 Discussion About the Passivation Surface Treatment ...........................48 3.3.2 Discussion About the Different MT Nucleation Templates ....................48 Conclusion ............................................................................................................... 49 Acknowledgment....................................................................................................... 49 References ............................................................................................................... 49
Abstract The following protocol describes a method to control the orientation and polarity of polymerizing microtubules (MTs). Reconstitution of specific geometries of dynamic MT networks is achieved using a ultraviolet (UV) micropatterning technique in combination with stabilized MT microseeds. The process is described in three Methods in Cell Biology, Volume 120 Copyright © 2014 Elsevier Inc. All rights reserved.
ISSN 0091-679X http://dx.doi.org/10.1016/B978-0-12-417136-7.00003-3
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main parts. First, the surface is passivated to avoid the non-specific absorption of proteins, using different polyethylene glycol (PEG)-based surface treatment. Second, specific adhesive surfaces (the micropatterns) are imprinted through a photomask using deep UVs. Lastly, MT microseeds are adhered to the micropatterns followed by MT polymerization.
INTRODUCTION Microtubules (MTs) are major components of the cytoskeleton and are implicated in many biological processes including cell migration, intracellular transport, and cell division (Keating & Borisy, 1999; Mimori-Kiyosue, 2011). MTs are hollow tubes formed by polymerization of tubulin dimers into 13 protofilaments and are polarized with their plus end polymerizing faster than their minus ends (Desai & Mitchison, 1997; Walker et al., 1988). MTs are highly dynamic polymers that undergo spontaneous transition from growth to shrinkage, this behavior is define as MT dynamic instability (Mitchison & Kirschner, 1984). Their nucleation in vivo is generally controlled by a nucleation complex composed of g-tubulin that caps the MT minus end and allows the plus end to dynamically explore the cytoplasm (Kollman, Merdes, Mourey, & Agard, 2011). In cells, MTs are organized into diverse patterns such as aster-like structures, parallel and antiparallel bundled networks. The diversity and reproducibility of these MT organizations are regulated by the control of MT spatial nucleation and by the activity of several microtubule-associated proteins (MAPs) and molecular motors (Dogterom & Surrey, 2013; Mimori-Kiyosue, 2011). The functions of MAPs and molecular motors have been intensively studied but there is a high redundancy in the functions of these proteins due to the critical role of MT organization in cellular processes. Although, in vivo systems have proven valuable to understanding the role of MAPs and motors, in vitro experiments are necessary to firmly assign their functions. For this purpose, many biomimetic systems have been developed using purified components (Bieling, Telley, & Surrey, 2010; Kapitein, Peterman, & Kwok, 2005; Tulin, McClerklin, Huang, & Dixit, 2012). However, effort to reconstitute MT networks in vitro has encountered many technical difficulties such as the nonspecific adsorption of proteins to surfaces and the lack of MT nucleation template such as the g-tubulin complex. Most of in vitro experimental procedures do not allow for the reconstitution of a MT network architecture representative of what is observed in vivo. Micropatterning techniques have revolutionized the study of the cytoskeleton organization as they enable the creation of reproducible cell shapes (Azioune, Carpi, Tseng, Thery, & Piel, 2010; Vignaud, Blanchoin, & The´ry, 2012). Several micropatterning techniques have been adapted for the reconstitution of MT networks in vitro. Examples include microprinting using polydimethylsiloxane (PDMS) stamps to spatially control the coating of biotinylated bovine serum albumin (BSA), or different MT nucleation templates (Dinarina et al., 2009; Ghosh, Hentrich, & Surrey, 2013; Shang et al., 2009) and the alignment of MT stabilized seeds on gold electrodes using electric fields (Uppalapati, Huang, Aravamuthan, Jackson, & Hancock, 2011).
3.1 Micropatterned Substrate Fabrication
Here, we present a novel method based on ultraviolet (UV) micropatterning that combines an efficient protein repellent surface treatment with a novel MT nucleation method from stabilized MT microseeds (Portran, Gaillard, Vantard, & Thery, 2013). We used a double passivation treatment consisting of a covalent attachment of silane-PEG on a glass substrate followed by a PLL-PEG coating to avoid both tubulin and MAPs adsorption (Bieling, Telley, & Surrey, 2010; Ionov, Synytska, Kaul, & Diez, 2010). We next describe the micropatterning design and procedure for glass passivation adapted to dynamic MTs. This method allows the observation of the global MT network formation and single MTs interaction by total internal reflection fluorescence (TIRF) microscopy (Portran, Gaillard, et al., 2013; Su et al., 2013).
3.1 MICROPATTERNED SUBSTRATE FABRICATION The first part of this section describes a glass surface passivation used to reduce tubulin and MAPs adsorption. In the second part, we will describe the micropattern design adapted to MTs and the method to produce micropatterns on the passivated glass surfaces.
3.1.1 Equipment • •
• • •
Software to design the photomask: Clewin (Any layout editor software can be used since they support .GDS, .CIF, or .DWF format). Photomasks can be purchased by photomask producers (Toppan). Photomasks must be transparent to wavelengths below 200 nm, in fused silica or synthetic quartz. Designing features with a limit size of 1–10 mm. Plasma cleaner (optional). UV ozone cleaner (model 42 series, jelight). Vacuum mask holder: A design was obtained from Azioune et al. (2010).
3.1.2 Materials • • • • • •
Glass staining jar (Dutscher) 100% Acetone Ethanol 96% Deionized and filtered water Hellmanex III (Hellma) 2% mPEG-Silane, 30 kDa (PSB-2014, from CreativePegWork)
3.1.3 Methods 3.1.3.1 Coverslip passivation Cleaning of coverslips and glasses in a glass-staining jar: 1. 30 min in 100% acetone. 2. 15 min in 96% ethanol. 3. Rinse three times in deionized and filtered water.
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4. 5. 6. 7.
2 h in 2% hellmanex III solution. Rinse abundantly with deionized and filtered water. Dry with pressurized filtered air. Stock in a sealed container away from dust.
Note: These coverslips and glasses can be kept for a few days but it is better to use them fresh. Silane-PEG coating: 1. Make a 1 mg/ml silane-PEG solution in 96% ethanol with 0.1% (v/v) HCl. Warm up to 50 C and stir the solution to solubilize the silane-PEG. Note: Silane-PEG can also be dissolved in acetone complemented by 5% H2O. 2. Incubate the cleaned glass surface in the Silane-PEG solution at room temperature (RT) for 18 h with gentle agitation. Optional: Prior to incubation in the silane-PEG solution, the glass surface can be activated by 2 min exposure to plasma in a plasma cleaner at 70 mW (Azioune, Storch, Bornens, The´ry, & Piel, 2009) or by piranha solution (Bieling, Telley, Hentrich, Piehler, & Surrey, 2010). 3. 4. 5. 6.
Wash the coverslips and glasses with 96% ethanol three times. Wash with deionized and filtered water three times. Dry with pressurized filtered air. Stock in a hermetic container protected from light and dust.
Note: The silane-PEG coated coverslips can be kept several months at 4 C but it is optimal to use them within the following weeks.
3.1.3.2 Micropatterning This section describes how to design and create the micropatterns on the passivated glass surface. Micropatterns design: Common micropattern shapes include the bar and the disk. Using these two basic shapes, MT asters and parallel/antiparallel MT organization can be reconstituted. Disk micropatterns allow radial nucleation of the MTs, and with bar micropatterns MTs parallel organization can be achieved. In order to reconstitute an anti-parallel MT network, two micropatterns (bars or disks) should be separated by 10–30 mm to allow MT interaction (Fig. 3.1). To avoid MTs overlap from two different micropatterns, micropatterns should be separate by at least 100 mm (Fig. 3.1). Micropattern thickness for MT microseeds (which length is 3 mm) on micropatterns bars, in this case micropatterns should be thick enough to allow the adsorption of MT seeds. The thickness of the micropatterns should be equal or superior to the MT seeds length. Micropattern surfaces are thus imprinted on the passivated glass surface using deep UV through the photomask as described below (Fig. 3.2):
3.2 Micropattern Functionalization for MTs Polymerization
FIGURE 3.1 Designing micropatterns. This scheme illustrates the basic geometrical dimensions that must be satisfied to ensure proper MT nucleation from the micropatterns (line width and micropattern size) and the minimum separation distance between two types of micropatterns (array step).
1. Wash the photomask with water and 100% isopropanol to remove any residue and dry it with pressurized filtered air. 2. Place the coverslips on the chrome face of the photomask. 3. Place on the vacuum holder and open vacuum to stick the coverslips to the mask. Note: The vacuum system allows a good contact between the photomask and the coverslip reducing the diffraction of the deep UVs. 4. Expose to deep UVs for 30–60 s. 5. Remove the coverslips using vacuum suction. Note: The micropatterned coverslips can be stored up to several weeks but proteins adherence is optimal within hours of micropatterning.
3.2 MICROPATTERN FUNCTIONALIZATION FOR MTs POLYMERIZATION This section describes the procedure to functionalize the micropatterns with neutravidin to further attached biotinylated MT microseeds and to observe MT elongation.
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FIGURE 3.2 Micropattern fabrication. This scheme summarizes the sequential steps for micropattern fabrication for MTs. Step 1: Clean glass coverslips (see washing procedure above). Step 2: Incubate the glass coverslips in a silane-PEG solution for 16 h. Step 3: Place the coverslip and the photomask on the mask holder. Place the sandwich under deep UV (180 nm) for 30–60 s to oxidize the silane-peg under transparent areas.
3.2.1 Materials • • • •
Micropatterned coverslips. mPEG-Silane, 30 kDa, passivated glasses. PLL20K-G35-PEG2K (Jenkem technology) solution at 0.1 mg/ml in 10 mM HEPES buffer, pH 7.4, stored at 4 C up to several months. Double-sided tape 70 mm thick (Lima).
Purified brain tubulin: • •
• • •
Tubulin is purified from cow brains according to Vantard, Peter, Fellous, Schellenbaum, and Lambert (1994). Purified tubulin is labeled using a standard procedure from Hyman et al. (1991) with either ATTO-565 (ATTO-TEC, AD 565-31), ATTO-488 (ATTO-TEC, AD 488-31), or with NHS-LC-LC-biotin (Pierce, EZ-Link NHS-LC-LC-Biotin). BRB80 (Brinkley Reassembly Buffer): 80 mM PIPES, 1 mM MgCl2, 1 mM EGTA, pH 6.9. Neutravidin (Thermo Scientific, 31000). GMPCPP 10 mM stock solution (JenaBioscience, NU-405).
3.2 Micropattern Functionalization for MTs Polymerization
• • • • • • • •
Taxol 10 mM stock in anhydrous dimethyl sulfoxide (DMSO) (Paclitaxel, Sigma, T-7191). GTP lithium salt (Sigma, G5884). BSA 10% (Sigma, A-7030) Catalase (Sigma, C-40). Glucose (Sigma, G8270). Glucose oxidase (Sigma, G-2133). Methyl Cellulose (Sigma, 1500 CP, M0555). Dithiothreitol (DTT) (Sigma, D9779).
3.2.2 Methods 3.2.2.1 MT seeds polymerization In order to obtain stabilized MT seeds, free tubulin is polymerized with Taxol and GMPCPP (a slowly hydrolysable GTP analog). These stabilizing agents enhance MT nucleation and stabilization, allowing the assembly of short stable MTs. 1. Mix 85% of biotin-labeled tubulin with 15% of fluorescently labeled tubulin in BRB80 at a final concentration of 50 mM. 2. Add 1 mM of GMPCPP and 20 mM of taxol. 3. Incubate for 5 min at 37 C. 4. Centrifuge on a tabletop centrifuge at 15,000 rpm for 5 min. 5. Discard supernatant and resuspend the MT microseeds in BRB80 with 1 mM GMPCPP and 20 mM taxol. Critical: Do not stock MT microseeds, directly flow the MT microseeds into the perfusion chamber to adhere them on the micropatterned surfaces.
3.2.2.2 Flow chamber assembly 1. Glass slides should be passivated using the same protocol as glass coverslips. 2. Using two bands of double-sided tape (70 mm, from Lima), assemble the perfusion chamber (Fig. 3.3), with the passivated glass slides and the micropatterned coverslips. 3. Flow 50 mg/ml of neutravidin, and incubate for 1 min. Critical: Do not use blocking agents such as BSA or casein before the incubation with neutravidin. 4. Wash the perfusion chamber with 10 volumes BRB80. 5. Flow PLL-g-PEG and incubate 20 s. 6. Wash the perfusion chamber with 20 volumes BRB80. 7. Flow the MT seeds and incubate for 2 min. 8. Wash the perfusion chamber with 20 volumes BRB80 with 0.5% BSA
3.2.2.3 MT seeds elongation Here we describe how to polymerize MTs from micropatterned-attached MT microseeds, by adding free tubulin and GTP (Fig. 3.4).
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FIGURE 3.3 Scheme representing the flow chamber assembly with a passivated coverglass and a micropatterned coverslip using two bands of double-sided tape.
FIGURE 3.4 Step 1: Incubate with neutravidin to coat the micropattern surface. Step 2: Incubate with PLL-PEG to enhance passivation of the surface. Step 3: Incubate the biotinylated MT seeds to adhere them on the neutravidin micropatterns. Step 4: Start MT elongation by the addition of free tubulin dimers and GTP.
1. Make the elongation Mix: • 10 mM of unlabeled tubulin and 2 mM of fluorescently labeled tubulin in BRB80 þ 1 mM GTP. • Add 0.025% of methyl cellulose at 1500 CP. • Add Oxygen scavenger: 2 mg/ml glucose, 80 mg/ml catalase, and 0.67 mg/ml glucose oxidase, 20 mM DTT. 2. Flow the elongation mix and seal the perfusion chamber with vitrex paste (Vitrex) or valap to avoid evaporation. 3. Observe at 30–37 C in TIRF microscopy.
3.2.2.4 Observation in TIRF microscopy MT dynamics can be visualized using an objective-based azimuthal ilas2 TIRF microscopy (Nikon eclipse Ti, modified by Roper scientific) and Evolve 512 camera (Photometrics) (Fig. 3.5A). Microscope stage was kept at 32 C using a warm stage controller (LINKAM MC60). Excitation was achieved using 491 and 561 nm lasers
3.2 Micropattern Functionalization for MTs Polymerization
FIGURE 3.5 (A) TIRF microscopy imaging of polymerizing MTs (in green) from MT microseeds (in red) from a bar micropattern (left panel) and from a disk micropattern (right panel). Scale bar ¼ 10 mm. (B and C) Schemes representing the different MT network types that can be achieved using micropatterns disks or bars. (B) Scheme representing the polarity and orientation of MTs. (C) Scheme representing the anti-parallel network organization using two micropatterns close to each other.
(Optical Insights). Time-lapse recording (one frame every 5 s) was performed for 30 min using Metamorph® software (version 7.7.5, Universal Imaging). Due to steric congestion, MT polymerization is polarized with the MT plus ends growing out of the micropatterns (Fig. 3.5A). MT polarity can be determined by TIRF microscopy by measuring MT polymerization speed (Portran, Gaillard, et al., 2013) as MTs plus ends polymerize faster than their minus ends (Walker et al., 1988). Using micropatterns disks or bars allows to obtain aster-like MT structures and parallel MT networks (Fig. 3.5B). Using two micropatterns close to each other create an antiparallel interaction between the polarized MTs (Fig. 3.5C).
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3.3 DISCUSSION Here, we have presented a method to spatially control MT nucleation and polarity on micropatterns on a PEG-treated surface. The passivated surface treatment described has been developed to study MAP interactions with dynamic MTs and can be further used for other types of experiments. Indeed, to study MT dynamic instability or MAP localization along the MT length, silane-PEG-Biotin can be used instead of the silane-PEG to attach biotinylated stabilized MT seeds on the surface (Portran, Zoccoler, et al., 2013; Stoppin-Mellet, Fache, Portran, Martiel, & Vantard, 2013). Furthermore, this micropatterning technique can be combined with a fluidic system to bend MTs in order to measure their rigidity (Portran, Zoccoler, et al., 2013).
3.3.1 Discussion about the passivation surface treatment The main challenge in microscopy is to avoid the direct interaction of the MTs with the surface, because it may modify their properties such as their dynamic instability. Therefore, multiple surface treatments have been developed to study MT dynamic instability and organization in presence of MAPs and molecular motors (Bieling, Telley, Hentrich, et al., 2010; Gell et al., 2010). The use of PEG-based surface treatment offers great advantages over non-covalent blocking methods, such as BSA (Vale et al., 1996) or casein (Howard, Hunt, & Baek, 1993), which may not achieve sufficient passivation. An efficient protein-repellent surface is essential to conserve the protein concentrations and avoid MT interactions with non-specific MAPs attached to the surface. Indeed, the PLL-PEG coating on a glass substrate exhibited excellent surface passivation, and has proven useful for single molecule tracking (Bieling, Telley, & Surrey, 2010; Gell et al., 2010). However, if PLL-PEG is efficient to prevent MAPs non-specific attachment on the surface, it is less efficient than a silane-PEG coating with regards to prevention of non-specific adsorption of tubulin on the surface (Ionov et al., 2010). The surface treatment described here has good repellent properties for tubulin and MAPs. Moreover, it provides a good stability and a high reproducibility since treated glass substrates may be stored for long periods.
3.3.2 Discussion about the different MT nucleation templates Many methods for micropatterning MTs have been published in the past few years (Ghosh et al., 2013; Shang et al., 2009; Uppalapati et al., 2011). Unfortunately, no universal solution exists as each method has drawbacks and advantages. The method described here is convenient as it is compatible with a surface treatment with excellent protein-repellent properties, in contrast to microprinting techniques with PDMS stamps (Dinarina et al., 2009; Ghosh et al., 2013; Shang et al., 2009). Moreover, the UV micropatterning may allow the attachment of any kind of MT nucleation template. Among the existing micropatterning techniques, the most physiological MT nucleation templates used would be the centrosomes or fragments of centrosomes
References
that can be purified from cell culture (Evans, Mitchison, & Kirschner, 1985; Shang et al., 2009). In contrast, several groups have initiated MT nucleation with plus end proteins that have been shown to enhance MT polymerization such as, XMAP215 (Ghosh et al., 2013; Popov, Severin, & Karsenti, 2002) or the MT binding domain of CLIP170 (H2) (Arnal, Heichette, Diamantopoulos, & Chretien, 2004). Using MT-stabilized seeds is the most common way to nucleate MTs, as they can be easily produced by polymerizing purified tubulin with stabilizing agents such as taxol and GMPCPP (Bieling, Telley, & Surrey, 2010; Gell et al., 2010; Tulin et al., 2012). Moreover, stabilized MT seeds have the advantage that they do not modify MT dynamic in contrast to plus end proteins (Ghosh et al., 2013; Popov et al., 2002).
CONCLUSION The micropatterning method described here aims to reconstitute MT structures with controlled nucleation and polarity. We have optimized the glass surface treatment to strongly reduce protein non-specific adsorption in order to reconstitute dynamic MT networks in presence of MAPs. Moreover, the use of MT-stabilized microseeds allows dense and polarized MT nucleation without affecting the parameter of MT dynamic instability. This micropatterning technique offers a great tool to describe the minimal requirements to reconstitute MT architecture (Portran, Gaillard, et al., 2013; Su et al., 2013) as observed in vivo. In the future, adding complexity to this system and using the previously developed actin filament micropatterning based on a similar methodology (Reymann et al., 2010) should allow the study of the crosstalk between actin filament and MTs. The development of these novel tools to manipulate actin and MT filaments promises a better understanding of the basic mechanisms involved in the formation and maintenance of the cytoskeleton architecture.
Acknowledgment I am grateful for support from Dr. Marylin Vantard and Dr. Manuel The´ry.
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Bieling, P., Telley, I. A., Hentrich, C., Piehler, J., & Surrey, T. (2010). Fluorescence microscopy assays on chemically functionalized surfaces for quantitative imaging of microtubule, motor, and þTIP dynamics. Methods in Cell Biology, 95, 555–580. Bieling, P., Telley, I. A., & Surrey, T. (2010). A minimal midzone protein module controls formation and length of antiparallel microtubule overlaps. Cell, 142, 420–432. Desai, A., & Mitchison, T. J. (1997). Microtubule polymerization dynamics. Annual Review of Cell and Developmental Biology, 13, 83–117. Dinarina, A., Pugieux, C., Corral, M. M., Loose, M., Spatz, J., Karsenti, E., et al. (2009). Chromatin shapes the mitotic spindle. Cell, 138, 502–513. Dogterom, M., & Surrey, T. (2013). Microtubule organization in vitro. Current Opinion in Cell Biology, 25(1), 23–29. Evans, L., Mitchison, T., & Kirschner, M. (1985). Influence of the centrosome on the structure of nucleated microtubules. Journal of Cell Biology, 100(4), 1185–1191. Gell, C., Bormuth, V., Brouhard, G. J., Cohen, D. N., Diez, S., Friel, C. T., et al. (2010). Microtubule dynamics reconstituted in vitro and imaged by single-molecule fluorescence microscopy. Methods in Cell Biology, 95, 221–245. Ghosh, S., Hentrich, C., & Surrey, T. (2013). Micropattern-controlled local microtubule nucleation, transport, and mesoscale organization. ACS Chemical Biology, 8(4), 673–678. Howard, J., Hunt, A. J., & Baek, S. (1993). Assay of microtubule movement driven by single kinesin molecules. Methods in Cell Biology, 39, 137–147. Hyman, A., Drechsel, D., Kellogg, D., Salser, S., Sawin, K., Steffen, P., Wordeman, L., & Mitchison, T. (1991). Preparation of modified tubulins. Methods in Enzymology, 196, 478–485. Ionov, L., Synytska, A., Kaul, E., & Diez, S. (2010). Protein-resistant polymer coatings based on surface-adsorbed poly(aminoethyl methacrylate)/poly(ethylene glycol) copolymers. Biomacromolecules, 11(1), 233–237. Kapitein, L. C., Peterman, E. J. G., & Kwok, B. H. (2005). The bipolar mitotic kinesin Eg5 moves on both microtubules that it crosslinks. Nature, 435, 114–118. Keating, T. J., & Borisy, G. G. (1999). Centrosomal and non-centrosomal microtubules. Biology of the Cell, 91, 321–329. Kollman, J. M., Merdes, A., Mourey, L., & Agard, D. A. (2011). Microtubule nucleation by g-tubulin complexes. Nature Reviews Molecular Cell Biology, 12(11), 709–721. Mimori-Kiyosue, Y. (2011). Shaping microtubules into diverse patterns: Molecular connections for setting up both ends. Cytoskeleton, 68, 603–618. Mitchison, T., & Kirschner, M. (1984). Dynamic instability of microtubule growth. Nature, 312, 237–242. Popov, A. V., Severin, F., & Karsenti, E. (2002). XMAP215 is required for the microtubulenucleating activity of centrosomes. Current Biology, 12(15), 1326–1330. Portran, D., Gaillard, J., Vantard, M., & Thery, M. (2013). Quantification of MAP and molecular motor activities on geometrically controlled microtubule networks. Cytoskeleton, 70(1), 12–23. Portran, D., Zoccoler, M., Gaillard, J., Stoppin-Mellet, V., Neumann, E., Arnal, I., et al. (2013). MAP65/Ase1 promote microtubule flexibility. Molecular Biology of the Cell, 24(12), 1964–1973. Reymann, A.-C., Martiel, J.-L., Cambier, T., Blanchoin, L., Boujemaa-Paterski, R., & Thery, M. (2010). Nucleation geometry governs ordered actin networks structures. Nature Materials, 9, 827–832.
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Shang, W., Crone, D. E., Yang, H., Dordick, J. S., Palazzo, R. E., & Siegel, R. W. (2009). Using centrosome fragments in the directed assembly of microtubules. Journal of Nanoscience and Nanotechnology, 9, 871–875. Stoppin-Mellet, V., Fache, V., Portran, D., Martiel, J. L., & Vantard, M. (2013). MAP65 coordinate microtubule growth during bundle formation. PLoS One, 8(2), e56808. Su, X., Arellano-Santoyo, H., Portran, D., Gaillard, J., Vantard, M., Thery, M., et al. (2013). Microtubule-sliding activity of a kinesin-8 promotes spindle assembly and spindle-length control. Nature Cell Biology, 15(8), 948–957. Tulin, A., McClerklin, S., Huang, Y., & Dixit, R. (2012). Single-molecule analysis of the microtubule cross-linking protein MAP65-1 reveals a molecular mechanism for contactangle-dependent microtubule bundling. Biophysical Journal, 102(4), 802–809. Uppalapati, M., Huang, Y. M., Aravamuthan, V., Jackson, T. N., & Hancock, W. O. (2011). “Artificial mitotic spindle” generated by dielectrophoresis and protein micropatterning supports bidirectional transport of kinesin-coated beads. Integrative Biology, 3(1), 57–64. Vale, R. D., Funatsu, T., Pierce, D. W., Romberg, L., Harada, Y., & Yanagida, T. (1996). Direct observation of single kinesin molecules moving along microtubules. Nature, 380, 451–453. Vantard, M., Peter, C., Fellous, A., Schellenbaum, P., & Lambert, A. M. (1994). Characterization of a 100-kDa heat-stable microtubule-associated protein from higher plants. European Journal of Biochemistry, 220, 847–853. Vignaud, T., Blanchoin, L., & The´ry, M. (2012). Directed cytoskeleton self-organization. Trends in Cell Biology, 22(12), 671–682. Walker, R. A., O’Brien, E. T., Pryer, N. K., Soboeiro, M. F., Voter, W. A., Erickson, H. P., et al. (1988). Dynamic instability of individual microtubules analyzed by video light microscopy: Rate constants and transition frequencies. Journal of Cell Biology, 107, 1437.
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3 Didier Portran
Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA 94305, USA
CHAPTER OUTLINE Introduction .............................................................................................................. 40 3.1 Micropatterned Substrate Fabrication...................................................................41 3.1.1 Equipment.......................................................................................41 3.1.2 Materials .........................................................................................41 3.1.3 Methods ..........................................................................................41 3.1.3.1 Coverslip Passivation ................................................................. 41 3.1.3.2 Micropatterning ......................................................................... 42 3.2 Micropattern Functionalization for MTs Polymerization .........................................43 3.2.1 Materials .........................................................................................44 3.2.2 Methods ..........................................................................................45 3.2.2.1 MT Seeds Polymerization........................................................... 45 3.2.2.2 Flow Chamber Assembly............................................................ 45 3.2.2.3 MT Seeds Elongation ................................................................. 45 3.2.2.4 Observation in TIRF Microscopy................................................. 46 3.3 Discussion..........................................................................................................48 3.3.1 Discussion About the Passivation Surface Treatment ...........................48 3.3.2 Discussion About the Different MT Nucleation Templates ....................48 Conclusion ............................................................................................................... 49 Acknowledgment....................................................................................................... 49 References ............................................................................................................... 49
Abstract The following protocol describes a method to control the orientation and polarity of polymerizing microtubules (MTs). Reconstitution of specific geometries of dynamic MT networks is achieved using a ultraviolet (UV) micropatterning technique in combination with stabilized MT microseeds. The process is described in three Methods in Cell Biology, Volume 120 Copyright © 2014 Elsevier Inc. All rights reserved.
ISSN 0091-679X http://dx.doi.org/10.1016/B978-0-12-417136-7.00003-3
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main parts. First, the surface is passivated to avoid the non-specific absorption of proteins, using different polyethylene glycol (PEG)-based surface treatment. Second, specific adhesive surfaces (the micropatterns) are imprinted through a photomask using deep UVs. Lastly, MT microseeds are adhered to the micropatterns followed by MT polymerization.
INTRODUCTION Microtubules (MTs) are major components of the cytoskeleton and are implicated in many biological processes including cell migration, intracellular transport, and cell division (Keating & Borisy, 1999; Mimori-Kiyosue, 2011). MTs are hollow tubes formed by polymerization of tubulin dimers into 13 protofilaments and are polarized with their plus end polymerizing faster than their minus ends (Desai & Mitchison, 1997; Walker et al., 1988). MTs are highly dynamic polymers that undergo spontaneous transition from growth to shrinkage, this behavior is define as MT dynamic instability (Mitchison & Kirschner, 1984). Their nucleation in vivo is generally controlled by a nucleation complex composed of g-tubulin that caps the MT minus end and allows the plus end to dynamically explore the cytoplasm (Kollman, Merdes, Mourey, & Agard, 2011). In cells, MTs are organized into diverse patterns such as aster-like structures, parallel and antiparallel bundled networks. The diversity and reproducibility of these MT organizations are regulated by the control of MT spatial nucleation and by the activity of several microtubule-associated proteins (MAPs) and molecular motors (Dogterom & Surrey, 2013; Mimori-Kiyosue, 2011). The functions of MAPs and molecular motors have been intensively studied but there is a high redundancy in the functions of these proteins due to the critical role of MT organization in cellular processes. Although, in vivo systems have proven valuable to understanding the role of MAPs and motors, in vitro experiments are necessary to firmly assign their functions. For this purpose, many biomimetic systems have been developed using purified components (Bieling, Telley, & Surrey, 2010; Kapitein, Peterman, & Kwok, 2005; Tulin, McClerklin, Huang, & Dixit, 2012). However, effort to reconstitute MT networks in vitro has encountered many technical difficulties such as the nonspecific adsorption of proteins to surfaces and the lack of MT nucleation template such as the g-tubulin complex. Most of in vitro experimental procedures do not allow for the reconstitution of a MT network architecture representative of what is observed in vivo. Micropatterning techniques have revolutionized the study of the cytoskeleton organization as they enable the creation of reproducible cell shapes (Azioune, Carpi, Tseng, Thery, & Piel, 2010; Vignaud, Blanchoin, & The´ry, 2012). Several micropatterning techniques have been adapted for the reconstitution of MT networks in vitro. Examples include microprinting using polydimethylsiloxane (PDMS) stamps to spatially control the coating of biotinylated bovine serum albumin (BSA), or different MT nucleation templates (Dinarina et al., 2009; Ghosh, Hentrich, & Surrey, 2013; Shang et al., 2009) and the alignment of MT stabilized seeds on gold electrodes using electric fields (Uppalapati, Huang, Aravamuthan, Jackson, & Hancock, 2011).
3.1 Micropatterned Substrate Fabrication
Here, we present a novel method based on ultraviolet (UV) micropatterning that combines an efficient protein repellent surface treatment with a novel MT nucleation method from stabilized MT microseeds (Portran, Gaillard, Vantard, & Thery, 2013). We used a double passivation treatment consisting of a covalent attachment of silane-PEG on a glass substrate followed by a PLL-PEG coating to avoid both tubulin and MAPs adsorption (Bieling, Telley, & Surrey, 2010; Ionov, Synytska, Kaul, & Diez, 2010). We next describe the micropatterning design and procedure for glass passivation adapted to dynamic MTs. This method allows the observation of the global MT network formation and single MTs interaction by total internal reflection fluorescence (TIRF) microscopy (Portran, Gaillard, et al., 2013; Su et al., 2013).
3.1 MICROPATTERNED SUBSTRATE FABRICATION The first part of this section describes a glass surface passivation used to reduce tubulin and MAPs adsorption. In the second part, we will describe the micropattern design adapted to MTs and the method to produce micropatterns on the passivated glass surfaces.
3.1.1 Equipment • •
• • •
Software to design the photomask: Clewin (Any layout editor software can be used since they support .GDS, .CIF, or .DWF format). Photomasks can be purchased by photomask producers (Toppan). Photomasks must be transparent to wavelengths below 200 nm, in fused silica or synthetic quartz. Designing features with a limit size of 1–10 mm. Plasma cleaner (optional). UV ozone cleaner (model 42 series, jelight). Vacuum mask holder: A design was obtained from Azioune et al. (2010).
3.1.2 Materials • • • • • •
Glass staining jar (Dutscher) 100% Acetone Ethanol 96% Deionized and filtered water Hellmanex III (Hellma) 2% mPEG-Silane, 30 kDa (PSB-2014, from CreativePegWork)
3.1.3 Methods 3.1.3.1 Coverslip passivation Cleaning of coverslips and glasses in a glass-staining jar: 1. 30 min in 100% acetone. 2. 15 min in 96% ethanol. 3. Rinse three times in deionized and filtered water.
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4. 5. 6. 7.
2 h in 2% hellmanex III solution. Rinse abundantly with deionized and filtered water. Dry with pressurized filtered air. Stock in a sealed container away from dust.
Note: These coverslips and glasses can be kept for a few days but it is better to use them fresh. Silane-PEG coating: 1. Make a 1 mg/ml silane-PEG solution in 96% ethanol with 0.1% (v/v) HCl. Warm up to 50 C and stir the solution to solubilize the silane-PEG. Note: Silane-PEG can also be dissolved in acetone complemented by 5% H2O. 2. Incubate the cleaned glass surface in the Silane-PEG solution at room temperature (RT) for 18 h with gentle agitation. Optional: Prior to incubation in the silane-PEG solution, the glass surface can be activated by 2 min exposure to plasma in a plasma cleaner at 70 mW (Azioune, Storch, Bornens, The´ry, & Piel, 2009) or by piranha solution (Bieling, Telley, Hentrich, Piehler, & Surrey, 2010). 3. 4. 5. 6.
Wash the coverslips and glasses with 96% ethanol three times. Wash with deionized and filtered water three times. Dry with pressurized filtered air. Stock in a hermetic container protected from light and dust.
Note: The silane-PEG coated coverslips can be kept several months at 4 C but it is optimal to use them within the following weeks.
3.1.3.2 Micropatterning This section describes how to design and create the micropatterns on the passivated glass surface. Micropatterns design: Common micropattern shapes include the bar and the disk. Using these two basic shapes, MT asters and parallel/antiparallel MT organization can be reconstituted. Disk micropatterns allow radial nucleation of the MTs, and with bar micropatterns MTs parallel organization can be achieved. In order to reconstitute an anti-parallel MT network, two micropatterns (bars or disks) should be separated by 10–30 mm to allow MT interaction (Fig. 3.1). To avoid MTs overlap from two different micropatterns, micropatterns should be separate by at least 100 mm (Fig. 3.1). Micropattern thickness for MT microseeds (which length is 3 mm) on micropatterns bars, in this case micropatterns should be thick enough to allow the adsorption of MT seeds. The thickness of the micropatterns should be equal or superior to the MT seeds length. Micropattern surfaces are thus imprinted on the passivated glass surface using deep UV through the photomask as described below (Fig. 3.2):
3.2 Micropattern Functionalization for MTs Polymerization
FIGURE 3.1 Designing micropatterns. This scheme illustrates the basic geometrical dimensions that must be satisfied to ensure proper MT nucleation from the micropatterns (line width and micropattern size) and the minimum separation distance between two types of micropatterns (array step).
1. Wash the photomask with water and 100% isopropanol to remove any residue and dry it with pressurized filtered air. 2. Place the coverslips on the chrome face of the photomask. 3. Place on the vacuum holder and open vacuum to stick the coverslips to the mask. Note: The vacuum system allows a good contact between the photomask and the coverslip reducing the diffraction of the deep UVs. 4. Expose to deep UVs for 30–60 s. 5. Remove the coverslips using vacuum suction. Note: The micropatterned coverslips can be stored up to several weeks but proteins adherence is optimal within hours of micropatterning.
3.2 MICROPATTERN FUNCTIONALIZATION FOR MTs POLYMERIZATION This section describes the procedure to functionalize the micropatterns with neutravidin to further attached biotinylated MT microseeds and to observe MT elongation.
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FIGURE 3.2 Micropattern fabrication. This scheme summarizes the sequential steps for micropattern fabrication for MTs. Step 1: Clean glass coverslips (see washing procedure above). Step 2: Incubate the glass coverslips in a silane-PEG solution for 16 h. Step 3: Place the coverslip and the photomask on the mask holder. Place the sandwich under deep UV (180 nm) for 30–60 s to oxidize the silane-peg under transparent areas.
3.2.1 Materials • • • •
Micropatterned coverslips. mPEG-Silane, 30 kDa, passivated glasses. PLL20K-G35-PEG2K (Jenkem technology) solution at 0.1 mg/ml in 10 mM HEPES buffer, pH 7.4, stored at 4 C up to several months. Double-sided tape 70 mm thick (Lima).
Purified brain tubulin: • •
• • •
Tubulin is purified from cow brains according to Vantard, Peter, Fellous, Schellenbaum, and Lambert (1994). Purified tubulin is labeled using a standard procedure from Hyman et al. (1991) with either ATTO-565 (ATTO-TEC, AD 565-31), ATTO-488 (ATTO-TEC, AD 488-31), or with NHS-LC-LC-biotin (Pierce, EZ-Link NHS-LC-LC-Biotin). BRB80 (Brinkley Reassembly Buffer): 80 mM PIPES, 1 mM MgCl2, 1 mM EGTA, pH 6.9. Neutravidin (Thermo Scientific, 31000). GMPCPP 10 mM stock solution (JenaBioscience, NU-405).
3.2 Micropattern Functionalization for MTs Polymerization
• • • • • • • •
Taxol 10 mM stock in anhydrous dimethyl sulfoxide (DMSO) (Paclitaxel, Sigma, T-7191). GTP lithium salt (Sigma, G5884). BSA 10% (Sigma, A-7030) Catalase (Sigma, C-40). Glucose (Sigma, G8270). Glucose oxidase (Sigma, G-2133). Methyl Cellulose (Sigma, 1500 CP, M0555). Dithiothreitol (DTT) (Sigma, D9779).
3.2.2 Methods 3.2.2.1 MT seeds polymerization In order to obtain stabilized MT seeds, free tubulin is polymerized with Taxol and GMPCPP (a slowly hydrolysable GTP analog). These stabilizing agents enhance MT nucleation and stabilization, allowing the assembly of short stable MTs. 1. Mix 85% of biotin-labeled tubulin with 15% of fluorescently labeled tubulin in BRB80 at a final concentration of 50 mM. 2. Add 1 mM of GMPCPP and 20 mM of taxol. 3. Incubate for 5 min at 37 C. 4. Centrifuge on a tabletop centrifuge at 15,000 rpm for 5 min. 5. Discard supernatant and resuspend the MT microseeds in BRB80 with 1 mM GMPCPP and 20 mM taxol. Critical: Do not stock MT microseeds, directly flow the MT microseeds into the perfusion chamber to adhere them on the micropatterned surfaces.
3.2.2.2 Flow chamber assembly 1. Glass slides should be passivated using the same protocol as glass coverslips. 2. Using two bands of double-sided tape (70 mm, from Lima), assemble the perfusion chamber (Fig. 3.3), with the passivated glass slides and the micropatterned coverslips. 3. Flow 50 mg/ml of neutravidin, and incubate for 1 min. Critical: Do not use blocking agents such as BSA or casein before the incubation with neutravidin. 4. Wash the perfusion chamber with 10 volumes BRB80. 5. Flow PLL-g-PEG and incubate 20 s. 6. Wash the perfusion chamber with 20 volumes BRB80. 7. Flow the MT seeds and incubate for 2 min. 8. Wash the perfusion chamber with 20 volumes BRB80 with 0.5% BSA
3.2.2.3 MT seeds elongation Here we describe how to polymerize MTs from micropatterned-attached MT microseeds, by adding free tubulin and GTP (Fig. 3.4).
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FIGURE 3.3 Scheme representing the flow chamber assembly with a passivated coverglass and a micropatterned coverslip using two bands of double-sided tape.
FIGURE 3.4 Step 1: Incubate with neutravidin to coat the micropattern surface. Step 2: Incubate with PLL-PEG to enhance passivation of the surface. Step 3: Incubate the biotinylated MT seeds to adhere them on the neutravidin micropatterns. Step 4: Start MT elongation by the addition of free tubulin dimers and GTP.
1. Make the elongation Mix: • 10 mM of unlabeled tubulin and 2 mM of fluorescently labeled tubulin in BRB80 þ 1 mM GTP. • Add 0.025% of methyl cellulose at 1500 CP. • Add Oxygen scavenger: 2 mg/ml glucose, 80 mg/ml catalase, and 0.67 mg/ml glucose oxidase, 20 mM DTT. 2. Flow the elongation mix and seal the perfusion chamber with vitrex paste (Vitrex) or valap to avoid evaporation. 3. Observe at 30–37 C in TIRF microscopy.
3.2.2.4 Observation in TIRF microscopy MT dynamics can be visualized using an objective-based azimuthal ilas2 TIRF microscopy (Nikon eclipse Ti, modified by Roper scientific) and Evolve 512 camera (Photometrics) (Fig. 3.5A). Microscope stage was kept at 32 C using a warm stage controller (LINKAM MC60). Excitation was achieved using 491 and 561 nm lasers
3.2 Micropattern Functionalization for MTs Polymerization
FIGURE 3.5 (A) TIRF microscopy imaging of polymerizing MTs (in green) from MT microseeds (in red) from a bar micropattern (left panel) and from a disk micropattern (right panel). Scale bar ¼ 10 mm. (B and C) Schemes representing the different MT network types that can be achieved using micropatterns disks or bars. (B) Scheme representing the polarity and orientation of MTs. (C) Scheme representing the anti-parallel network organization using two micropatterns close to each other.
(Optical Insights). Time-lapse recording (one frame every 5 s) was performed for 30 min using Metamorph® software (version 7.7.5, Universal Imaging). Due to steric congestion, MT polymerization is polarized with the MT plus ends growing out of the micropatterns (Fig. 3.5A). MT polarity can be determined by TIRF microscopy by measuring MT polymerization speed (Portran, Gaillard, et al., 2013) as MTs plus ends polymerize faster than their minus ends (Walker et al., 1988). Using micropatterns disks or bars allows to obtain aster-like MT structures and parallel MT networks (Fig. 3.5B). Using two micropatterns close to each other create an antiparallel interaction between the polarized MTs (Fig. 3.5C).
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3.3 DISCUSSION Here, we have presented a method to spatially control MT nucleation and polarity on micropatterns on a PEG-treated surface. The passivated surface treatment described has been developed to study MAP interactions with dynamic MTs and can be further used for other types of experiments. Indeed, to study MT dynamic instability or MAP localization along the MT length, silane-PEG-Biotin can be used instead of the silane-PEG to attach biotinylated stabilized MT seeds on the surface (Portran, Zoccoler, et al., 2013; Stoppin-Mellet, Fache, Portran, Martiel, & Vantard, 2013). Furthermore, this micropatterning technique can be combined with a fluidic system to bend MTs in order to measure their rigidity (Portran, Zoccoler, et al., 2013).
3.3.1 Discussion about the passivation surface treatment The main challenge in microscopy is to avoid the direct interaction of the MTs with the surface, because it may modify their properties such as their dynamic instability. Therefore, multiple surface treatments have been developed to study MT dynamic instability and organization in presence of MAPs and molecular motors (Bieling, Telley, Hentrich, et al., 2010; Gell et al., 2010). The use of PEG-based surface treatment offers great advantages over non-covalent blocking methods, such as BSA (Vale et al., 1996) or casein (Howard, Hunt, & Baek, 1993), which may not achieve sufficient passivation. An efficient protein-repellent surface is essential to conserve the protein concentrations and avoid MT interactions with non-specific MAPs attached to the surface. Indeed, the PLL-PEG coating on a glass substrate exhibited excellent surface passivation, and has proven useful for single molecule tracking (Bieling, Telley, & Surrey, 2010; Gell et al., 2010). However, if PLL-PEG is efficient to prevent MAPs non-specific attachment on the surface, it is less efficient than a silane-PEG coating with regards to prevention of non-specific adsorption of tubulin on the surface (Ionov et al., 2010). The surface treatment described here has good repellent properties for tubulin and MAPs. Moreover, it provides a good stability and a high reproducibility since treated glass substrates may be stored for long periods.
3.3.2 Discussion about the different MT nucleation templates Many methods for micropatterning MTs have been published in the past few years (Ghosh et al., 2013; Shang et al., 2009; Uppalapati et al., 2011). Unfortunately, no universal solution exists as each method has drawbacks and advantages. The method described here is convenient as it is compatible with a surface treatment with excellent protein-repellent properties, in contrast to microprinting techniques with PDMS stamps (Dinarina et al., 2009; Ghosh et al., 2013; Shang et al., 2009). Moreover, the UV micropatterning may allow the attachment of any kind of MT nucleation template. Among the existing micropatterning techniques, the most physiological MT nucleation templates used would be the centrosomes or fragments of centrosomes
References
that can be purified from cell culture (Evans, Mitchison, & Kirschner, 1985; Shang et al., 2009). In contrast, several groups have initiated MT nucleation with plus end proteins that have been shown to enhance MT polymerization such as, XMAP215 (Ghosh et al., 2013; Popov, Severin, & Karsenti, 2002) or the MT binding domain of CLIP170 (H2) (Arnal, Heichette, Diamantopoulos, & Chretien, 2004). Using MT-stabilized seeds is the most common way to nucleate MTs, as they can be easily produced by polymerizing purified tubulin with stabilizing agents such as taxol and GMPCPP (Bieling, Telley, & Surrey, 2010; Gell et al., 2010; Tulin et al., 2012). Moreover, stabilized MT seeds have the advantage that they do not modify MT dynamic in contrast to plus end proteins (Ghosh et al., 2013; Popov et al., 2002).
CONCLUSION The micropatterning method described here aims to reconstitute MT structures with controlled nucleation and polarity. We have optimized the glass surface treatment to strongly reduce protein non-specific adsorption in order to reconstitute dynamic MT networks in presence of MAPs. Moreover, the use of MT-stabilized microseeds allows dense and polarized MT nucleation without affecting the parameter of MT dynamic instability. This micropatterning technique offers a great tool to describe the minimal requirements to reconstitute MT architecture (Portran, Gaillard, et al., 2013; Su et al., 2013) as observed in vivo. In the future, adding complexity to this system and using the previously developed actin filament micropatterning based on a similar methodology (Reymann et al., 2010) should allow the study of the crosstalk between actin filament and MTs. The development of these novel tools to manipulate actin and MT filaments promises a better understanding of the basic mechanisms involved in the formation and maintenance of the cytoskeleton architecture.
Acknowledgment I am grateful for support from Dr. Marylin Vantard and Dr. Manuel The´ry.
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