Covalent immobilization of microtubules on glass surfaces for molecular motor force measurements and other single-molecule assays

Abstract

Rigid attachment of microtubules (MTs) to glass cover slip surfaces is a prerequisite for a variety of microscopy experiments in which MTs are used as substrates for MT-associated proteins, such as the molecular motors kinesin and cytoplasmic dynein. We present an MT-surface coupling protocol in which aminosilanized glass is formylated using the cross-linker glutaraldehyde, fluorescence-labeled MTs are covalently attached, and the surface is passivated with highly pure beta-casein. The technique presented here yields rigid MT immobilization while simultaneously blocking the remaining glass surface against nonspecific binding by polystyrene optical trapping microspheres. This surface chemistry is straightforward and relatively cheap and uses a minimum of specialized equipment or hazardous reagents. These methods provide a foundation for a variety of optical tweezers experiments with MT-associated molecular motors and may also be useful in other assays requiring surface-immobilized proteins.

Fig. 1

Protocol scheme. Each pathway summarizes the major steps in preparing a slide chamber with MTs attached and the surface passivated against nonspecific microsphere binding

Fig. 2

Glass preparation for aminosilanization. (a) Borosilicate glass cover slips are sonicated in an alkaline (pH ~11.5) phosphate detergent to remove gross surface contaminants (and possibly a very thin surface layer of the silicon dioxide network that forms the glass structure). Next, plasma cleaning removes any residual organic contaminants and converts surface siloxanes to silanol groups that are more reactive with aminopropyltriethoxysilanes. (b) Prior to plasma cleaning, 20 μL of ddH₂O deposited on the cover slip surface forms a bead (left), which, when viewed from the side (right), forms a dome shape with a non-negligible contact angle, θ_c (even if one attempts to spread the drop over the surface). (c) Following plasma cleaning, 20 μL of ddH₂O flows evenly over the highly hydrophilic glass surface and does not form a bead (left). The contact angle is greatly reduced (and difficult to observe; right)

Fig

3 Aminopropyltriethoxysilane structures and reaction scheme. (a) Chemical structures of 3-aminopropyltriethoxysilane (APTES) and N-(2-aminoethyl)-3-aminopropyltriethoxysilane (AEAPTES). (b) General reaction scheme for aminopropyltriethoxysilanes, shown for the specific case of AEAPTES. Exposure to water leads to hydrolysis of the ethoxy groups, yielding two alcohols, a silanol and an ethanol leaving group. Silanols can undergo condensation reactions either with each other (water condensation) or with unhydrolyzed silanes (ethanol condensation) to form siloxane bonds and yield oligomers. The water released by the condensation reaction can participate in hydrolysis of additional ethoxy groups. Thus, even small amounts of water can catalyze silane polymerization. Uncontrolled polymerization yields complex, disordered networks that form a viscous sol–gel [36]. Note that the reaction scheme shown here is a simplified, conceptual summary and that hydrolysis and condensation can potentially occur on different parts of each molecule simultaneously. Silane polymerization is catalyzed by the addition of ammonia (not shown). (c) Aminopropyltriethoxysilanes for five-membered ring structures (left APTES, right AEAPTES), allowing the amino group to intramolecularly catalyze silane polymerization, even in the absence of water

Fig. 4

Aminopropyltriethoxysilane functionalization of glass using APTES (R=–(CH₂)₃–) or AEAPTES (R=–(CH₂)₃–NH(CH₂)₂–). An appropriately cleaned glass surface containing a high density of silanol groups is exposed to the aminosilane in nearly anhydrous acetone, yielding a diverse mixture of free silanes and silanols in solution, which then physisorb onto the surface via hydrogen bonding and/or ionic interactions (several configurations in addition to the ones shown are possible). The addition of heat drives the formation of siloxane bonds between the physisorbed silanes/silanols and the glass surface by supplying energy and removing condensation products (water and ethanol) by evaporation. This reaction is catalyzed by the terminal amine group. Rinsing in ethanol and water removes any remaining physisorbed aminosilane deposits and leads to hydrolysis of remaining ethoxy groups on the bound silanes, converting them to silanols. A final heating/drying helps these silanol groups form intramolecular siloxane linkages (siloxane bonds may also form via reaction of adjacent ethoxy and silanol groups, as during the initial bonding to the surface). This conceptual scheme does not illustrate several concurrent pathways that also lead to stable binding of aminosilanes to the glass surface (e.g., aminosilane oligomerization in solution, followed by physisorption and binding to the surface). The final product of this treatment is a glass surface densely covered in covalently attached amines

Fig. 5

Slide chamber preparation. (a) Parafilm is applied to a glass slide and (b) burnished with a clean pipette tip so it adheres to the glass. (c) The paper backing is removed with a pair of sharp-nosed tweezers (paper backing is pseudo-colored pink in this image to enhance visibility). (d) An aminosilanized glass cover slip is carefully placed on top of the exposed film strips (the cover slip is pseudo-colored pink and outlined in yellow in this image to enhance visibility) and lightly pressed down (not shown), and (e) the slide chamber is heated briefly (cover-slip-side-up) until the Parafilm becomes transparent. (f) The cover slip is then gently pressed with a tweezers in order to form a tight bond with the parafilm. After treating the chamber with glutaraldehyde (not shown), (g) microtubule solution and then trapping assay solution are introduced from one end of the chamber while simultaneously using a filter paper “wick” on the opposite end to help draw the solution through (in the photograph, a blue dye is used instead of trapping solution in order to enhance visibility). (h) The chamber is then sealed using a cotton-tipped applicator saturated in vacuum grease. By twisting the applicator at the entrance of the chamber, grease is swept into the mouth of the chamber, forming a perfect seal. (i) Using a template during steps (a-d) helps ensure consistent chamber volumes and cover slip placement. The template provided has a chamber width of 4 mm, yielding a volume of ~10 μL (Color figure online)

Fig. 6

Glutaraldehyde treatment of amino-functionalized glass. The bifunctional glutaraldehyde binds surface amine groups via a hydrolysis reaction, thus functionalizing the surface with formyl groups that will bind surface-exposed Iysines on proteins. Extensive rinsing removes any free glutaraldehyde that could yield unwanted reactions in the final assay. Finally, the glass is thoroughly dried and stored under vacuum

Fig. 7

Labeling of tubulin with Cy3 dye. Tubulin is first polymerized into MTs. Cy3 conjugated to the reactive N-hydroxysuccinimide (NHS) is then added, which facilitates attachment of the dye to amine groups on the surface of the MT. After pelleting the MTs and removing excess dye, the MTs are resuspended and depolymerized in the cold. The insoluble fraction is then pelleted and the supernatant transferred to a new tube in which the tubulin is repolymerized, pelleted, and depolymerized again, followed by aliquotting and snap freezing. This strategy ensures that the tubulin is labeled in regions other than the key interfaces required for polymerization and that the final product contains only tubulin capable of cyclic polymerization and depolymerization (and thus unperturbed by the attached dye)

Fig. 8

MT preparation. (a) Addition of GTP to free tubulin (αβ-tubulin dimers) induces MT polymerization (promoted by addition of glycerol [40, 41] and 37°C temperature). When preparing fluorescent MTs, the small amount <5 %) of tubulin labeled with the organic fluorophore Cy3 incorporates randomly into the MT lattice and is distributed sparsely enough that the dye molecules do not affect motor interaction with the MTs in the optical trapping assay. Initially, MTs exhibit dynamic instability [42, 43]. Paclitaxel greatly enhances polymerization and stabilizes the MTs [44-49] (the DMSO in which paclitaxel is dissolved also enhances MT polymerization [50]). (b) Removal of residual-free tubulin and very short MT fragments is accomplished by sedimentation through a 60 %-glycerol “cushion.” First, MTs are layered carefully on top of the cushion. Following centrifugation, the free tubulin and very short MTs remaining in the supernatant are removed. The MT pellet is then resuspended in buffer. These MTs are stable for days to weeks at room temperature

Fig. 9

MT immobilization in aminosilane-/glutaraldehyde-treated slide chambers. (a) The negatively charged MT is attracted to the positively charged glass surface. Following incubation, reactive formyl groups attached to the glass bind to amines on surface-exposed Iysines on the MT, thus covalently linking the MT to the cover slip. (b) End-on view of MT attached to glass, with remaining surface passivated with β-casein (introduced to the chamber at 2 mg/mL). The β-casein, an amphiphile capable of adsorption on a variety of surfaces, forms a bilayer on the glass surface that prevents unwanted interactions with trapping microspheres, and its lysine residues react with any remaining formyl groups. (c) The β-casein N-terminal hydrophilic region is negatively charged, allowing favorable interactions with the positively charged glass surface. The top layer extends this hydrophilic region into the solution, forming a “brush” layer on the surface that prevents trapping beads from sticking. (d) Fluorescence image of Cy3-labeled MTs covalently bound to the cover slip in a microscope flow chamber. They are very well aligned with the long axis of the chamber due to combination of the laminar flow induced when filling the chamber and the highly favorable initial adhesion to the positively charged glass. Scale bar: 10 11m