«CHAPTER 14 Studying Kinesin Motors by Optical 3D-Nanometry in Gliding Motility Assays Bert Nitzsche*, Volker Bormuth*, Corina Bräuer*, Jonathon ...»
Studying Kinesin Motors by Optical
3D-Nanometry in Gliding Motility Assays
Bert Nitzsche*, Volker Bormuth*, Corina Bräuer*, Jonathon
Howard*, Leonid Ionov*, Jacob Kerssemakers†, Till Korten*,
Cecile Leduc‡, Felix Ruhnow*, and Stefan Diez*
Max Planck Institute of Molecular Cell Biology and Genetics, 01307 Dresden, Germany
Kavli Institute of Nanoscience, Delft University of Technology, 2628 CJ Delft, The Netherlands
Centre de Physique Moleculaire Optique et Hertzienne, Universit Bordeaux 1, CNRS (UMR 5798), e 33405 Talence cedex, France Abstract I. Introduction II. Setup of Gliding Motility Assays A. Surface Treatment and Flow-Chamber Preparation B. Microtubule Preparation C. Surface Immobilization of Motor Proteins D. Attachment of Fluorescent Markers to Microtubules E. Imaging of Microtubule Motility III. Analysis of Microtubule and Quantum Dot Movements A. Quick Motility Evaluation in Two Dimensions B. Nanometer Tracking in Two Dimensions C. Resolving Nanometer Distances in the Third Dimension IV. Future Directions. Acknowledgments. Reagents References Abstract Recent developments in optical microscopy and nanometer tracking have facilitated our understanding of microtubules and their associated proteins. Using fluorescence microscopy, dynamic interactions are now routinely observed in vitro on the METHODS IN CELL BIOLOGY, VOL. 95 978-0-12-374815-7 Copyright Ó 2010 Elsevier Inc. All rights reserved. DOI: 10.1016/S0091-679X(10)95014-0 248 Bert Nitzsche et al.
level of single molecules, mainly using a geometry in which labeled motors move on surface-immobilized microtubules. Yet,we think that the historically older gliding geometry, in which motor proteins bound to a substrate surface drive the motion microtubules, offers some unique advantages. (1) Motility can be precisely followed by coupling multiple fluorophores and/or single bright labels to the surface of microtubules without disturbing the activity of the motor proteins. (2) The number of motor proteins involved in active transport can be determined by several strategies. (3) Multimotor studies can be performed over a wide range of motor densities.
These advantages allow for studying cooperativity of processive as well as nonprocessive motors. Moreover, the gliding geometry has proven to be most promising for nanotechnological applications of motor proteins operating in synthetic environments. In this chapter we review recent methods related to gliding motility assays in conjunction with 3D-nanometry. In particular, we aim to provide practical advice on how to set up gliding assays, how to acquire high-precision data from microtubules and attached quantum dots, and how to analyze data by 3D-nanometer tracking.
Optically following the live action of motor proteins at work has been a longstanding goal of biologists. However, observing the motors directly using light microscopy is technically much more challenging than imaging the huge polymeric filaments of the cytoskeleton along which the motors move. Even the narrow, 6-nm wide actin filaments can be imaged by dark-field (Nagashima and Asakura, 1980) or epi-fluorescence (Yanagida et al., 1984) microscopy. Historically, this facilitated the development of the so-called “upside-down” motility assays early on. In these “gliding” assays, the motors are bound to a planar substrate (usually a glass coverslip), and the movement of the filaments across the surface (Fig. 1) is followed by time-resolved microscopy (Kron and Spudich, 1986). By reducing the motor density on the surface, gliding assays even provide the possibility to obtain recordings from individual motor proteins (Howard et al., 1989). Yet, it has always been an attractive idea to visualize the movement of the motors rather than that of the filaments. In order to do so “stepping” motility assays, in which the filaments are bound to the substrate and the motor movement along the immobilized filaments is imaged, have been developed. This was first done by binding the motors to large, micron-sized beads, which could be followed by video microscopy (Sheetz and Spudich, 1983;
Spudich et al., 1985; Yanagida et al., 1984) or held in an optical trap (Rief et al., 2000; Schnitzer et al., 2000; Svoboda et al., 1993). Later on, Funatsu et al. pushed the sensitivity of the fluorescence microscope to the limit of being able to visualize individual motor molecules labeled with the cyanine-based fluorophores (Funatsu et al., 1995). Using total internal reflection fluorescence (TIRF) microscopy, the processive movement of individual kinesin-1 molecules along microtubules was visualized by labeling the motors with Cy3 (Vale et al., 1996) or with the green fluorescent protein (GFP) (Pierce et al., 1997). Since then, the number of singlemolecule fluorescence measurements of kinesin and dynein motors interacting with microtubules has vastly expanded. Nowadays, not only the stepping of motors is observed in these assays, but also diffusion and (de)polymerizing activities of motors
14. Studying Kinesin Motors by Optical 3D-Nanometry
Fig. 1 Schematic diagram of a gliding motility assay in which reconstituted microtubules, tagged with semiconductor nanocrystals (quantum dots), are propelled over a kinesin-coated substrate surface.
(See Plate no. 16 in the Color Plate Section.) (and other microtubule-associated proteins) (Bieling et al., 2007; Brouhard et al., 2008; Fink et al., 2009; Helenius et al., 2006; Varga et al., 2006, 2009).
Despite all the recent developments and groundbreaking results obtained in stepping motility assays, we think that there are still a number of compelling reasons to revisit the historically older gliding motility assays: (1) Fluorescently labeled filaments are bright objects that can be tracked with high precision. (2) Photostable optical reporters, such as quantum dots (QDs), can easily be attached to the filaments without interfering with the operation of individual motors. (3) Several strategies to determine the number of motors involved in transport are feasible. (4) The collective behavior involving multiple processive or nonprocessive motors can be studied over a wide range of motor densities. We will report methods and protocols that allow for experiments taking advantage of these benefits.
immobilization as the most critical steps in setting up a gliding motility assay.
Unfortunately, there is no general protocol that works reliably with all types of motor proteins. Yet, there are certain concepts that, when adapted by some minor, but crucial, modifications, function with many different motor proteins. In the end, every gliding motility assay has to be optimized for the exact motor protein used, and evidence that the assay works is only provided by the facts that (1) the experiments are reproducible and (2) the gliding velocities are consistent with in vivo activity of the motor. Below we will list a number of example procedures and general hints on how to establish a “successful” gliding motility assay.
A. Surface Treatment and Flow-Chamber Preparation Depending on the specific experiment, we routinely treat glass coverslips or silicon wafers with various cleaning and coating procedures to adjust the surface properties. The treated glass coverslips (for epi-fluorescence and TIRF measurements) or silicon wafers [for fluorescence-interference contrast (FLIC) measurements] then provide the surface of the flow chambers where the gliding motility experiments are performed in. Generally, surface preparation should be performed very carefully in a clean and dust-free environment to guarantee the reproducibility and the high quality of the experimental results.
Here we describe a simple, but effective, cleaning procedure (easy-clean), a method to passivate surfaces to prevent protein adsorption [based on polyethylene glycol (PEG)], a method to render surfaces highly hydrophobic (based on dichlorodimethylsilane), and an approach to generate surfaces, which gradually [in one dimension (1D)] change their capacity to bind proteins.
1. Easy-Clean Procedure Load the glass coverslips or silicon wafer chips into porcelain or polytetrafluoroethylene (PTFE) racks. Place the racks into a glass container and fill with mucasol (1:20 dilution in deionized water) such that all coverslips/chips are completely covered. Sonicate (using Bransonic 2510, Branson, Danbury, CT, USA) for 15 min and rinse with deionized water for 2 min. Remove the water from the container using a (e.g., 1 ml) pipette tip connected to the vacuum line. Fill the container with ethanol, again immersing the coverslips/chips completely, sonicate for 10 min, and rinse with nanopure water for 2 min in the container. Take the racks out of the container and carefully blow dry the coverlips/chips using nitrogen.
When used under the microscope, these surfaces are mildly hydrophilic. Being usually devoid of any fluorescent particles, they are even suited for single-molecule fluorescence measurements.
Tip: We store the coverslips/chips (prepared by the easy-clean procedure as well as by the methods listed below) in a dry container, which is sealed such that no dust can get onto the surfaces.
2. Protein-Repelling Surfaces (Based on PEG-Silane) To passivate surfaces, we use a procedure adapted from Papra et al. (2001) as follows: Load the glass coverslips or silicon wafer chips into porcelain or PTFE
14. Studying Kinesin Motors by Optical 3D-Nanometry
3. Hydrophobic Surfaces (Based on Dichlorodimethylsilane) This procedure is described in Chapter 13 by Gell et al., this volume. We use hydrophobic surfaces to strongly adsorb antibodies (as adapter proteins for specific motor molecules) in combination with the possibility to block the rest of these surfaces against nonspecific protein binding by casein or Pluronics (see below).
5. Flow-Chamber Preparation The general procedure we use to assemble our flow chambers is described in Chapter 13 by Gell et al., this volume. Apart from double-sided Scotch tape to confine the flow channels, we also use Parafilm or Nescofilm as an alternative. Flowchamber assembly is then as follows: Cut the Para-/Nescofilm in stripes using a (razor) blade or a laser cutter (Speedy 100C 25W, Trotec, Marchtrenk, Austria).
Place the prepared Para-/Nescofilm stripes on top of a 22 Â 22 mm coverslip and cut off (using a razor blade) those parts of the stripes that protrude over the edges of the coverslip. Place the top coverslip (18 Â 18 mm or silicon chip) in a central location on the stripes and transfer the assembly (22 Â 22 coverslip facing down) to a heating plate with a temperature of %120°C for Parafilm or %150°C for Nescofilm. The two coverslips (or coverslip/silicon chip) will be firmly joined by the melting Para-/ Nescofilm. This process can be well monitored by a change in the optical properties of the Para-/Nescofilm. Gently applying a bit of pressure to the top coverslip/chip during the melting process ensures a tight sealing and eliminates air bubbles that might have formed above or beneath the stripes. Without delay, place the flow chamber onto the surface of a metal (e.g., aluminum) block for fast cooling.
Mounting the assembly in the appropriate holder (see also Chapter 13 by Gell et al., this volume) is the last step before the channels (volume approximately 3 Â 18 Â 0.1 mm % 5 µl) of the flow chamber can be filled with solutions.
Tip: When using a pegylated coverslip (with an extremely hydrophilic surface) as one side of the flow chamber, double-sided Scotch tape and parafilm stripes frequently lift-off the pegylated surface and hence the channels become leaky. We therefore recommend using Nescofilm in conjunction with pegylated surfaces.
Tip: When assembling the flow chambers, take care to not expose bare surfaces to dust. Apply the same caution as during the surface preparation.
B. Microtubule Preparation For gliding motility assays, microtubules are commonly reconstituted from purified tubulin. Thereby, their physical structure, including the number of protofilaments and the stiffness, strongly depends on the assembly conditions (Meurer-Grob et al., 2001; Pierson et al., 1978; Ray et al., 1993). Below, we will describe the generation of stable (i.e., nondynamic) microtubules using the slowly hydrolyzable GTP-analogue GMP-CPP with/without further stabilization using Taxol. In order to allow for the attachment of streptavidin-coated QDs as fluorescent markers, part of the tubulin used in the polymerization reaction is biotinylated.
1. Biotinylated GMP-CPP Microtubules Supplement 100 µl BRB80 (80 mM PIPES/KOH pH 6.9, 1 mM MgCl2, 1 mM EGTA) by 2 µM tubulin, 4 mM MgCl2, and 1 mM GMP-CPP. Tubulin may be a mixture of 5–50% fluorescently labeled (e.g., Alexa 488 or TAMRA), 2–50% biotinylated, and 50–93% unlabeled tubulin. (Note: Because very high tubulin labeling ratios can lead to artifacts in the interaction of motor proteins with the filaments, we never use more than 50% labeled tubulin in the polymerization reaction) Allow the microtubules to assemble for ! 2 h at 37°C. Centrifuge the
14. Studying Kinesin Motors by Optical 3D-Nanometry
2. Biotinylated Double-Stabilized Microtubules Prepare biotinylated microtubules using GMP-CPP (as described above). However, resuspending the microtubules after the centrifugation step, supplement the BRB80 with 10 µM Taxol and keep this amount of Taxol present in all subsequent solutions. Microtubules stabilized this way are stable for weeks at room temperature.
C. Surface Immobilization of Motor Proteins We use two types of strategies to immobilize motor proteins on surfaces: nonspecific and specific binding. In the simplest case (nonspecific binding) motor proteins are allowed to adsorb to surfaces precoated with other “space filling” proteins (Howard et al., 1993). This nonspecific approach works for some kinesin motors (e.g., kinesin-1). For other motor proteins, however, procedures targeting specific sequences of the motor proteins can significantly improve the quality of motility assays. Thereby, bioactive linker molecules (e.g., specific antibodies or streptavidin) that are directed toward specified regions distal to the motor domain (e.g., purification tags, GFP or biotinylated sites) are first attached to the surface.