Chapter 13 - Microtubule Dynamics Reconstituted In Vitro and Imaged by Single-Molecule Fluorescence Microscopy

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Abstract

In vitro assays that reconstitute the dynamic behavior of microtubules provide insight into the roles of microtubule-associated proteins (MAPs) in regulating the growth, shrinkage, and catastrophe of microtubules. The use of total internal reflection fluorescence microscopy with fluorescently labeled tubulin and MAPs has allowed us to study microtubule dynamics at the resolution of single molecules. In this chapter we present a practical overview of how these assays are performed in our laboratory: fluorescent labeling methods, strategies to prolong the time to photo-bleaching, preparation of stabilized microtubules, flow-cells, microtubule immobilization, and finally an overview of the workflow that we follow when performing the experiments. At all stages, we focus on practical tips and highlight potential stumbling blocks.

Introduction

Microtubules are highly dynamic polymers that undergo spontaneous transitions from growing to shrinking phases (Mitchison and Kirschner, 1984). This behavior, termed dynamic instability, is coupled to the hydrolysis of guanosine triphosphate (GTP) and is regulated by many proteins (Howard and Hyman, 2009), including depolymerizing kinesins (Helenius et al., 2006, Varga et al., 2009), polymerases (Brouhard et al., 2008), and plus-tip proteins (Akhmanova and Steinmetz, 2008). The discovery of dynamic instability was contingent on the ability to visualize individual microtubules. Bulk assays such as turbidity, which detects only the total amount of polymer, are insensitive to the dramatic length changes that individual microtubules undergo when a solution containing tubulin and GTP is under steady-state conditions (Mitchison and Kirschner, 1984).

With advances in fluorescence microscopy, especially the development of total internal reflection fluorescence (TIRF) microscopy (Axelrod, 2008, Axelrod et al., 1984, Funatsu et al., 1995) and the discovery of genetically encoded fluorescent proteins (Zhang et al., 2002), the visualization of individual molecules is becoming routine. The principle underlying single-molecule fluorescence is visualization by localization. When a fluorophore is free in solution it diffuses very quickly (∼100 µm2/s), and within the typical exposure time of a camera (0.1 s), the fluorophore will be “smeared out” over an area of several square microns, corresponding to many camera pixels, which are typically ∼0.01 µm2. However, when the molecule binds, for example to a microtubule, its rate of diffusion slows dramatically. All the fluorescence is now localized to a small number of pixels during the exposure time of the camera, giving a signal that exceeds the background, even if there are many fluorophores free in solution. TIRF microscopy is important because by exciting only those molecules near the surface where binding takes place, the background is reduced. However, other techniques that reduce the out-of-focus fluorescence, such as confocal microscopy, can also be used for single-molecule studies (Gell et al., 2006).

Single-molecule fluorescence assays have provided many new insights into the movement of motor proteins. For example, the sizes of the steps taken by myosin-V along actin filaments (Yildiz et al., 2003) and kinesin-1 along microtubules (Yildiz et al., 2004) have been measured. Single-molecule assays have also provided new insights into the regulation of microtubule dynamics: kinesin-13 targets the microtubule end through diffusion on the lattice prior to capture at the end (Helenius et al., 2006); XMAP-215 is a processive depolymerase that “surfs” on the growing end of a microtubule as it adds many tubulin dimers (Brouhard et al., 2008); EB1 binds only transiently to the comet tail that it forms at the growing end of a microtubule (Bieling et al., 2007).

The application of single-molecule techniques to study microtubule dynamics is technically much more challenging than its application to motor proteins. The reason is that motors move quickly, often with speeds on the order of 1 µm/s, and therefore make many elementary steps in a second. By contrast, the dynamics of microtubules is slow. Growth is often only on the order of microns per minute, and the transitions from growth to shrinkage (catastrophe) and shrinkage to growth (rescue) take place at rates less than 1/min. Thus, microtubule dynamics must be observed over timescales of many minutes. This leads to several problems. The main one is that the fluorescent molecules will slowly accumulate on surfaces. Nonspecific binding to the chamber surfaces leads to an increase in the background, making detection of the fluorophores interacting specifically with the microtubules more difficult to observe. Furthermore, nonspecific binding leads to a reduction in the concentration of molecules in solution, causing an apparent decrease in activity. This chapter focuses on experimental procedures to reduce nonspecific binding to surfaces, as well as to increase the fluorescence lifetime of the fluorophores.

Section snippets

Single-Molecule TIRF Microscopy

Within the last decade, TIRF microscopy has emerged as a key tool in biophysics, particularly in single-molecule fluorescence studies (Gell et al., 2006, Selvin and Ha, 2007). Incorporated into conventional fluorescence microscopes, TIRF allows imaging limited to a thin layer (∼100 nm) above a glass substrate (for reviews see Axelrod et al., 1984, Gell et al., 2009, Thompson and Steele, 2007). This removes the distraction of out-of-focus fluorescent material, thereby providing sufficient

List of Reagents

In this section, we present an alphabetical list of the reagents used throughout this chapter. We give details of product codes and manufacturers where possible, comment on storage and handling as well as details of preparation.

  • Alexa Fluor 488 (Invitrogen, Molecular Probes, Karlsruhe, Germany, A30005), used for tubulin labeling (see Hyman et al., 1991), we use the tetrafluorophenol conjugate.

  • Antibeta-tubulin SAP4G5 (Sigma, Munich, Germany, T7816), typically a 1:50–1:200 dilution from the stock

Choice of Fluorophore/Protein Labeling

The right choice of fluorescent label is important for the success of a dynamic microtubule experiment. It depends on the experimental approach, number of components in the assay, and available instrumentation. In this section, we highlight some of the considerations that affect the choice of labeling method and dye. We refer the reader to Bane et al. (this volume) and also the following papers (Gell et al., 2006, Hunter et al., 2003, Hyman et al., 1991, Selvin and Ha, 2007) for more detailed

(Anti-)blinking/Photo-Toxicity/Photo-Bleaching Cocktails

Many fluorescent molecules used for single-molecule TIRF display unwanted blinking on the millisecond to second timescale, often, but not exclusively, due to the dye being excited into a triplet state (Aitken et al., 2008, Rasnik et al., 2006). Population of dye triplet states is also thought to be an important precursor to irreversible loss of fluorescence (photo-bleaching) (Aitken et al., 2008, Rasnik et al., 2006). Dye triplet states are effectively quenched by molecular oxygen, but the

Preparation of GMPCPP-Stabilized Microtubules

Stabilized microtubules for use as substrates for microtubule depolymerization assays, or as seeds upon which to nucleate the growth of microtubule extensions, are prepared by polymerization of tubulin in the presence of the slowly hydrolyzed GTP analogue, GMPCPP (Hyman et al., 1992). The distribution of microtubule lengths obtained, and the propensity of the microtubules to spontaneously depolymerize, can be tailored by altering the concentration of tubulin used in the polymerization reaction

Glass Treatment and Sample Chamber Preparation

In this section, we describe the preparation and construction of sample chambers for studying microtubules in a single-molecule TIRF microscope. The aim is to produce clean, low-fluorescence glass surfaces, formed into simple flow cells, that allow convenient immobilization of microtubules or microtubule seeds and that can be effectively passivated to prevent unwanted nonspecific absorption (see Section VIII).

Binding of Microtubules and Passivation of Surfaces

Stabilized microtubules are bound to the surface via a spacer protein that attaches nonspecifically to the silanized coverglass surface, but specifically to the microtubule (see Fig. 1). The spacer protein holds the microtubule away from the surface, reducing unwanted surface interactions. Immobilized microtubules can be used as seeds from which to examine polymerization, substrates for depolymerization studies, and substrates for studying the mechanism of MAPs. The use of a spacer, in contrast

Dynamic Microtubule Assays

In this section, we outline an assay that can be used to study the behavior of MAPs on dynamic microtubules. Firstly, we present the basic workflow for these types of assays and discuss the important components of the reaction mixture that will be perfused into the channel containing the microtubule seeds. Secondly, we discuss several important variables that must be considered and controlled for accurate experiments. Finally, we discuss the basic type of analysis carried out on these types of

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