Continuous and time-shared multiple optical tweezers for the study of single motor proteins

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Abstract

We present a comparison between continuous (CW) and time-shared (TS) multiple optical tweezers applied to the study of the interaction between a single motor protein (myosin) with its track (an actin filament). In the experimental assay, named “three-bead assay”, a single actin filament is stretched between two beads trapped in a CW or in a TS double trap. The actin filament is presented to a single myosin molecule lying on a third bead attached to the coverslide. The CW double trap is obtained by splitting a single laser source into two orthogonally polarized beams, while the TS one is obtained by rapidly scanning a single laser beam with acusto-optic modulators. When using the CW traps, position detection of the left or right bead is obtained by means of a single quadrant detector photodiode (QDP) placed in the back focal plane of the condenser and selected with a polarizer; when using the TS traps, the position of multiple traps with the same QDP can be collected independently using triggered and synchronized generation and acquisition. The two techniques are thoroughly analysed and compared, evidencing advantages and disadvantages of each one.

Introduction

During the last decade, optical tweezers have become a powerful tool for manipulating single biomolecules and for investigating the mechanic and kinetic properties of proteins and biopolymers.

Since their development [1], optical tweezers have undergone many technical advances. Among these, multiple optical tweezers have been proved to be especially useful in experiments where polymers need to be manipulated at the single-molecule level [2].

Multiple optical tweezers can be divided into two major classes: time-shared (TS) and continuous (CW). The first class is obtained by rapidly moving a single optical trap between different positions in the sample plane; if the time taken to scan the different trap positions is much smaller than the diffusion time of the trapped particles, the laser beam works as stable multiple optical tweezers. Faster scans imply that more traps can be generated simultaneously and/or that more efficient trapping can be achieved. TS traps have been obtained by deflecting the laser beam using galvano mirrors [3], piezoelectric mirrors [4], or acousto-optic modulators (AOMs) [5], [6], [7]; depending on the technique used, scanning rates can reach respectively 10–50, 1–2, and 10–200 kHz. Traps generation is usually computer-controlled so that the number of traps, their position, and stiffness can all be modified in real time. TS traps are also easy to be constructed and aligned, since only one laser beam is needed.

CW multiple optical tweezers are obtained by simply dividing a beam into two or more optical paths and then recombining the beams before the objective [8]; alternatively, two or more laser sources can be combined together [6]. This approach is simple to be realized when two traps are needed, but becomes more complicated when more than two traps are required.

A double optical tweezers assay largely used to study interactions between a single myosin motor and a single actin filament is the “three-bead assay”, developed by Finer et al. in 1994 [8]. In this assay, double optical tweezers are used to catch and stretch an actin filament between two trapped beads, creating a configuration usually named “dumbbell”. The actin filament is presented to a third bead stuck to the coverslide surface, which on average carries one myosin molecule. Interactions between actin and myosin are detected from noise reduction in the bead position signal and from data analysis the mechanic and kinetic properties of the molecule are derived [9].

Myosin is a family of motor proteins almost ubiquitous in eukaryotic cells. The chemical energy contained in an ATP molecule is converted during a chemo-mechanical cycle in which the myosin motor produces its movement (working stroke). In particular, myosin II is a non-processive molecular motor responsible for muscle contraction. During the last decade, its properties at the single-molecule level have been investigated using both CW [8], [9], [10], [11], [12], [13] and TS [14], [15] double optical tweezers. The same experimental assay has been used to study cardiac V1 and V3 myosin [16], smooth muscle myosin [17], [18], myosin I [19], and myosin V [20], [21].

We have built a multiple optical tweezers setup that can work both as CW and TS double optical tweezers. CW traps are obtained by splitting an infrared laser source into two orthogonally polarized beams, while TS traps are obtained by using two crossed AOMs. Position detection of the CW or TS tweezers is achieved using a single quadrant detector photodiode (QDP) placed in the back-focal plane of the condenser. Separate detection of the TS traps is possible using an acquisition board synchronized and triggered to the generation signal, as explained in the next section.

We have compared the two techniques in a three-bead assay. We show that, using CW traps, the stability of the system is within 1 nm and the working stroke of the molecule can be determined with sub-nanometer accuracy, along with its kinetic properties. On the other hand, in the TS configuration, the dumbbell oscillates at the switching frequency with amplitude that depends on the traps stiffness and position and on the switching frequency. This oscillation does not change the kinetics of the molecule significantly, but must be taken into account when evaluating the working stroke using back focal plane detection.

Section snippets

The experimental apparatus

The experimental apparatus (Fig. 1) was set up around a custom made optical microscope. The mechanical structure was designed in order to obtain high mechanical stability and to allow using custom optics. Two linear manual translators (M-014 Physik Instrumente) and a piezoelectric stage (P-527.2 CL Physik Instrumente), allow gross (25 mm stroke) and fine (1 nm resolution) movements of the sample in the xy plane, respectively. The objective (Nikon Plan-Apo 60X, NA 1.2, WD 0.2 mm, water immersion)

Dumbbell dynamic in CW and TS optical tweezers

In this section, the motion of the dumbbell in a CW and in a TS optical tweezers are compared. The two traps are supposed to have the same stiffness kt/2; the combined stiffness of the double trap (kt∼0.05 pN/nm) is chosen to be much smaller than the myosin stiffness (km∼1 pN/nm) [26]. The actin filament can be considered almost inextensible [27], its stiffness being ka∼44 pN/nm for a 1 μm long filament. The links between the beads and the filament can reach 1–3 pN/nm when the tension on the

CW and TS three-bead assay

We have compared CW and TS recordings of the position signals when a single myosin molecule was interacting with a single actin filament. Preparation of proteins and sample cell has been performed by standard methods [28]. During a single acquisition, 100 s of the position signal from one or both beads were recorded, depending if CW or TS tweezers were utilized, respectively. During acquisition, the displacement between the actin filament and the myosin molecule was stabilized by means of a

Conclusions

We have presented an experimental apparatus that is capable of working of both as CW or as TS double optical tweezers. Position detection is achieved using a single QDP placed in the back focal plane of the condenser. When using the CW traps, left or right trap detection is selected by means of a polarizer; when using the TS configuration, detection of multiple traps is achieved with a triggered and synchronized generation/acquisition, during which n acquired points from the left trap position

Acknowledgements

The authors are grateful to M. Canepari, P. Cacciafesta, and R. Bottinelli for the biochemical preparations used in the acto-myosin experiments. Work under Contract HPRI-CT-1999-00111 CE, partially supported by “SINPHYS” PAIS 2002 project of INFM.

Note added in proof: During processing of this manuscript, novel silicon QDPs designed specifically for near-infrared lasers (1064 nm) have become available (UDT SPOT-15-YAG). We have verified that using these photodiodes, bandwidths up to 100 kHz can

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