Eukaryotic cell systems synthesize specialized proteins, so-called motorproteins, able to generate mechanical force, necessary for realization of different intracellular transport and motility processes. A prominent member of the group of motorproteins is kinesin. Kinesin represents a molecular motor working in association with microtubules, which can be characterized as 1-20 µm long and 25 nm thick proteinaceous hollow-cylindrical assemblies. Kinesin molecules are long (about 100 nm) filamentous structures, consisting of two globular heads, a stalk, and a tail. The head and the tail domain contain the microtubule- and the cargo-binding site, respectively. The energy required for force generation is supplied by hydrolysis of adenosine triphosphate (ATP). The hydrolytic reaction is performed in a special enzymatic centre, localized in the kinesin head domain (1). From mechanical point of view, in a cellular transport unit microtubules serve as stator and the kinesin, which carries a certain cargo (vesicle), as slide (Fig. 1). Gliding direction is determined by microtubule polarity.
Fig. 1 Linear motoric unit of a nanoactuatoric machinery based on kinesin/microtubule association
Under defined in vitro conditions, taxol-stabilized microtubules are able to glide over kinesin-coated glass surfaces (2). They follow more or less linear tracks with velocities of about 0.6-1.2 µm/s (Fig. 2).
In such a system, the kinesin bound tightly to a glass slide as "cargo", whose mass is much greater than that of microtubules, fulfils stator function and the microtubules are the sliding part. Gliding velocity is regulated by different factors, including ATP and Mg2+ concentration, pH, and kinesin density (3,4). So far, no data have been available concerning temperature dependence and temperature stability of kinesin-dependent microtubule force generation.
Therefore, we studied microtubule gliding across a kinesin-coated glass surface within the temperature interval between 6 and 45 °C. Kinesin and microtubules were prepared from porcine brain according to Kuznetsov and Gelfand (5), mixed with ATP, and transferred to glass slides. Microtubule gliding was visualized by video-enhanced differential interference contrast microscopy (6), using a Zeiss Axiophot microscope equipped with a Hamamatsu Chalnicon video camera and a Hamamatsu Argus 20 image processor. Gliding velocity of individual microtubules was directly measured using the Argus software. The experiments were performed in a closed chamber in which the temperature gradient within the sample was minimized by cooling or warming both the microscope and the sample.
The velocity of microtubule gliding steadily increased (about 10-fold) within the range from 5°C to about 40°C (Fig. 3).
The temperature dependence of gliding velocity was found to coincide strictly with the temperature dependence of kinesin ATPase activity.
Microtubules were found to glide even at 40 to 45 °C. However, an enhanced tendency of detachment of microtubules from kinesin-coated glass was observed. Therefore, measurements at these temperatures were difficult (indicated by the high standard deviation). At 45 °C, only a few microtubules gliding across the kinesin-coated surface could be observed, suggesting denaturation of at least one component of the force-generating system. To find out which component was damaged, we preincubated either the kinesin or the microtubules for 15 min at temperatures > 37 °C and studied them in the gliding assay at room temperature. It was found that the taxol-stabilized microtubules endure temperatures up to 60 °C. In contrast, the kinesin was more sensitive to high temperature and became irreversibly damaged during preincubation above 43 °C.
The results presented here demonstrate that temperature is an essential parameter which might be used to regulate force generation of a kinesin-microtubule-based nanoactoric device within the limits between 5 and 45 °C.
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