Force generation of organelle transport measured in vivo by an infrared laser trap

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Abstract
Organelle transport along microtubules is believed to be mediated by organelle-associated force-generating molecules. Two classes of microtubule-based organelle motors have been identified: kinesin and cytoplasmic dynein. To correlate the mechanochemical basis of force generation with the in vivo behaviour of organelles, it is important to quantify the force needed to propel an organelle along microtubules and to determine the force generated by a single motor molecule. Measurements of force generation are possible under selected conditions in vitro, but are much more difficult using intact or reactivated cells. Here we combine a useful model system for the study of organelle transport, the giant amoeba Reticulomyxa, with a novel technique for the non-invasive manipulation of and force application to subcellular components, which is based on a gradient-force optical trap, also referred to as 'optical tweezers'. We demonstrate the feasibility of using controlled manipulation of actively translocating organelles to measure direct force. We have determined the force driving a single organelle along microtubules, allowing us to estimate the force generated by a single motor to be 2.6 x 10(-7) dynes.