Single particle tracking study of organelles transport in Xenopus Melanophores

VALERIA LEVI, PHD1, ANNA S. SERPINSKAYA2, VLADIMIR GELFAND2, ENRICO GRATTON

Salt Lake City, Utah

Congreso; 50th annual meeting of the Biophysical Society; 2006

Organelle transport is essential for a variety of cellular functions. Xenopus melanophores have black pigment organelles or melanosomes which, in response to hormonal signals, disperse in the cytoplasm or aggregate in the perinuclear region. Melanosomes are moved by two microtubule motors, kinesin-II and cytoplasmic dynein and the actin motor myosin V. We explored organelle transport along microtubules and actin filaments by using a new fast-tracking routine designed for a microscope under bright field illumination which has spatial precision of 2 nm and temporal resolution of 10 ms. Melanophores were incubated with nocodazole or latrunculin B to depolymerize microtubules and actin filaments, respectively. In these conditions, the transport is driven only by myosin V or microtubule-motors. Melanosomes moving along microtubules presented velocity distributions with several peaks that could not be fit with single Gaussian functions. We postulated that the melanosome velocity depends linearly on the number of active motors. According to this model, 1 to 3 dynein molecules transport each melanosome in the minus-end direction. The transport in the plus-end direction is driven by 1 to 2 copies of kinesin-2. The active dynein molecules attached to each melanosome increases during aggregation suggesting that this is a mechanism to regulate the net direction of melanosome transport. The model also shows that multiple motors of the same polarity cooperate during the melanosome transport while motors of opposite polarity do not compete. Melanosome moving along actin filaments presented complex trajectories in which we could identify regions of fast-motion lasting less than 100 ms. The histogram of distances traveled in these regions shows a maximum at 36 nm, compatible with the step size of the motor in vitro.