Quantitative measurements and modeling of cargo–motor interactions during fast transport in the living axon
Video S1. Negatively charged beads injected into a freshly dissected squid giant axon and imaged within 10 minutes after injection with a 10x objective captured at 4 second intervals on a Zeiss 510 laser scanning confocal microscope with phase (grayscale) and fluorescence (red and green) channels. Yellow indicates both red and green fluorescence are present. Note the expansion of red (negatively charged beads) in the anterograde (to the right) direction, and no expansion of the green signal. Also note no changes on the retrograde (left) side of the injection bolus (yellow). (3.812Mb)
Video S2. High magnification of the axon shown in video S1 (63X, 4 second time lapse, 100 frames play-back at 12 fps) on the anterograde side of the injection. Note the rapid movements of the red (negatively charged) beads, with no moves apparent in the green channel. This video is not aligned, yet the movement of axoplasm is minimal. (6.478Mb)
Seamster, Pamela E.
Bearer, Elaine L.
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The kinesins have long been known to drive microtubule-based transport of sub-cellular components, yet the mechanisms of their attachment to cargo remain a mystery. Several different cargo-receptors have been proposed based on their in vitro binding affinities to kinesin-1. Only two of these—phosphatidyl inositol, a negatively charged lipid, and the carboxyl terminus of the amyloid precursor protein (APP-C), a trans-membrane protein—have been reported to mediate motility in living systems. A major question is how these many different cargo, receptors and motors interact to produce the complex choreography of vesicular transport within living cells. Here we describe an experimental assay that identifies cargo–motor receptors by their ability to recruit active motors and drive transport of exogenous cargo towards the synapse in living axons. Cargo is engineered by derivatizing the surface of polystyrene fluorescent nanospheres (100 nm diameter) with charged residues or with synthetic peptides derived from candidate motor receptor proteins, all designed to display a terminal COOH group. After injection into the squid giant axon, particle movements are imaged by laser-scanning confocal time-lapse microscopy. In this report we compare the motility of negatively charged beads with APP-C beads in the presence of glycine-conjugated non-motile beads using new strategies to measure bead movements. The ensuing quantitative analysis of time-lapse digital sequences reveals detailed information about bead movements: instantaneous and maximum velocities, run lengths, pause frequencies and pause durations. These measurements provide parameters for a mathematical model that predicts the spatiotemporal evolution of distribution of the two different types of bead cargo in the axon. The results reveal that negatively charged beads differ from APP-C beads in velocity and dispersion, and predict that at long time points APP-C will achieve greater progress towards the presynaptic terminal. The significance of this data and accompanying model pertains to the role transport plays in neuronal function, connectivity, and survival, and has implications in the pathogenesis of neurological disorders, such as Alzheimer's, Huntington and Parkinson's diseases.
Author Posting. © IOP Publishing, 2012. This article is posted here by permission of IOP Publishing for personal use, not for redistribution. The definitive version was published in Physical Biology 9 (2012): 055005, doi:10.1088/1478-3975/9/5/055005.