Visualization and quantification of vesicle trafficking on a three-dimensional cytoskeleton network in living cells

Racine V, Sachse M, Salamero J, Fraisier V, Trubuil A, Sibarita JB.
J Microsc.. 2007 Mar; 225(Pt 3): 214-28
DOI: JMI1723 [pii]10.1111/j.1365-2818.2007.01723.x

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Recent progress in biology and microscopy has made it possible to acquire
multidimensional data on rapid cellular activities. Unfortunately, the data
analysis needed to describe the observed biological process still remains a major
bottleneck. We here present a novel method of studying membrane trafficking by
monitoring vesicular structures moving along a three-dimensional cytoskeleton
network. It allows the dynamics of such structures to be qualitatively and
quantitatively investigated. Our tracking method uses kymogram analysis to
extract the consistent part of the temporal information and to allow the
meaningful representation of vesicle dynamics. A fully automatic extension of
this method, together with a statistical tool for dynamic comparisons, allows the
precise analysis and comparison of overall speed distributions and directions. It
can handle typical complex situations, such as a dense set of vesicles moving at
various velocities, fusing and dissociating with each other or with other cell
compartments. The overall approach has been characterized and validated on
synthetic data. We chose the Rab6A protein, a GTPase involved in the regulation
of intracellular membrane trafficking, as a molecular model. The application of
our method to GFP-Rab6A stable cells acquired using fast four-dimensional
deconvolution video-microscopy gives considerable cellular dynamic information
unreachable using other techniques.

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