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The major research goal of the Dixit lab is to understand how the cytoskeleton and its
associated proteins control plant cell form and function. We are particularly interested in the
mechanisms that control patterning of the cortical microtubule cytoskeleton because this process
regulates plant cell morphogenesis. We have shown that encounters between cortical microtubules
foster their co-alignment and that this process favors polar microtubule bundling (i.e., with
microtubule plus-ends facing in the same direction). Monte Carlo simulations have verified that
deterministic modifications of the stochastic microtubule dynamics, as a consequence of
microtubule encounters, are necessary and sufficient for cortical array organization. Our
current work in this area focuses on the microtubule plus-end complex to understand how cortical
microtubules sense and regulate the outcome of encounters. In addition, we are using a
computational approach to identify the critical parameters governing cortical microtubule
pattern formation. This approach relies on feedback between experimental data and computational
predictions and takes advantage of the wealth of Arabidopsis mutants affecting cortical
microtubule organization and live-cell imaging for this purpose. The long-term goal of this
project is to obtain a systems level understanding of the relationship between the cytoskeleton
and plant morphogenesis.
The Dixit lab is also very interested in the microtubule-based motor protein, kinesin. Plants
possess an incredible diversity of kinesins, many of which represent plant-specific subfamilies
of unknown function. The wealth of natural sequence variation among the plant kinesins also
affords unique opportunities to investigate fundamental structure-function relationships in
terms of motor activity. For this work, we have focused on the subgroup of Arabidopsis kinesins
that possess both microtubule and actin binding domains and therefore potentially coordinate the
activities of the microtubule and actin cytoskeleton. We are using GFP fusions, reverse genetics
and overexpression studies to characterize the function of these kinesins in whole plants. In
addition, we are exploiting the power of single molecule analyses (using TIRF microscopy) along
with ensemble assays (such as filament gliding assays) to investigate the biophysical properties
of these motors and to conduct detailed structure-function analyses. One of the long-term goals
of this project is to study the role of motor protein diversity in plant structural and
functional evolution using comparative genomics and proteomics strategies.
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(A) Confocal image of cortical microtubules in Arabidopsis epidermal cells. (B) Maximum projection of pseudo-colored time-lapse images of EB1-GFP from a living tobacco BY2 cell. This image illustrates the growth vector of individual cortical microtubules going sequentially from blue to green to yellow to red. (C) Single GFP-labeled kinesin molecules (3 molecules can be seen in this sequence) observed moving along a rhodamine-labeled microtubule using total internal reflection fluorescence microscopy. The dotted line indicates the movement of one of the kinesin molecules over 3 seconds.
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