Washington University in St. Louis
Campus Box 1137
One Brookings Drive
St. Louis, MO 63130-4899
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.
Photo caption: (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.
Fishel EA and Dixit R (2013). Role of nucleation in cortical microtubule array organization: variations on a theme. Plant Journal, doi: 10.1111/tpj.12166
Tulin A, McClerklin S, Huang Y and Dixit R (2012). Single-molecule analysis of the microtubule crosslinking protein MAP65-1 reveals a molecular mechanism for contact-angle-dependent microtubule bundling. Biophysical Journal, 102: 802-809.
Eren EC, Gautam N and Dixit R (2012). Computer simulation and mathematical models of the noncentrosomal plant cortical microtubule cytoskeleton. Cytoskeleton, 69: 144-154.
Dixit R (2012). Putting a bifunctional motor to work: insights into the role of plant KCH kinesins. New Phytologist, 193: 543-545.
Zhu C and Dixit R (2012). Functions of the Arabidopsis kinesin superfamily of microtubule-based motor proteins. Protoplasma. DOI 10.1007/s00709-011-0343-9
Zhu C and Dixit R (2011). Single molecule analysis of the Arabidopsis FRA1 kinesin shows that it is a functional motor protein with unusually high processivity. Molecular Plant, 4: 879-885.
Sun F, Zhu C, Dixit R and Cavalli V (2011). Sunday Driver /JIP3 binds kinesin heavy chain directly and enhances its motility. EMBO J, 30:3416-3429.
Eren EC, Dixit R and Gautam N (2010) A three-dimensional computer simulation model reveals the mechanisms for self-organization of plant cortical microtubules into oblique arrays. Molecular Biology of the Cell, 21: 2674-2684.
Dixit R and Ross JL (2010). Studying plus-end tracking at single molecule resolution using TIRF microscopy. In, Methods in Cell Biology. Microtubules, In vitro. 95: 543-554. Eds. John J. Correia and Les Wilson. Elsevier.
Ross JL and Dixit R (2010). Multiple color single molecule TIRF imaging and tracking of MAPs and motors. In, Methods in Cell Biology. Microtubules, In vitro. 95: 521-542. Eds. John J. Correia and Les Wilson. Elsevier.