Quantification of Cytoskeletal Dynamics in Time-Lapse Recordings.

The cytoskeleton is key to many essential processes in a plant cell, e.g., growth, division, and defense. Contrary to what "skeleton" implies, the cytoskeleton is highly dynamic, and is able to re-organize itself continuously. The advent of live-cell microscopy and the development of genetically encoded fluorophores enabled detailed observation of the organization and dynamics of the cytoskeleton. Despite the biological importance of the cytoskeletal dynamics, quantitative analyses remain laborious endeavors that only a handful of research teams regularly conduct. With this protocol, we provide a standardized step-by-step guide to analyze the dynamics of microtubules. We provide example data and code for post-processing in Fiji that enables researchers to modify and adapt the routine to their needs. More such tools are needed to quantitatively assess the cytoskeleton and thus to better understand cell biology. © 2019 by John Wiley & Sons, Inc.

[1]  R. Cyr,et al.  Encounters between Dynamic Cortical Microtubules Promote Ordering of the Cortical Array through Angle-Dependent Modifications of Microtubule Behaviorw⃞ , 2004, The Plant Cell Online.

[2]  K. Schwarzerová,et al.  Multifunctional Microtubule-Associated Proteins in Plants , 2016, Front. Plant Sci..

[3]  Michael Unser,et al.  A pyramid approach to subpixel registration based on intensity , 1998, IEEE Trans. Image Process..

[4]  G. Borisy,et al.  Contribution of plus and minus end pathways to microtubule turnover. , 1999, Journal of cell science.

[5]  T. Mitchison,et al.  Microtubule polymerization dynamics. , 1997, Annual review of cell and developmental biology.

[6]  S. Shaw,et al.  CLASP Facilitates Transitions between Cortical Microtubule Array Patterns1 , 2018, Plant Physiology.

[7]  D. Ehrhardt,et al.  A Mechanism for Reorientation of Cortical Microtubule Arrays Driven by Microtubule Severing , 2013, Science.

[8]  K. Ueda,et al.  Visualization of microtubules in living cells of transgenicArabidopsis thaliana , 1999, Protoplasma.

[9]  Teh-hui Kao,et al.  A GFP–MAP4 Reporter Gene for Visualizing Cortical Microtubule Rearrangements in Living Epidermal Cells , 1998, Plant Cell.

[10]  E. Deinum,et al.  Efficient event-driven simulations shed new light on microtubule organization in the plant cortical array , 2014, Front. Physics.

[11]  Johannes E. Schindelin,et al.  Fiji: an open-source platform for biological-image analysis , 2012, Nature Methods.

[12]  J. Mathur,et al.  A Novel Localization Pattern for an EB1-like Protein Links Microtubule Dynamics to Endomembrane Organization , 2003, Current Biology.

[13]  J. Doonan,et al.  EB1 reveals mobile microtubule nucleation sites in Arabidopsis , 2003, Nature Cell Biology.

[14]  J. Sedbrook,et al.  CLASP stabilization of plus ends created by severing promotes microtubule creation and reorientation , 2018, The Journal of cell biology.

[15]  Katie Porter,et al.  From filaments to function: The role of the plant actin cytoskeleton in pathogen perception, signaling and immunity. , 2016, Journal of integrative plant biology.

[16]  T. Hashimoto,et al.  Low concentrations of propyzamide and oryzalin alter microtubule dynamics in Arabidopsis epidermal cells. , 2004, Plant & cell physiology.

[17]  L. Blanchoin,et al.  Actin dynamics in plant cells: a team effort from multiple proteins orchestrates this very fast-paced game. , 2010, Current opinion in plant biology.

[18]  S. Diez,et al.  Dynamic Actin Patterns and Arp2/3 Assembly at the Substrate-Attached Surface of Motile Cells , 2004, Current Biology.

[19]  Elisabeth Lipka,et al.  Mechanisms of plant cell division , 2015, Wiley interdisciplinary reviews. Developmental biology.

[20]  Bang Wong,et al.  Points of view: Color blindness , 2011, Nature Methods.

[21]  Gaudenz Danuser,et al.  plusTipTracker: Quantitative image analysis software for the measurement of microtubule dynamics. , 2011, Journal of structural biology.

[22]  Marten Postma,et al.  PlotsOfData—A web app for visualizing data together with their summaries , 2019, PLoS biology.

[23]  S. Shaw,et al.  Microtubule Array Patterns Have a Common Underlying Architecture in Hypocotyl Cells1 , 2017, Plant Physiology.

[24]  D. Ehrhardt,et al.  Arabidopsis cortical microtubules position cellulose synthase delivery to the plasma membrane and interact with cellulose synthase trafficking compartments , 2009, Nature Cell Biology.

[25]  E. Chang,et al.  Establishment of polarity during organization of the acentrosomal plant cortical microtubule array. , 2005, Molecular biology of the cell.

[26]  David Zwicker,et al.  Tracking single particles and elongated filaments with nanometer precision. , 2011, Biophysical journal.

[27]  David W Ehrhardt,et al.  Morlin, an inhibitor of cortical microtubule dynamics and cellulose synthase movement , 2007, Proceedings of the National Academy of Sciences.

[28]  L. Amos,et al.  Microtubule structure and its stabilisation. , 2004, Organic & biomolecular chemistry.

[29]  René Schneider,et al.  A Mechanism for Sustained Cellulose Synthesis during Salt Stress , 2015, Cell.

[30]  E. Deinum,et al.  Modelling the role of microtubules in plant cell morphology. , 2013, Current opinion in plant biology.

[31]  A. Fernie,et al.  Cellulose-Microtubule Uncoupling Proteins Prevent Lateral Displacement of Microtubules during Cellulose Synthesis in Arabidopsis. , 2016, Developmental cell.

[32]  S. Shaw,et al.  Microtubule dynamics and organization in the plant cortical array. , 2006, Annual review of plant biology.

[33]  René Schneider,et al.  Cellulose and callose synthesis and organization in focus, what's new? , 2016, Current opinion in plant biology.

[34]  Jonathon Howard,et al.  Microtubule dynamics reconstituted in vitro and imaged by single-molecule fluorescence microscopy. , 2010, Methods in cell biology.

[35]  Sidney L. Shaw,et al.  Sustained Microtubule Treadmilling in Arabidopsis Cortical Arrays , 2003, Science.

[36]  R. Hawkins,et al.  Survival of the aligned: ordering of the plant cortical microtubule array. , 2009, Physical review letters.

[37]  Murray Evaluating the performance of fluorescence microscopes , 1998, Journal of microscopy.