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Assessing the utility of photoswitchable fluorescent proteins for tracking intercellular protein movement in the Arabidopsis root.
PLoS One. 2011; 6(11):e27536.Plos

Abstract

One way in which cells communicate is through the direct transfer of proteins. In plants, many of these proteins are transcription factors, which are made by one cell type and traffic into another. In order to understand how this movement occurs and its role in development, we would like to track this movement in live, intact plants in real-time. Here we examine the utility of the photoconvertible proteins, Dendra2 and (to a lesser extent) EosFP as tags for studying intracellular and intercellular protein movement in the Arabidopsis root. To this end, we made fusions between Dendra2 and six mobile transcription factors. Our results show that Dendra2 is an effective tool for studying protein movement between plant cells. Interestingly, we found that Dendra2 could not simply be swapped into existing constructs that had originally contained GFP. Most of the fusions made in this way failed to produce a fluorescent fusion. In addition we found that the optimal settings for photoconversion of Dendra2 in stably transformed roots were different from what has been published for photoconversion in transient assays in plants or in animal cells. By modifying the confocal setting, we were able to photoconvert Dendra2 in all cell layers in the root. However the efficiency of photoconversion was affected by the position of the cell layer within the root, with more internal tissues requiring more energy. By examining the Dendra2 fusions, we confirmed the mobility of the SHORT-ROOT (SHR) and CAPRICE (CPC) transcription factors between cells and we further discovered that SHR movement in stele and CPC movement in the epidermis are non-directional.

Authors+Show Affiliations

Department of Biology, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America.No affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, Non-P.H.S.

Language

eng

PubMed ID

22132108

Citation

Wu, Shuang, et al. "Assessing the Utility of Photoswitchable Fluorescent Proteins for Tracking Intercellular Protein Movement in the Arabidopsis Root." PloS One, vol. 6, no. 11, 2011, pp. e27536.
Wu S, Koizumi K, Macrae-Crerar A, et al. Assessing the utility of photoswitchable fluorescent proteins for tracking intercellular protein movement in the Arabidopsis root. PLoS ONE. 2011;6(11):e27536.
Wu, S., Koizumi, K., Macrae-Crerar, A., & Gallagher, K. L. (2011). Assessing the utility of photoswitchable fluorescent proteins for tracking intercellular protein movement in the Arabidopsis root. PloS One, 6(11), e27536. https://doi.org/10.1371/journal.pone.0027536
Wu S, et al. Assessing the Utility of Photoswitchable Fluorescent Proteins for Tracking Intercellular Protein Movement in the Arabidopsis Root. PLoS ONE. 2011;6(11):e27536. PubMed PMID: 22132108.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Assessing the utility of photoswitchable fluorescent proteins for tracking intercellular protein movement in the Arabidopsis root. AU - Wu,Shuang, AU - Koizumi,Koji, AU - Macrae-Crerar,Aurora, AU - Gallagher,Kimberly L, Y1 - 2011/11/23/ PY - 2011/08/09/received PY - 2011/10/19/accepted PY - 2011/12/2/entrez PY - 2011/12/2/pubmed PY - 2012/4/3/medline SP - e27536 EP - e27536 JF - PloS one JO - PLoS ONE VL - 6 IS - 11 N2 - One way in which cells communicate is through the direct transfer of proteins. In plants, many of these proteins are transcription factors, which are made by one cell type and traffic into another. In order to understand how this movement occurs and its role in development, we would like to track this movement in live, intact plants in real-time. Here we examine the utility of the photoconvertible proteins, Dendra2 and (to a lesser extent) EosFP as tags for studying intracellular and intercellular protein movement in the Arabidopsis root. To this end, we made fusions between Dendra2 and six mobile transcription factors. Our results show that Dendra2 is an effective tool for studying protein movement between plant cells. Interestingly, we found that Dendra2 could not simply be swapped into existing constructs that had originally contained GFP. Most of the fusions made in this way failed to produce a fluorescent fusion. In addition we found that the optimal settings for photoconversion of Dendra2 in stably transformed roots were different from what has been published for photoconversion in transient assays in plants or in animal cells. By modifying the confocal setting, we were able to photoconvert Dendra2 in all cell layers in the root. However the efficiency of photoconversion was affected by the position of the cell layer within the root, with more internal tissues requiring more energy. By examining the Dendra2 fusions, we confirmed the mobility of the SHORT-ROOT (SHR) and CAPRICE (CPC) transcription factors between cells and we further discovered that SHR movement in stele and CPC movement in the epidermis are non-directional. SN - 1932-6203 UR - https://www.unboundmedicine.com/medline/citation/22132108/Assessing_the_utility_of_photoswitchable_fluorescent_proteins_for_tracking_intercellular_protein_movement_in_the_Arabidopsis_root_ L2 - http://dx.plos.org/10.1371/journal.pone.0027536 DB - PRIME DP - Unbound Medicine ER -