Tracking transcription factor mobility and interaction in Arabidopsis roots with fluorescence correlation spectroscopy

Abstract

To understand complex regulatory processes in multicellular organisms, it is critical to be able to quantitatively analyze protein movement and protein-protein interactions in time and space. During Arabidopsis development, the intercellular movement of SHORTROOT (SHR) and subsequent interaction with its downstream target SCARECROW (SCR) control root patterning and cell fate specification. However, quantitative information about the spatio-temporal dynamics of SHR movement and SHR-SCR interaction is currently unavailable. Here, we quantify parameters including SHR mobility, oligomeric state, and association with SCR using a combination of Fluorescent Correlation Spectroscopy (FCS) techniques. We then incorporate these parameters into a mathematical model of SHR and SCR, which shows that SHR reaches a steady state in minutes, while SCR and the SHR-SCR complex reach a steady-state between 18 and 24 hr. Our model reveals the timing of SHR and SCR dynamics and allows us to understand how protein movement and protein-protein stoichiometry contribute to development.DOI: http://dx.doi.org/10.7554/eLife.14770.001

Root stem cell niche organizer specification by molecular convergence of PLETHORA and SCARECROW transcription factor modules

“…Note that our cooperative combinatorial model does not exclude the existence of either additional shared targets or many unshared PLT and SCR target genes. Unshared targets can serve broader roles in root development, such as the progression of cell division and differentiation in the root PLT gradient (Mähönen et al 2014) and the regulation of division and differentiation of the cortical-endodermal lineage by SCR and its binding partner, SHR (Di Laurenzio et al 1996;Helariutta et al 2000;Sabatini et al 2003;Cui et al 2007;Cruz-Ramírez et al 2012;Clark et al 2016;Long et al 2017). The many additional nonoverlapping functions are consistent with the observation that the published regulated targets of the PLT and SCR pathways are quite distinct (Levesque et al 2006;Moreno-Risueno et al 2015;Santuari et al 2016).…”

Cell-Fate Specification in Arabidopsis Roots Requires Coordinative Action of Lineage Instruction and Positional Reprogramming

“…In contrast, SHR direct target SCARECROW (SCR) was unable to influence the division pattern when misexpressed alone (Fig. 1, A and C), which is consistent with the previous studies showing that SCR forms a protein complex with SHR to promote periclinal cell division (Heidstra et al, 2004;Cui et al, 2007;Long et al, 2015b;Clark et al, 2016).…”

A matter of time — How transient transcription factor interactions create dynamic gene regulatory networks

“…Typically, entire plants tissues (such as the shoot or root tissue) are utilized as starting material, whereby different cell layers are not discriminated. Consequently, if TF binding activity is specific to a specific cell layer (for example TFs TMO5 [85] or SHR [86] in the Arabidopsis root), such interactions will be diluted across the cell population.…”

“…Undoubtedly, some degree of error is introduced by the technical limitations of ChIP itself [99], which may contribute to false positives. Also, TF binding is not always gene proximal, it can occur non-specifically open chromatin regions [36, 49, 74, 77–80], and regulation may require additional TF partners [85, 86]. But it is unlikely that these limitations explain all those TF binding events detected by ChIP that are not found to have an effect on transcription.…”