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

  • Clark N
  • Hinde E
  • Winter C
  • et al.
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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.Stem cells are a specific type of cell found in both plants and animals. These cells can divide to produce daughter cells that can take on the role of any of the different tissues and organs within the plant or animal. A plant known as Arabidopsis is often used as a model in scientific research. In Arabidopsis, two proteins called SHORTROOT and SCARECROW are known to control the ability of stem cells in the roots to divide.SHORTROOT is made in cells at the center of the root known as the vasculature. From there, it moves to the next cell layer (called the endodermis) where it interacts with SCARECROW to form a protein complex. Here, Clark et al. investigated how quickly SHORTROOT moves between cells, the direction it moves in, and how it interacts with SCARECROW.The experiments used a new imaging technique called scanning fluorescence correlation spectroscopy to track the movements of SHORTROOT molecules in the root. This technique relies on the protein of interest (in this case, SHORTROOT) being attached to a fluorescent protein so that it is visible when the cells are examined. In plants that had lower levels of SCARECROW, SHORTROOT moves between cells more quickly and in an unrestricted manner. This suggests that SCARECROW forms a complex with SHORTROOT to restrict its movement in the endodermis. The experiments also show that SHORTROOT is only able to leave the endodermis to return to the vasculature when SCARECROW levels are lower than normal.Clark et al. developed a model to map the behavior of SHORTROOT and SCARECROW in the root and predict how the levels of these proteins change over time. One of the next steps following on from this work would be to test whether other proteins restrict the movement of SHORTROOT, perhaps by studying mutant plants in which SHORTROOT is less able to move.

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Clark, N. M., Hinde, E., Winter, C. M., Fisher, A. P., Crosti, G., Blilou, I., … Sozzani, R. (2016). Tracking transcription factor mobility and interaction in Arabidopsis roots with fluorescence correlation spectroscopy. ELife, 5. https://doi.org/10.7554/elife.14770

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