Tracking transcription factor mobility and interaction in Arabidopsis roots with fluorescence correlation spectroscopy.
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.
Department of Plant and Microbial Biology, North Carolina State University, Raleigh, United States.|Biomathematics Graduate Program, North Carolina State University, Raleigh, United States.
Natalie M
0000-0003-0988-321X
NM
Clark
Laboratory for Fluorescence Dynamics, University of California, Irvine, Irvine, United States.
Elizabeth
E
Hinde
Department of Biology, Howard Hughes Medical Institute, Duke University, Durham, United States.
Cara M
CM
Winter
Department of Plant and Microbial Biology, North Carolina State University, Raleigh, United States.
Adam P
AP
Fisher
Department of Biology, Howard Hughes Medical Institute, Duke University, Durham, United States.
Giuseppe
G
Crosti
Plant Developmental Biology, Wageningen University, Wageningen, Netherlands.
Ikram
I
Blilou
Laboratory for Fluorescence Dynamics, University of California, Irvine, Irvine, United States.
Enrico
E
Gratton
Department of Biology, Howard Hughes Medical Institute, Duke University, Durham, United States.
Philip N
0000-0001-5302-758X
PN
Benfey
Department of Plant and Microbial Biology, North Carolina State University, Raleigh, United States.
Rosangela
R
Sozzani
D017360:Arabidopsis / Q000201:enzymology*; D029681:Arabidopsis Proteins / Q000032:analysis*; D008962:Models, Theoretical; D018517:Plant Roots / Q000201:enzymology*; D025941:Protein Interaction Mapping; D062211:Spatio-Temporal Analysis; D013050:Spectrometry, Fluorescence; D013997:Time Factors; D014157:Transcription Factors / Q000032:analysis*; D014158:Transcription, Genetic
D029681:Arabidopsis Proteins; C100978:SCR protein, Arabidopsis; C410510:SHORT ROOT protein, Arabidopsis; D014157:Transcription Factors
<i>a. thaliana</i>; developmental biology; diffusion coefficient; oligomeric state; plant biology; protein movement; protein stoichiometry; spatio-temporal correlation; stem cells