title: Quantification and cluster analysis of actin cytoskeletal structures in plant cells: role of actin bundling in stomatal movement during diurnal cycles in Arabidopsis guard cells.
abstract: Manual evaluation of cellular structures is a popular approach in cell biological studies. However, such approaches are laborious and are prone to error, especially when large quantities of image data need to be analyzed. Here, we introduce an image analysis framework that overcomes these limitations by semi-automatic quantification and clustering of cytoskeletal structures. In our framework, cytoskeletal orientation, bundling and density are quantified by measurement of newly-developed, robust metric parameters from microscopic images. Thereafter, the microscopic images are classified without supervision by clustering based on the metric patterns. Clustering allows us to collectively investigate the large number of cytoskeletal structure images without laborious inspection. Application of this framework to images of GFP-actin binding domain 2 (GFP-ABD2)-labeled actin cytoskeletons in Arabidopsis guard cells determined that microfilaments (MFs) are radially oriented and transiently bundled in the process of diurnal stomatal opening. The framework also revealed that the expression of mouse talin GFP-ABD (GFP-mTn) continuously induced MF bundling and suppressed the diurnal patterns of stomatal opening, suggesting that changes in the level of MF bundling are crucial for promoting stomatal opening. These results clearly demonstrate the utility of our image analysis framework.
authors.affiliation: Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwanoha Kashiwa, Chiba 277-8562, Japan.
authors.forename: Takumi
authors.initials: T
authors.lastname: Higaki
authors.forename: Natsumaro
authors.initials: N
authors.lastname: Kutsuna
authors.forename: Toshio
authors.initials: T
authors.lastname: Sano
authors.forename: Noriaki
authors.initials: N
authors.lastname: Kondo
authors.forename: Seiichiro
authors.initials: S
authors.lastname: Hasezawa
mesh_terms: D008841:Actin Cytoskeleton / Q000235:genetics / Q000378:metabolism; D000199:Actins / Q000378:metabolism*; D017360:Arabidopsis / Q000378:metabolism* / Q000502:physiology; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism*; D002940:Circadian Rhythm / Q000235:genetics / Q000502:physiology*; D016000:Cluster Analysis; D003599:Cytoskeleton / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence; D054046:Plant Stomata / Q000378:metabolism*
chemical_list: D000199:Actins; D029681:Arabidopsis Proteins; D049452:Green Fluorescent Proteins