Tobacco Arp3 is localized to actin-nucleating sites in vivo

• Published in: Journal of experimental botany 2009-02, Vol.60 (2), p.603-614
• Main Authors: Maisch, Jan, Fišerová, Jindřiška, Fischer, Lukáš, Nick, Peter
• Format: Article
• Language: English
• Subjects: Actin; Actin Cytoskeleton - metabolism; actin-related protein 3 (ARP3); Actin-Related Protein 3 - chemistry; Actin-Related Protein 3 - isolation & purification; Actin-Related Protein 3 - metabolism; Actins; Actins - metabolism; Amino Acid Sequence; Animal cells; Auxins; Biological and medical sciences; Cell Division; Cell lines; Cell Nucleus - metabolism; Cells; Fluorescence; Fundamental and applied biological sciences. Psychology; Green Fluorescent Proteins - metabolism; Luminescent Proteins - metabolism; Membrane Glycoproteins - metabolism; Microfilament Proteins - metabolism; Microfilaments; Molecular Sequence Data; Nicotiana - cytology; Nicotiana - metabolism; Nucleation; Phylogeny; Plant cells; Plant Proteins - metabolism; Plants; Protein Transport; Recombinant Fusion Proteins - metabolism; Red Fluorescent Protein; RESEARCH PAPER; Research Papers; tobacco BY-2
• ISSN: 0022-0957; 1460-2431
• DOI: 10.1093/jxb/ern307

The polarity of actin is a central determinant of intracellular transport in plant cells. To visualize actin polarity in living plant cells, the tobacco homologue of the actin-related protein 3 (ARP3) was cloned and a fusion with the red fluorescent protein (RFP) was generated. Upon transient expression of these fusions in the tobacco cell line BY-2 (Nicotiana tabacum L. cv. Bright Yellow 2), punctate structures were observed near the nuclear envelope and in the cortical plasma. These dots could be shown to decorate actin filaments by expressing RFP-ARP3 in a marker line, where actin was tagged by GFP (green fluorescent protein)-FABD (fimbrin actin-binding domain 2). When actin filaments were disrupted by latrunculin B or by prolonged cold treatment, and subsequently allowed to recover, the actin filaments reformed from the RFP-ARP3 structures, that therefore represented actin nucleation sites. The intracellular distribution of these sites was followed during the formation of pluricellular files, and it was observed that the density of RFP-ARP3 increased in the apex of the polarized, terminal cells of a file, whereas it was equally distributed in the central cells of a file. These findings are interpreted in terms of position-dependent differences of actin organization.