Vacuolar membrane dynamics revealed by GFP‐AtVam3 fusion protein

Abstract

Background: The plant vacuole is a multifunctional organelle that has various physiological functions. The vacuole dynamically changes its function and shape, dependent on developmental and physiological conditions. Our current understanding of the dynamic processes of vacuolar morphogenesis has suffered from the lack of a marker for observing these processes in living cells. Results: We have developed transgenic Arabidopsis thaliana expressing a vacuolar syntaxin‐related molecule (AtVam3/SYP22) fused with green fluorescent protein (GFP). Observations using confocal laser scanning microscopy demonstrated that the plant vacuole contained a dynamic membrane system that underwent a complex architectural remodelling. Three‐dimensional reconstitution and time‐lapse analysis of GFP‐fluorescence images revealed that cylindrical and sheet‐like structures were present in the vacuolar lumen and were moving dynamically. The movement, but not the structure itself, was abolished by cytochalasin D, an inhibitor of actin polymerization. This moving structure, which sometimes penetrated through the vacuolar lumen, possessed a dynamic membrane architecture similar to the previously recognized ‘transvacuolar strand.’ Conclusion: We propose two possible models for the formation of the vacuolar lumenal structure. Membrane structures including protruding tubules and reticular networks have recently been recognized in many other organelles, and may be actively involved in intra‐ and/or inter‐organelle signalling.

Increase in Invaginated Vacuolar Membrane Structure Caused by Plant Cell Expansion by Genotoxic Stress Induced by DNA Double-Strand Breaks

“…The tubular vacuoles are mainly detected around mitotic spindles and young cell plates during M phase of BY-2 cells (Kutsuna and Hasezawa 2002) and in mitotic cells of the meristematic regions of the root tips of A. thaliana (Seguí-Simarro and Staehelin 2006). Bulbs, which are spherical vesicles in the vacuolar lumen, were detected in rapidly expanding cells of young leaves and hypocotyls of A. thaliana (Kutsuna and Hasezawa 2005, Saito et al 2002, Uemura et al 2002. The cytoplasmic strand is the structure that is transverse within the vacuole like a tunnel (Hoffmann andNebenführ 2004, Ruthardt et al 2005).…”

Overexpression of theArabidopsisSyntaxin PEP12/SYP21 Inhibits Transport from the Prevacuolar Compartment to the Lytic Vacuole in Vivo

“…This protein is well established as a tonoplast marker (Sato et al, 1997;Uemura et al, 2002Uemura et al, , 2004 and would serve to test whether membrane traffic from the PVC to the vacuole is blocked under SYP21 overexpression. As expected from published findings, the majority of the protoplasts expressing YFP-SYP22 show clear tonoplast localization in the absence of SYP21 overexpression ( Figure 6C).…”

“…As expected from published findings, the majority of the protoplasts expressing YFP-SYP22 show clear tonoplast localization in the absence of SYP21 overexpression ( Figure 6C). The presence of internal subregional structures, previously named bulbs, exhibiting a stronger fluorescence than the outer vacuolar membrane are also a typical feature of tonoplast staining (Mitsuhashi et al, 2000;Saito et al, 2002;Uemura et al, 2002).…”

KATAMARI1/MURUS3 Is a Novel Golgi Membrane Protein That Is Required for Endomembrane Organization in Arabidopsis

“…Vacuoles, which are the most prominent compartment in the plant cell, also have a dynamic and complex membrane structure. Studies using a vacuolar membrane-targeted GFP found that spherical structures (bulbs) consisting of a double membrane were often observed within the lumen of vacuoles and were connected with the vacuolar membrane Uemura et al, 2002). These bulbs were observed to move around within or along the outline of the membrane, mediated by the actin filaments but not by the microtubules (Uemura et al, 2002).…”