Expression of a functional yellow fluorescent protein fusion to cellulose synthase (CESA) in transgenic Arabidopsis plants allowed the process of cellulose deposition to be visualized in living cells. Spinning disk confocal microscopy revealed that CESA complexes in the plasma membrane moved at constant rates in linear tracks that were aligned and were coincident with cortical microtubules. Within each observed linear track, complex movement was bidirectional. Inhibition of microtubule polymerization changed the fine-scale distribution and pattern of moving CESA complexes in the membrane, indicating a relatively direct mechanism for guidance of cellulose deposition by the cytoskeleton.

Plant cell cortical microtubule arrays attain a high degree of order without the benefit of an organizing center such as a centrosome. New assays for molecular behaviors in living cells and gene discovery are yielding insight into the mechanisms by which acentrosomal microtubule arrays are created and organized, and how microtubule organization functions to modify cell form by regulating cellulose deposition. Surprising and potentially important behaviors of cortical microtubules include nucleation from the walls of established microtubules, and treadmilling-driven motility leading to polymer interaction, reorientation, and microtubule bundling. These behaviors suggest activities that can act to increase or decrease the local level of order in the array. The SPIRAL1 (SPR1) and SPR2 microtubule-localized proteins and the radial swollen 6 (rsw-6) locus are examples of new molecules and genes that affect both microtubule array organization and cell growth pattern. Functional tagging of cellulose synthase has now allowed the dynamic relationship between cortical microtubules and the cell-wall-synthesizing machinery to be visualized, providing direct evidence that cortical microtubules can organize cellulose synthase complexes and guide their movement through the plasma membrane as they create the cell wall.

Morlin (7-ethoxy-4-methyl chromen-2-one) was discovered in a screen of 20,000 compounds for small molecules that cause altered cell morphology resulting in swollen root phenotype in Arabidopsis. Live-cell imaging of fluorescently labeled cellulose synthase (CESA) and microtubules showed that morlin acts on the cortical microtubules and alters the movement of CESA. Morlin caused a novel syndrome of cytoskeletal defects, characterized by cortical array reorientation and compromised rates of both microtubule elongation and shrinking. Formation of shorter and more bundled microtubules and detachment from the cell membrane resulted when GFP::MAP4-MBP was used to visualize microtubules during morlin treatment. Cytoskeletal effects were accompanied by a reduction in the velocity and redistribution of CESA complexes labeled with YFP::CESA6 at the cell cortex. Morlin caused no inhibition of mouse myoblast, bacterial or fungal cell proliferation at concentrations that inhibit plant cell growth. By contrast, morlin stimulated microtubule disassembly in cultured hippocampal neurons but had no significant effect on cell viability. Thus, morlin appears to be a useful new probe of the mechanisms that regulate microtubule cortical array organization and its functional interaction with CESA.

Brassinosteroids (BRs) are essential hormones for plant growth and development. BRs regulate gene expression by inducing dephosphorylation of two key transcription factors, BZR1 and BZR2/BES1, through a signal transduction pathway that involves cell-surface receptors (BIRl1 and BAK1) and a GSK3 kinase (BIN2). How BR-regulated phosphorylation controls the activities of BZR1/BZR2 is not fully understood. Here, we show that BIN2-catalyzed phosphorylation of BZR1/BZR2 not only inhibits DNA binding, but also promotes binding to the 14-3-3 proteins. Mutations of a BIN2-phosphorylation site in BZR1 abolish 14-3-3 binding and lead to increased nuclear localization of BZR1 protein and enhanced BR responses in transgenic plants. Further, BR deficiency increases cytoplasmic localization, and BR treatment induces rapid nuclear localization of BZR1/BZR2. Thus, 14-3-3 binding is required for efficient inhibition of phosphorylated BR transcription factors, largely through cytoplasmic retention. This study demonstrates that multiple mechanisms are required for BR regulation of gene expression and plant growth.

Background: In premitotic plant cells, the future division plane is predicted by a cortical ring of microtubules and F-actin called the preprophase band (PPB). The PPB persists throughout prophase, but is disassembled upon nuclear-envelope breakdown as the mitotic spindle forms. Following nuclear division, a cytokinetic phragmoplast forms between the daughter nuclei and expands laterally to attach the new cell wall at the former PPB site. A variety of observations suggest that expanding phragmoplasts are actively guided to the former PPB site, but little is known about how plant cells "remember" this site after PPB disassembly.

In animals and yeast, CLASP proteins are microtubule plus-end tracking proteins (+TIPS) involved in the regulation of microtubule plus-end dynamics and stabilization. Here we show that mutations in the Arabidopsis CLASP homolog result in various plant growth reductions, cell form defects and reduced mitotic activity. Analysis of Arabidopsis plants that carry a YFP: AtCLASP fusion construct regulated by the AtCLASP native promoter showed similarities to the described localization of the animal CLASP proteins, but also prominent differences including punctate and preferential localization along cortical microtubules. Colocalization studies of YFP: AtCLASP and CFP:EB1b also showed that AtCLASP is enriched at the plus ends of microtubules where it localizes behind the AtEB1b protein. Moreover, AtCLASP overexpression causes abnormal cortical microtubule bundling and array organization. Cortical microtubule arrays have evolved to be prominent in plants, and our findings suggest that plant CLASP proteins may have adopted specific functions in regulating cortical microtubule properties and cell growth.

The gravitropism defective 2 (grv2) mutants of Arabidopsis thaliana were previously characterized as exhibiting shoot agravitropism resulting from mutations in a homolog of the Caenorhabditis elegans RECEPTOR-MEDIATED ENDOCYTOSIS-8 (RME-8) gene, which is required in C. elegans for endocytosis. A fluorescent protein fusion to the GRV2 protein localized to endosomes in transgenic plants, and vacuolar morphology was altered in grv2 mutants. A defect in vacuolar membrane dynamics provides a mechanistic explanation for the gravitropic defect, and may also account for the presence of an enlarged vacuole in early embryos, together with a nutrient requirement during seedling establishment. The GRV2-positive endosomes were sensitive to Wortmannin but not brefeldin A (BFA), consistent with GRV2 operating late in the endocytic pathway, prior to delivery of vesicles to the central vacuole. The specific enlargement of GRV2:YFP structures by Wortmannin, together with biochemical data showing that GRV2 co-fractionates with pre-vacuolar markers such as PEP12/SYP21, leads us to conclude that in plants GRV2/RME-8 functions in vesicle trafficking from the multivesicular body/pre-vacuolar compartment to the lytic vacuole.

Phototropin 1 (phot1) is a photoreceptor for phototropism, chloroplast movement, stomatal opening, leaf expansion, and solar tracking in response to blue light. Following earlier work with PHOT1::GFP (Sakamoto and Briggs, 2002), we investigated the pattern of cellular and subcellular localization of phot1 in 3 - 4 d old etiolated seedlings of Arabidopsis thalinana. As expressed from native upstream sequences, the PHOT1:: GFP fusion protein is expressed strongly in the abaxial tissues of the cotyledons and in the elongating regions of the hypocotyl. It is moderately expressed in the shoot/root transition zone and in cells near the root apex. A fluorescence signal is undetectable in the root epidermis, root cap, and root apical meristem itself. The plasma membranes of mesophyll cells near the cotyledon margin appear labeled uniformly but cross-walls created by recent cell divisions are more strongly labeled. The pattern of labeling of individual cell types varies with cell type and developmental stage. Blue-light treatment causes PHOT1:: GFP, initially relatively evenly distributed at the plasma membrane, to become reorganized into a distinct mosaic with strongly labeled punctate areas and other areas completely devoid of fluorescence - a phenomenon best observed in cortical cells in the hypocotyl elongation region. Concomitant with or following this reorganization, PHOT1:: GFP moves into the cytoplasm in all cell types investigated except for guard cells. It disappears from the cytoplasm by an unidentified mechanism after several hours in darkness. Neither its appearance in the cytoplasm nor its eventual disappearance in darkness is prevented by the translation inhibitor cycloheximide, although the latter process is retarded. We hypothesize that blue-light-induced phot1 relocalization modulates blue-light-activated signal transduction.

Live cell imaging and genetic studies are demonstrating that cortical microtubule arrays in plant cells are dynamic structures in which microtubule (MT) bundles play a key role in creating array organization and function. Steps important for creating and organizing these arrays include recruitment of nucleation complexes to the cell cortex and to the lattices of previously established MTs, association of newly created MTs to the cell cortex, release of MTs from sites of nucleation, transport of released MTs by polymer treadmilling, and subsequent interactions between treadmilling MTs. The results of MT interactions include induced catastrophe, severing, and the capture and reorientation of growing polymer ends by bundling interactions. Together, these properties predict a capacity for self-ordering that is likely to play an important role in establishing the parallel organization of the arrays.