Interaction and cross-talk between microtubules and actin microfilaments are important for numerous processes during plant growth and development, including the control of cell elongation and tissue expansion, but little is known about the molecular components of this interaction. Plant kinesins with the calponin-homology domain (KCH) were recently identified and associated with a putative role in microtubule-microfilament cross-linking. The putative biological role of the rice KCH member OsKCH1 is addressed here using a combined approach with Tos17 kch1 knock-out mutants on the one hand, and a KCH1 overexpression line generated in tobacco BY-2 cells. It is shown that OsKCH1 is expressed in a development and tissue-specific manner in rice and antagonistic cell elongation and division phenotypes as a result of knock-down and overexpression are reported. Further, the dynamic repartitioning of OsKCH1 during the cell cycle is described and it is demonstrated that KCH overexpression delays nuclear positioning and mitosis in BY-2 cells. These findings are discussed with respect to a putative role of KCHs as linkers between actin filaments and microtubules during nuclear positioning.

Interaction and cross-talk between microtubules and actin microfilaments are important for the cell axis and polarity during plant cell growth and development, but little is known about the molecular components of this interaction. Plant kinesins with a calponin-homology domain (KCHs) were recently identified and associated with a putative role in microtubule-microfilament cross-linking. KCHs belong to a distinct branch of the minus end-directed kinesin subfamily and so far have only been identified in land plants including the mosses. Here we report the identification of a new KCH from rice (Oryza sativa), OsKCH1, and show that OsKCH1 is associated with cortical microtubules and actin microfilaments in vivo. Furthermore, OsKCH1 is shown to bind to micro-tubules and actin microfilaments in vitro in a domain-dependent way. Additionally, this unique type of kinesin is shown to oligomerize both in vivo and in vitro. These findings are discussed with respect to a general role for KCHs as linkers between actin filaments and microtubules in both cell elongation and division.

Abbreviations: BY-2 Nicotiana tabacum L. cv. Bright Yellow 2, CPP cell-permeating peptide, FITC fluorescein isothiocyanate, QDs Fluorescent semiconductor quantum dots Fluorescence microscopy has developed into a key technology of the postgenomic era in biology, because it combines structural information with molecular specificity. However, the resolution of this approach is limited by bleaching and optical cross-reference of the fluorescent labels. Fluorescent semiconductor quantum dots (QDs) provide excellent bleaching stability and tunable emission spectra, and therefore would be an excellent alternative to overcome these limitations. However, to apply them to cell biology, three challenges have to be met: bioconjugation to molecular probes that confer the specificity of the label, passage through the external barriers of the cell, and suppression of toxic side effects of the nanoparticles. In plant cells that are ensheathed by a cellulosic cell wall, these challenges are especially prominent. Moreover, plants are located at the start of the food chain and thus of high relevance for the ecotoxicological assessment of nanomaterials. We have therefore explored the application of nanoparticles to plant cell biology. We have first evaluated different strategies to visualize microtubules by QDs in vitro and in cellula. By using silica-coated QDs coupled to anti-tubulin antibodies we were able to image microtubules in tobacco BY-2 cells by direct immunofluorescence making use of the superior bleaching stability of the nanoparticle label. To adapt this tool for in vivo imaging, we have successfully employed Trojan Peptoids as vehicles into living tobacco cells. We want to extend this strategy not only to use functionalized nanoparticles for life-cell imaging, but also to adapt them as tool to manipulate intracellular architecture.B406

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