Dr David Collings - Research
1) Interactions between actin microfilaments and microtubules in the co-ordination of regulated cell expansion and elongation in the roots of Arabidopsis thaliana.
Plant cells contain a cytoskeleton comprising dynamic arrays of microfilaments and microtubules.
In interphase cells, roles for microtubules in controlling cell wall organisation and the direction of cell expansion, and hence plant morphogenesis, have long been recognised.
Directed cell elongation, which drives the development of plants tissues, is controlled by the cell wall containing transversely-oriented cellulose microfibrils that constrain its radial expansion. Transversely-aligned cortical microtubules lie adjacent to the plasma membrane and direct the deposition of cellulose microfibrils in the cell wall.
In interphase cells, plant microfilaments form several distinct arrays including large subcortical microfilament bundles that drive cytoplasmic streaming and a cortical array associated with the plasma membrane. Microfilaments, and especially the cortical array, are dynamic showing rapid polymerisation and depolymerisation.
Fluorescently-labelled or tagged cortical microfilaments, lying parallel to microtubules, have been reported in fixed and living cells. Many electron micrographs also show microfilaments running along the sides of and cross-bridged to cortical microtubules. These microtubule-associated microfilaments seem ubiquitous in plants, occurring in many different species and cell types, including in the elongating cells of roots.
Despite this extensive literature on microtubule-associated microfilaments, little research has been conducted on the proteins that mediate these direct interactions between the microfilaments and microtubules.
Figure 1. The mor1 Arabidopsis mutant, which encodes a defective microtubule associated protein, is temperature-sensitive (it shows a phenotype at 29oC but not at 21oC). The effects of cytoskeletal disruption on root growth were quantified by measurements of the diameter of the root near the tip, with disruption causing the root to swell. At the restrictive temperature, mor1 is hypersensitive to concentrations of the microtubule disrupting drug oryzalin that do not affect wild-type plants nor the mor1 mutant at its permissive temperature. Similarly, mor1 is also hypersensitised to the actin microfilament disrupting drug latrunculin, thus demonstrating that there is some interplay between the microtubules and microfilaments. Data is from Collings et al. (2006).
Experiments demonstrate that microfilaments and microtubules interact in plant cells contributing to the control of cell elongation and tissue expansion (Collings and Allen 2000, Collings 2007). For example, drugs targeting microtubules can cause microfilaments to undergo re-organisation (Collings and Wasteneys 2005) while disruption of microfilaments in Arabidopsis roots with the drug latrunculin inhibits elongation and induces expansion, with plants with disrupted microtubules being hypersensitised to the effects of latrunculin (Collings et al. 2006) (Fig. 1). My future research will use this latrunculin hypersensitivity response to investigate how microfilaments and microtubules interact and control cell expansion.
Proposed research and importance:
Numerous research strategies are planned for further investigations of the role of the cytoskeleton in cell expansion and root growth. These projects combine cell biology (confocal and electron microscopy), molecular biology and biochemistry. By answering these questions, this project will greatly enhance understanding of the organisation and functions of microfilaments in plants, and contribute to a greater understanding of how plant growth is controlled and controllable. This research might be applicable to industry, as the control of cell expansion and elongation are fundamental to the formation of wood.
1) Screening for microfilament-related cell expansion mutants using a semi-automated version of the latrunculin hypersensitivity assay. A trial run of this screen identified a novel microtubule-related mutant from a pool of root swelling mutants. This mutant was mapped to chromosome V and identified as β-tubulin-3, a component of microtubules. This further reinforces that interactions exist between the microtubules and microfilaments. A further mutant has been identified that is hypersensitive to microfilament disruption but which does not appear to be related to the microtubule cytoskeleton. The identity of this mutant remains unknown.
To screen for further latrunculin-hypersensitive mutants, EMS mutagenised seed will be assayed. Putative mutants will be grown and their seed collected for re-screening and further phenotypic analysis. Mutants that show consistent phenotypes related to microfilament disruption will then be identified by gene mapping. This screen should reveal numerous new mutations that modulate the microfilament cytoskeleton in Arabidopsis roots.
2) Analysis of genes up- and down-regulated during latrunculin hypersensitivity. During the latrunculin-hypersensitivity response, genes will be up- and down-regulated. Although many of these may be related to physical growth responses (wall formation etc), some genes may be cytoskeletal. A microarray analysis has already been run in which several cytoskeletal genes were identified as being down-regulated, including two that are potential linkers between the microtubule and microfilament cytoskeletons. These nature of these genes, and the proteins that they encode, requires further investigation. T-DNA knockout lines of these genes will be assayed for drug hypersensitivity and their cytoskeleton investigated by immunofluorescence and through crossing to microfilament- and microtubule-tagged GFP reporter lines.
3) Observations of microtubule-associated microfilaments. From the 1970s through to the 1990s, numerous papers used to electron microscopy to report the existence of microtubule-associated filaments. The diameter and structure of these filaments are consistent with them being actin microfilaments but although there is some immunological evidence (Lancelle and Hepler 1991), this has not been confirmed. A return to conventional transmission electron microscopy, and further characterisation of these structures in Arabidopsis roots and other plant tissues is required. Ideally, these characterisations should be directly linked to observations by field emission scanning electron microscopy (FESEM), to immunofluorescence, and to and GFP imaging in living tissues.
4) Actin knockout mutants. Although numerous mutants have been identified in plants tubulin genes (Ishida et al 2007), mutations modifying plant actin genes are limited to T-DNA knockouts. Of the three different actins expressed in vegetative (non-reproductive) tissues in higher plants (actin2, actin7 and actin8), knockouts are currently available for actin2 and actin7. Initial characterisations show that while act2 is hypersensitive to latrunculin, act7 has a reduced sensitivity. further characterisation is necessary, and the role of these different protein isoforms in cell elongation needs to be determined.
References:
Collings,D.A. (2007) Crossed-wires: Interactions, cross-talk and signal exchange between the microtubule and microfilament networks in plants. In Plant Microtubules – Development and Flexibility, P.Nick, ed (Berlin: Springer-Verlag), in press.
Collings,D.A. and Allen,N.S. (2000) Cortical actin interacts with the plasma membrane and microtubules. In Actin: A Dynamic Framework for Multiple Plant Cell Functions, C.J.Staiger, F.Baluška, D.Volkmann, and P.W.Barlow, eds (Dordrecht: Kluwer Academic), pp. 145-163.
Collings,D.A., Lill,A.W., Himmelspach,R., and Wasteneys,G.O. (2006) Drug sensitisation studies show actin microfilaments and microtubules interact during root elongation in Arabidopsis thaliana. New Phytol. 170:275-290.
Collings,D.A. and Wasteneys,G.O. (2005) Actin microfilament and microtubule distribution patterns in the expanding root of Arabidopsis thaliana. Can.J.Bot. 83:579-590.
Ishida,T., Kaneko,Y., Iwano,M. and Hashimoto, T. (2007) Helical microtubule arrays in a collection of twisting mutants of Arabidopsis thaliana. Proc.Nat.Acad.Sci. 104: 8544-8549.
Lancelle,S.A. and Hepler,P.K. (1991) Association of actin with cortical microtubules revealed by immunogold localization in Nicotiana pollen tubes. Protoplasma 165:167-172.