The Actin Cytoskeleton

by Elison B. Blancaflor

Plant Biology Division, The Samuel Roberts Noble Foundation, Ardmore, Oklahoma, USA.

F-actin
The actin cytoskeleton plays a critical role in plant development by regulating a number of fundamental cellular processes including cell division, cell expansion, organelle motility and vesicle trafficking [1,2]. To function properly, the dynamics and organization of the actin cytoskeleton is mediated by a multitude of accessory proteins that are responsible for specifying whether cellular actin exists in the filamentous (F-actin) or monomeric, globular form (G-actin). Furthermore these accessory proteins are involved in actin nucleation, cross-linking of individual actin filaments to form actin bundles and facilitating actin interactions with other cellular components [2,3,4]. It is partly through this tight spatial and temporal control of actin organization that plant cells are able to achieve their highly polarized growth patterns and diverse shapes.
Fluorescent methods to visualize actin organization have contributed substantially to understanding actin function and organization during plant development. Prior to the discovery of fluorescent proteins (FPs), imaging of actin in plant cells at the light microscopic level has relied on fluorescently labeled phalloidin, a toxin from the basiodiomycete fungus Amanita phalloides that binds to F-actin and indirect immunofluorescence using actin specific antibodies. Although improvements in fixation and sample preparation methods continue to provide invaluable tools for studies on the plant actin cytoskeleton [5,6], these approaches are limited in terms of their ability to depict the intricate dynamics characteristic of actin in living cells. As with other cellular structures, studies on actin in plants has benefited significantly from the introduction of FPs. The first reported use of FPs to visualize actin organization in living plants was in 1998 where GFP was fused to the actin binding domain of mouse talin (GFP-mTalin; [7]). In addition to talin, GFP fusions to plant actin regulatory proteins have been demonstrated to bind F-actin in vivo. For example, translational GFP fusions to the headpiece of Arabidopsis villin decorated filamentous structures in suspension cells and seedlings [8]. Moreover, GFP linked to actin depolymerizing factor (ADF) localized to distinct actin cables in pollen tubes [9]. Despite the availability of GFP-ADF and GFP-Villin, the GFP-mTalin construct was the most widely used live reporter for documenting F-actin organization in plants at least until GFP fusions to the actin binding domain 2 (ABD2) of Arabidopsis fimbrin 1 were introduced (GFP-ABD2; [10,11,12,13]. In addition to Arabidopsis, GFP-ABD2 was used to image changes in F-actin organization in root hairs [14] and in response to colonization by mycorrhizal fungi in Medicago truncatula hairy roots [15]. Moreover, stable expression of GFP-ABD2 revealed F-actin-dependent vacuolar dynamics and the existence of novel mitotic structures in tobacco Bright Yellow-2 cells [16,17]. Recently, a new variant of GFP-ABD2 was described that exhibited a dramatic increase in fluorescence when stably expressed in Arabidopsis plants. This modification to the GFP-ABD2 probe involved the simple addition of GFP molecules to both the C and N termini of ABD2 [18].
Although, live F-actin reporters based on FPs have rapidly gained popularity, it is important to be mindful of potential problems and artifacts resulting from the over-expression of these side binding cytoskeletal reporters. For instance, in a study of pollen tube actin organization, GFP-Talin, GFP-ADF and GFP-ABD2 were compared with rapidly frozen pollen tubes and each of the FP reporters produced different actin labeling patterns from that of optimally fixed material [19]. Furthermore, Arabidopsis plants harboring the GFP-Talin construct exhibited defects in plant morphology possibly through interfering with the activity of other actin regulatory proteins [20] while expression of ABD2 with GFP tags on both the N and C termini (GFP-ABD2-GFP) exhibited subtle defects in cell morphology [18]. Recently, GFP-Talin expressing lines affected the efficiency of auxin transport while organelle motility in some GFP-ABD2 lines were found to be slightly inhibited [21]. Given the pivotal role that the cytoskeleton plays in defining plant morphology, the problems outlined above are not that surprising. However such observations highlight the need to develop additional fluorescent actin reporters and to use the currently available actin FP reporters with caution.

Recently a green to red photo-convertible probe, mEosFp::FABD-mTn has been reported [26]. This new addition introduces a high degree of precision in labelling F-actin locally for following actin dynamics and interactions with microtubules and other organelles.

mEosFPmTn
Fluorescent protein probes for the actin cytoskeleton
 FP probes for Actin

References

 

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2. Hussey, P.J., Ketelaar, T. and Deeks, M.J. (2006) Control of actin cytoskeleton in plant cell growth. Annu. Rev. Plant Biol. 57, 109-125.

3. Blancaflor, E.B., Wang, Y.-S. and Motes, C.M. (2006) Organization and function of the actin cytoskeleton in developing root cells. Int. Rev. Cytol. 252, 219-264.

4. Staiger, C.J. and Blanchoin, L. (2006) Actin dynamics: old friends with new stories. Curr. Opin. Plant Biol. 9, 554-562.

5. 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.

6. Lovy-Wheeler, A., Wilsen, K.L., Baskin, T.I. and Hepler, P.K. (2005) Enhanced fixation reveals the apical cortical fringe of actin filaments as a consistent feature of the pollen tube. Planta, 221, 95-104.

7. Kost, B., Spielhofer, P. and Chua, N.H. (1998) A GFP-mouse talin fusion protein labels plant actin filaments in vivo and visualizes the actin cytoskeleton in growing pollen tubes. Plant J. 16, 393–401.

8. Klahre U, Friederich E, Kost B, Louvard D, Chua N-H (2000) Villin-Like Actin-Binding Proteins Are Expressed Ubiquitously in Arabidopsis. Plant Physiology 122: 35-48.

9. Chen CY, Wong EI, Vidali L, Estavillo A, Hepler PK, Wu H-m, Cheung AY (2002) The Regulation of Actin Organization by Actin-Depolymerizing Factor in Elongating Pollen Tubes. Plant Cell 14:2175-2190.

10. Sheahan MB, Rose RJ, McCurdy DW (2004) Organelle inheritance in plant cell division: the actin cytoskeleton is required for unbiased inheritance of chloroplasts, mitochondria and endoplasmic reticulum in dividing protoplasts. Plant J. 37: 379-390.

11. Sheahan, M.B., Staiger, C.J., Rose, R.J. and McCurdy, D.W. (2004). A green fluorescent protein fusion to actin-binding domain 2 of Arabidopsis fimbrin highlights new features of a dynamic actin cytoskeleton in live plant cells. Plant Physiol. 136, 3968-3978.

12. Voigt, B., Timmers, A.C.J., Samaj, J., Muller, J., Balu_ka, F. and Menzel, D. (2005) GFP-ABD2 fusion construct allows in vivo visualization of the dynamic actin cytoskeleton in all cells of Arabidopsis seedlings. Eur. J. Cell Biol. 84, 595-608.

13. Wang, Y.-S., Motes, C.M., Mohamalawari, D.R. and Blancaflor, E.B. (2004) Green fluorescent protein fusions to Arabidopsis fimbrin 1 for spatio-temporal imaging of F-actin dynamics in roots. Cell Motil. Cytoskelet. 59, 79-93.

14. Timmers, A.C., Vallotton, P., Heym, C., Menzel, D. (2007) Microtubule dynamics in root hairs of Medicago truncatula. Eur. J. Cell Biol. 86, 69-83.

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16. Sano, T., Higaki, T., Oda, Y., Hayashi, T. and Hasezawa, S. (2005). Appearance of actin microfilament ' twin peaks' in mitosis and their function in cell plate formation, as visualized in tobacco BY-2 cells expressing GFP-fimbrin. Plant J. 44, 595-605.

17. Higaki, T., Kutsuna, N., Okubo, E., Sano, T. and Hasezawa, S. (2006) Actin microfilaments regulate vacuolar structures and dynamics: dual observation of actin microfilaments and vacuolar membrane in living tobacco BY-2 Cells. Plant Cell Physiol. 47, 839-852.

18. Wang Y-S, Yoo C-M, Blancaflor EB (2007) Improved imaging of actin filaments in transgenic Arabidopsis plants expressing a green fluorescent protein fusion to the C and N termini of the fimbrin actin binding domain 2. New Phytologist (in press)

19. Wilsen, K.L., Lovy-Wheeler, A., Voigt, B., Menzel, D., Kunkel, J.G. and Hepler, P.K. (2006) Imaging the actin cytoskeleton in growing pollen tubes. Sex Plant Reprod. 19, 51-62.

20. Ketelaar, T., Anthony, R.G. and Hussey, P.J. (2004) Green fluorescent protein-mTalin causes defects in actin organization and cell expansion in Arabidopsis and inhibits actin depolymerizing factor's actin depolymerizing activity in vitro. Plant Physiol. 136, 3990-3998.

21. Holweg, C.L. (2007) Living markers for actin block myosin-dependent motility of plant organelles and auxin. Cell Motil. Cytoskeleton, 64, 69-81.

22. Finka, A., Schaefer, D.G., Saidi Y., Goloubinoff, P. and Zryd, J.-P. (2007) In vivo visualization of F-actin structures during the development of the moss Physcomitrella patens. New Phytol. 174, 63-76.

23. Takemoto, D., Jones, DA. and Hardham, A.R. (2003) GFP-tagging of cell components reveals the dynamics of subcellular re-organization in response to infection of Arabidopsis by oomycete pathogens. Plant J. 33, 775-92.

24. Motes, C.M., Pechter, P., Yoo, C.-M., Wang, Y.-S., Chapman, K.D. and Blancaflor, E.B. (2005). Differential effects of two phospholipase D inhibitors, 1-butanol and N-acylethanolamine, on in vivo cytoskeletal organization and Arabidopsis seedling growth. Protoplasma, 226, 109-123.

25. Liu, J.-Z., Blancaflor, E.B. and Nelson, R.S. (2005) The tobacco mosaic virus 126 kD protein, a constituent of the virus replication complex, alone or within the complex aligns with and traffics along microfilaments. Plant Physiol. 138, 1853-1865.

26. Schenkel, M. , Sinclair, A.M. , Johnstone, D. , Bewley, J.D.  and Mathur, J. (2008) Visualizing the actin cytoskeleton in living plant cells using a photo-convertible mEos::FABD-mTn fluorescent fusion protein. Plant Methods. 4.21.