Hostname: page-component-78c5997874-mlc7c Total loading time: 0 Render date: 2024-11-10T14:05:51.016Z Has data issue: false hasContentIssue false
  • English
  • Français

Development of a live cell imaging method for mitochondria in Trebouxia photobionts

Published online by Cambridge University Press:  30 May 2017

Mark R. BRICKLEY
Mark R. BRICKLEY*
Affiliation:
Biovital Research, Unit 7, Camelot Court, Somerton Business Park, Somerton, Somerset TA11 6NP, UK. Email: mbrickley@mac.com
Get access Rights & Permissions [Opens in a new window]

Abstract

The Trebouxia photobiont freshly isolated from Xanthoria parietina (L.) Th. Fr. was used to develop a live cell chondriome (mitochondrial DNA) labelling method. In the initial phase six candidate dyes were tested and compared for mitochondrial labelling utility as assessed by the signal to noise ratio (SNR) of the mitochondrial signal to the adjacent cellular background in standardized confocal images of 30 labelled cells. DIOC7, JC-1 and MitoTracker orange (MTO) dyes showed some labelling ability. MTO had significantly higher utility than the other dyes. In a second phase, MTO concentration was optimized. The final labelling protocol was a 30 minute incubation with 1 μM of MTO. The resultant labelling was suitable for both widefield and confocal microscopy. Both 2D thresholding and 3D volume construction are demonstrated using the resultant data. The protocol can therefore be utilized for both qualitative research and for quantitative measurement of the chondriome in Trebouxia photobionts. This will facilitate a wide range of mitochondrial investigations in lichenology.

Keywords

chondriomedyeslichensmicroscopyMitoTracker orangephotobiont
Type
Articles
Information
The Lichenologist , Volume 49 , Issue 3 , May 2017 , pp. 275 - 286
Copyright
© British Lichen Society, 2017 

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

References

Ascaso, C., Brown, D. H. & Rapsch, S. (1985) Ultrastructural studies of desiccated lichens. In Lichen Physiology and Cell Biology (D. H. Brown, ed.): 259–274. New York: Plenum Press.CrossRefGoogle Scholar
Brickley, M. (2012) Mitochondria in freshwater algae. Ph.D. thesis, Harper Adams University College.Google Scholar
Brickley, M. R., Weise, V., Hawes, C. & Cobb, A. (2010) Morphology and dynamics of mitochondria in Mougeotia . European Journal of Phycology 45: 258–266.CrossRefGoogle Scholar
Brickley, M. R., Cobb, A. H. & Lawrie, E. (2012) Use of a potentiometric vital dye to determine the effect of the herbicide bromoxynil octanoate on mitochondrial bioenergetics in Chlamydomonas rheinhardtii . Pest Management Science 68: 580–586.Google Scholar
Casano, L. M., Del Campo, E. M., Garcia-Breijo, F. J., Reig-Arminana, J., Gasulla, F., Del Hoyo, A., Guera, A. & Barreno, E. (2011) Two Trebouxia algae with different physiological performances are ever-present in lichen thalli of Ramalina farinacea. Coexistence versus competition? Environmental Microbiology 13: 806–818.Google Scholar
Chung, P. J., Kim, Y. S., Park, S. H., Nahm, B. H. & Kim, J. K. (2009) Subcellular localization of rice histone deacetylases in organelles. FEBS Letters 583: 2249–2254.Google Scholar
De la Torre, R., Sancho, L. G., Horneck, G., Ríos, A. D. L., Wierzchos, J., Olsson-Francis, K., Cockell, C. S., Rettberg, P., Berger, T., De Vera, J.-P. et al. (2010) Survival of lichens and bacteria exposed to outer space conditions – Results of the Lithopanspermia experiments. Icarus 208: 735–748.Google Scholar
Ehara, T., Tesuaki, O. & Eiji, H. (1995) Behaviour of mitochondria in sychronized cells of Chlamydomonas rheinhardtii (Chlorophyta). Journal of Cell Science 108: 499–507.Google Scholar
Ettl, H. & Gärtner, G. (1984) About the significance of cytology in the taxonomy of algae, demonstrated in Trebouxia (Chlorellales, Chlorophyceae). Plant Systematics and Evolution 148: 135–147.Google Scholar
Fricker, M. (2001) Fluorescent probes for living plant cells. In Plant Cell Biology: Practical Approach Series (C. Hawes & B. Satiat-Jeunmaitre, eds): 35–84. Oxford, UK: Oxford University Press.Google Scholar
Friedel, T. & Budel, B. (2008) Photobionts. In Lichen Biology (T. H. Nash III, ed.): 19–26. Cambridge, UK: Cambridge University Press.Google Scholar
Gasulla, F., De Nova, P. G., Esteban-Carrasco, A., Zapata, J. M., Barreno, E. & Guera, A. (2009) Dehydration rate and time of desiccation affect recovery of the lichenic algae Trebouxia erici: alternative and classical protective mechanisms. Planta 231: 195–208.Google Scholar
Green, T. G. A., Nash, T. H. & Lange, O. L. (2008) Physiological ecology of carbon dioxide exchange. In Lichen Biology (T. H. Nash III, ed.): 152–181. Cambridge, UK: Cambridge University Press.Google Scholar
Honegger, R. (2003) The impact of different long-term storage conditions on the viability of lichen-forming ascomycetes and their green algal photobiont, Trebouxia spp. Plant Biology 5: 324–330.Google Scholar
Jacobs, J. B. & Ahmadjian, V. (1969) The ultrastructure of lichens. I. A general survey. Journal of Phycology 5: 227–240.Google Scholar
Kranner, I., Beckett, R., Hochman, A. & Nash, T. H. III (2008) Desiccation tolerance in lichens: a review. Bryologist 111: 576–593.Google Scholar
Liu, Z., Bushnell, W. R. & Brambl, R. (1987) Potentiometric cyanine dyes are sensitive probes for mitochondria in intact plant cells. Plant Physiology 84: 1385–1390.Google Scholar
McNally, J. G., Karpova, T., Cooper, J. & Conchello, J. A. (1999) Three-dimensional imaging by deconvolution microscopy. Methods 19: 373–385.Google Scholar
Muggia, L., Grube, M. & Tretiach, M. (2008) A combined molecular and morphological approach to species delimitation in black-fruited, endolithic Caloplaca: high genetic and low morphological diversity. Mycological Research 112: 36–49.CrossRefGoogle ScholarPubMed
Nash, T. H. III (Ed.) (2008) Lichen Biology. Cambridge, UK: Cambridge University Press.Google Scholar
Nyati, S., Werth, S. & Honegger, R. (2013) Genetic diversity of sterile cultured Trebouxia photobionts associated with the lichen-forming fungus Xanthoria parietina visualized with RAPD-PCR fingerprinting techniques. Lichenologist 45: 825–840.Google Scholar
Poot, M., Zhang, Y. & Kramer, J. (1996) Analysis of mitochondrial morphology and function with novel fixable fluorescent stains. Journal of Histochemistry and Cytochemistry 44: 1363–1372.Google Scholar
Schindelin, J., Arganda-Carreras, I., Frise, E., Kaynig, V., Longair, M., Pietzsch, T., Preibisch, S., Rueden, C., Saalfeld, S., Schmid, B. et al. (2012) Fiji: an open-source platform for biological-image analysis. Nature Methods 9: 676–682.Google Scholar
Škaloud, P. & Peksa, O. (2008) Comparative study of chloroplast morphology and ontogeny in Asterochloris (Trebouxiophyceae, Chlorophyta). Biologia 63: 873–880.Google Scholar
Vannini, G. L., Pancaldi, S., Poli, E. & Easulo, M. P. (1988) Rhodamine 123 as a vital stain for mitochondria of plant cells. Plant, Cell and Environment 11: 123–127.CrossRefGoogle Scholar
Vonesch, C. & Cristofani, R. T. (2014) Deconvolution lab. Available at: http://bigwww.epfl.ch/algorithms/deconvolutionlab/ [Accessed 28/7/2016].Google Scholar
Zandt, B.-J., Liu, J. H., Veruki, M. L. & Hartveit, E. (2017) AII amacrine cells: quantitative reconstruction and morphometric analysis of electrophysiologically identified cells in live rat retinal slices imaged with multi-photon excitation microscopy. Brain Structure and Function 222: 151–182.Google Scholar