Summary
In plant cells the cell wall is a formidable obstacle in many physiological studies such as patch-clamp measurements and cell labelling with antibodies. Enzymatic digestion of the cell wall, in order to release a protoplast, has a number of disadvantages; therefore we worked out an alternative method to gain access to the plasma membrane. The wall of specialized cells from three higher plant species and one unicellular alga were perforated using the focussed UV light of a nitrogen laser. In order to enhance the absorption of the UV light by the walls, a dye was used that binds specifically to cell wall components. Extrusion of the protoplast or parts thereof was controlled by a regulated gradual decrease of the osmolarity of the solution surrounding the cells. Cytoplasmic streaming and chloroplast circulation were maintained in the protoplasts, demonstrating their viability after the wall perforation with the laser. Continuous deposition of new cell wall material by the polar tip of pollen tubes after surgical removal of the wall at the tip is another demonstration of the viability of the cells. Formation of high resistance seals between the plasma membrane and a patch pipet was surprisingly difficult. The role of ‘Hechtian strands’ and continuing synthesis of cell wall material in seal formation is further investigated. Other applications for the surgical laser are: fusion of two cells or vacuoles, analysis of the composition of specific parts of the cell wall, and release of the vacuole from an identified cell type for patchclamp studies.
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Abbreviations
- CFW:
-
calcofluor white
- PM:
-
plasma membrane
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De Boer, A.H., Van Duijn, B., Giesberg, P. et al. Laser microsurgery: a versatile tool in plant (electro) physiology. Protoplasma 178, 1–10 (1994). https://doi.org/10.1007/BF01404115
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DOI: https://doi.org/10.1007/BF01404115