Chapter 58 Fluorescence Labeling of Flagellar Membranes

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Abstract

Lipophilic fluorescent dyes are used for examining cell structure and investigating the structure and function of specific organelles. One of these dyes, 3, 3′-dihexyloxacarbocyanide iodide [DiOC6(3)] is used to investigate the properties of the endoplasmic reticulum in living and fixed cells. DiOC6(3) diffuses through the plasma membrane and enters cell membranes, but in living cells the dye becomes concentrated within specific organelles. The concentration of DiOC6(3) determines the organelles who appear stained. This selectivity of DiOC6(3) staining reflects accumulation of the positively charged dye molecules in subcellular membranes that have negative membrane potential. The sensitivity of DiOC6(3) accumulations to membrane potential is used to measure plasma membrane potential in mammalian cells. When Chlamydomonas reinhardtii cells are exposed to low concentrations of DiOC6(3), flagella rather than mitochondria are specifically stained. Staining of mitochondria is observed only when the DiOC6(3) concentrations is increased, and even then, the flagella continue to stain. At the highest DiOC6(3) concentrations tested, other cellular membranes including the chloroplast membrane, also became labeled. If the staining characteristics of DiOC6(3)reflect its membrane potential-sensitive partitioning into membranes, flagellar ion pumps are to maintain the higher negative membrane potential across the flagellar membranes than that of the rest of the plasma membrane, or even that of mitochondria. It is conceivable that this membrane potential may have functional roles in controlling flagellar length, autotomy, or aspects of flagellar beating. There is description of medium and culture conditions, preparation and storage of DiOC6(3), staining of Chlamydomonas with DiOC6(3), microscopy, staining patterns and prospects. Long-chain dyes, once incorporated into a membrane, the dye is retained within that membrane. DiOC6(3) staining may be revealing physiologically relevant information concerning flagellar membrane potential. Use of an emission filter that blockslight from 555 to 1.2 nm eliminates chlorophyll autofluorescence, a necessity photography on a standard light microscope.

Original languageEnglish (US)
Pages (from-to)413-418
Number of pages6
JournalMethods in cell biology
Volume47
Issue numberC
DOIs
StatePublished - Jan 1 1995

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