Use of a fluorescent membrane probe to identify zooxanthellae in hospite among dissociated endoderm cell culture from coral

Abstract

Preparation of homogeneous endoderm cells and culture is a prerequisite to understanding the cellular and molecular mechanism of endosymbiosis in the cnidarian-dinoflagellate association. During the cell isolation from the stony coral Euphyllia glabrescens, various amounts of symbiotic endoderm cells were found to release their symbionts (Symbiodinium spp., or zooxanthellae in generic usage) into the culture. Due to the bulky occupation by zooxanthellae inside the endoderm cell, the symbiotic endoderm cells, or zooxanthellae in hospite, are difficult to be distinguished from released zooxanthellae by microscopic examination. We now report a method for this identification using a fluorescent analogue of sphingomyelin, N-[5-(5,7-dimethyl boron dipyrromethene difluoride)-1-pentanoyl]-D-erythro-sphingosylphosphorylcholine (C(5)-DMB-SM). Incubation of symbiotic endoderm cells with C(5)-DMB-SM-defatted bovine serum albumin (DF-BSA) complex results in bright fluorescent membrane staining. Nevertheless, the membrane staining of free-living or released zooxanthellae by this complex is significantly decreased or even diminished. This method has provided a fast and reliable assay to identify symbiotic endoderm cells and will greatly accelerate the progress of endosymbiosis research.

Increased susceptibility of algal symbionts to photo-inhibition resulting from the perturbation of coral gastrodermal membrane trafficking

“…To focus on these specific cells, the current study prepared large quantities of homogeneous "symbiotic gastrodermal cells" (SGCs). The process of isolating SGCs followed a previous report that performed cell dissociation from inside-out tentacles of E. glabrescens [15]. Figure 1 shows differences in membrane structures between SGCs ( Figure 1AD) and cultured Symbiodinium (i.e., free-living clade C1 Symbiodinium; Figure 1A′D′).…”

“…Colonies were immediately transferred in seawater-filled coolers to Taiwan's National Museum of Marine Biology and Aquarium, placed in an upright position in an outdoor aquarium with flowthrough seawater (exchange rate ~2% per hour), and left to recover before use. The "inside-out" manipulation of amputated tentacles and isolation of symbiotic gastrodermal cells (SGCs) were performed according to a published procedure [15]. Intactness of the gastrodermal cell membranes in the SGCs was then examined sensu [15].…”

“…The "inside-out" manipulation of amputated tentacles and isolation of symbiotic gastrodermal cells (SGCs) were performed according to a published procedure [15]. Intactness of the gastrodermal cell membranes in the SGCs was then examined sensu [15]. Cells were suspended in filtered seawater (FSW) at a final concentration of ~1×10 6 2×10 6 mL 1 and were immediately used for experiments.…”

“…The band-pass filter collected oxidized DCF fluorescence, while the long-pass filter collected fluorescence of both DCF and Symbiodinium chlorophyll in the SGC. As a consequence, the fluorescent intensity (gray level/pixel area) of oxidized DCF, representing the cellular ROS levels, was then quantified using the long-pass FITC image as a mask with quantitative imaging processing software (Metamorph Image Processing System; Molecular Devices, Downingtown, PA, USA) as previously described [15,18]. This procedure was repeated on images collected from four to five different cell populations in the same irradiation treatment.…”

“…Incubation of SGCs with the BODIPY-SM/DF-BSA complex solution enables incorporation of the BODIPY-SM into the outer leaflet of the SGC plasma membrane [15]. Breakage or permeability changes in the plasma membrane will result in the diffusion of this lipid analogue into the cytosol.…”