Autofluorescence mediated red spherulocyte sorting provides insights into the source of spinochromes in sea urchins

Abstract

Red spherule cells (RSCs) are considered one of the prime immune cells of sea urchins, but their detailed biological role during immune responses is not well elucidated. Lack of pure populations accounts for one of the major challenges of studying these cells. In this study, we have demonstrated that live RSCs exhibit strong, multi-colour autofluorescence distinct from other coelomocytes, and with the help of fluorescence-activated cell sorting (FACS), a pure population of live RSCs was successfully separated from other coelomocytes in the green sea urchin, Strongylocentrotus droebachiensis. This newly developed RSCs isolation method has allowed profiling of the naphthoquinone content in these cells. With the use of ultra high-performance liquid chromatography, UV absorption spectra, and highresolution tandem mass spectrometry, it was possible to identify sulphated derivatives of spinochrome C, D, E and spinochrome dimers, which suggests that the RSCs may play an important biological role in the biogenesis of naphthoquinone compounds and regulating their bioactivity.

Spinochrome Identification and Quantification in Pacific Sea Urchin Shells, Coelomic Fluid and Eggs Using HPLC-DAD-MS

“…Spinochromes easily fragment via the removal of H 2 O, CO, and CO 2 [ 48 , 49 ]. In the experimental sections in previous reports by Brasseur et al [ 12 , 45 , 46 , 47 ], it was indicated the mass spectrometric equipment used allows the obtaining of high-resolution mass spectra and of MS/MS fragmentation of quinonoid pigments.…”

Regulation of dynamic pigment cell states at single-cell resolution

“…Coelomocytes, however, expressed fmos from clade 1, but not the other fmos ( Figure 5E ). These cells are diverse in morphology and putative function, of which the red spherule cells are known to produce echinochrome and spinochrome ( Coates et al, 2018 ; Hira et al, 2020 ). In the pigment biosynthesis pathway, the enzyme pks1 is upstream of the fmos in the biosynthetic pathway of echinochrome.…”

“…We used the available data of S. purpuratus quantitative RNA expression through development (http://www.spbase.org:3838/quantdev/) as an additional tool to determine the temporal expression of representative genes from the 4 clades of fmos. (Coates et al, 2018;Hira et al, 2020). In the pigment biosynthesis pathway, the enzyme pks1 is upstream of the fmos in the biosynthetic pathway of echinochrome.…”

“…From an intense red sea star ( Fromia milleporella ) to a black and white striped brittle star ( Ophiactis savignyi ) to the variegated sea urchin ( Lytechinus variegatus), pigment is a pervasive feature of this phylum. Because of the molecular techniques now available for echinoderms, mechanisms controlling pigmentation in these animals are being revealed ( Hira et al, 2020 ; Liu et al, 2019 ; Wessel et al, 2020 ; Yaguchi et al, 2020 ). The purple sea urchin, Strongylocentrotus purpuratus , provides a molecularly tractable model organism to dissect the developmental importance of pigmented cells, and the biosynthesis of their pigment.…”

“…A known function of sea urchin larval pigment cells includes an essential role in the innate immune defense system ( Buckley and Rast, 2017 ; Ch Ho et al, 2016 ; Hibino et al, 2006 ; Hira et al, 2020 ; Kiselev et al, 2013 ; Ransick and Davidson, 2006 ; Schrankel et al, 2016 ; Solek et al, 2013 ). When larvae are exposed to bacteria, pigment cells migrate from the ectoderm to the gut, a site for invading microbes, and interact with other immune cells ( Ch Ho et al, 2016 ).…”