Fig. 4: Co-localization of endocytotic calcium vesicles with pHves in the calcifying primary mesenchyme cells (PMCs) of the sea urchin larva.
From: Proton channels govern vesicular carbonate chemistry in mineralizing cells of a marine calcifier
Simultaneous labeling of calcium (calcein) and pH (carboxy-SNARF) in the vesicles of the PMC bodies and syncytial cords. Scale bar: 25 µm (A) and 10 µm (A′, A“). B Percentage of calcein and SNARF-positive vesicles within the total number of either dye-containing vesicles. Values presented for individual cells as mean ± SEM (N = 54), ****P < 0.0001, unpaired t-test. C An example of calcium-rich spicule and vesicles that are partially co-localized with SNARF in the PMC body and syncytial extensions (arrows). Scale bar: 10 µm. D High magnification image of one PMC with predominantly alkaline vesicles and their co-localization with calcium-rich vesicles (arrows). Scale bar: 10 µm. E Pearson´s R co-localization analysis of calcein-positive vesicles with alkaline and acidic vesicles, where +1, 0, and −1 indicates 100%, 50%, and 0% co-localization, respectively. Values presented for individual cells as mean ± SEM (N = 26 acidic; N = 23 alkaline), ****P < 0.0001, unpaired t-test. F, F′ Representative images of SNARF-positive vesicles along the spicules of 5 dpf-old control and re-calcifying larvae. Scale bar: 25 µm. G Abundance of SNARF-containing vesicles in control and re-calcifying PMCs. Values presented for individual cells as mean ± SEM (N = 34 control; N = 36 re-calcification), ***P = 0.0002, ****P < 0.0001, unpaired t-test. H, H′ Representative traces and measurements of pHves changes in response to changes in pHe in control and re-calcifying PMCs. Values presented for individual cells as mean ± SEM (N = 25), *P = 0.0469, ***P = 0.0006, ****P < 0.0001, unpaired t-test. I, I′ Representative traces and measurements of pHi changes in response to changes in pHe in control and re-calcifying PMCs. Values presented for individual cells as mean ± SEM (N = 25 control; N = 30 re-calcification), ****P < 0.0001, unpaired t-test. J Representative traces and measurements of changes in PMC plasma membrane depolarization intensity in control and re-calcifying larvae. Values presented for individual cells as mean ± SEM (N = 53 control; N = 38 re-calcification), unpaired t-test.
