Spatial compartmentalization of free radical formation and mitochondrial heterogeneity in bivalve gills revealed by live-imaging techniques, supplementary material

Live-imaging techniques (LIT) utilize target-specific fluorescent dyes to visualize biochemical processes using confocal and multiphoton scanning microscopy, which are increasingly employed as non-invasive approach to physiological in-vivo and ex-vivo studies. Here we report application of LIT to bivalve gills for ex-vivo analysis of gill physiology and mapping of reactive oxygen (ROS) and nitrogen (RNS) species formation in the living tissue. Our results indicate that H2O2, HOO· and ONOO- radicals (assessed through C-H2DFFDA staining) are mainly formed within the blood sinus of the filaments and are likely to be produced by hemocytes as defense against invading pathogens. The oxidative damage in these areas is controlled by enhanced CAT (catalase) activities recorded within the filaments. The outermost areas of the ciliated epithelial cells composing the filaments, concentrated the highest mitochondrial densities (MTK Deep Red 633 staining) and the most acidic pH values (as observed with ageladine-a). These mitochondria have low (depolarized) membrane potentials (D psi m) (JC-1 staining), suggesting that the high amounts of ATP required for ciliary beating may be in part produced by non-mitochondrial mechanisms, such as the enzymatic activity of an ATP-regenerating kinase. Nitric oxide (NO, DAF-2DA staining) produced in the region of the peripheral mitochondria may have an effect on mitochondrial electron transport and possibly cause the low membrane potential. High DAF-2DA staining was moreover observed in the muscle cells composing the wall of the blood vessels where NO may be involved in regulating blood vessel diameter. On the ventral bend of the gills, subepithelial mucus glands (SMG) contain large mucous vacuoles showing higher fluorescence intensities for O2·- (DHE staining) than the rest of the tissue. Given the antimicrobial properties of superoxide, release of O2·- into the mucus may help to avoid the development of microbial biofilms on the gill surface. However, cells of the ventral bends are paying a price for this antimicrobial protection, since they show significantly higher oxidative damage, according to the antioxidant enzyme activities and the carbonyl levels, than the rest of the gill tissue. This study provides the first evidence that one single epithelial cell may contain mitochondria with significantly different membrane potentials. Furthermore, we provide new insight into ROS and RNS formation in ex-vivo gill tissues which opens new perspectives for unraveling the different ecophysiological roles of ROS and RNS in multifunctional organs such as gills.

活细胞成像技术（Live-imaging techniques, LIT）利用靶标特异性荧光染料，结合共聚焦与多光子扫描显微镜技术可视化生物化学过程，此类技术正日益被用作生理体内与离体研究的非侵入性手段。本研究报道了将活细胞成像技术应用于双壳类鳃组织的案例，用于鳃生理功能的离体分析，以及活体组织中活性氧（ROS, reactive oxygen species）与活性氮（RNS, reactive nitrogen species）生成的定位成像。研究结果显示，经C-H2DFFDA染色检测的过氧化氢（H₂O₂）、过氧羟自由基（HOO·）与过氧亚硝基阴离子（ONOO⁻）主要生成于鳃丝的血窦中，且大概率由血细胞产生以抵御入侵病原体。上述区域的氧化损伤可通过鳃丝中检测到的增强型过氧化氢酶（CAT, catalase）活性得到调控。构成鳃丝的纤毛上皮细胞的最外层区域，拥有最高的线粒体密度（经MTK Deep Red 633染色检测）与最低的pH值（通过ageladine-a观测得到）。这些线粒体的膜电位（ΔΨm）较低（去极化状态，经JC-1染色检测），这提示纤毛摆动所需的大量三磷酸腺苷（ATP），有一部分可能通过非线粒体途径生成，例如ATP再生激酶的酶促活性。外周线粒体区域产生的一氧化氮（NO, nitric oxide，经DAF-2DA染色检测），可能会影响线粒体电子传递过程，并可能导致膜电位降低。此外，在构成血管壁的肌细胞中也观察到了较强的DAF-2DA染色信号，一氧化氮可能参与调控血管管径。在鳃的腹侧弯曲处，上皮下黏液腺（SMG, subepithelial mucus glands）含有大型黏液泡，经二氢乙锭（DHE, dihydroethidium）染色检测的超氧阴离子（O₂·⁻）荧光强度显著高于其他组织区域。鉴于超氧阴离子具有抗菌活性，将O₂·⁻释放至黏液中，或可避免鳃表面形成微生物生物膜。然而，鳃腹侧弯曲处的细胞为这种抗菌保护付出了代价：根据抗氧化酶活性与羰基化蛋白水平检测结果，其氧化损伤程度显著高于鳃组织的其他区域。本研究首次证实，单个上皮细胞内可存在膜电位差异显著的线粒体。此外，本研究还为离体鳃组织中ROS与RNS的生成机制提供了新的认识，为阐明ROS与RNS在鳃等多功能器官中的多样生态生理功能开辟了新的研究方向。