Deep conservation of mitochondrial HSP60 structure with lineage-specific and context-dependent regulation reflects thermal resilience in cnidarians

Heat shock proteins (HSPs) are ubiquitous molecular chaperones that safeguard proteostasis under stress. We first investigated the expression dynamics of the mitochondrial chaperonin HSP60 across diverse cnidarians to understand its stress-responsive regulation. Using immunoblotting, we quantified HSP60 expression in Pocillopora acuta (reef-building coral), Exaiptasia diaphana (sea anemone), and Cassiopea xamachana (upside-down jellyfish). In P. acuta, HSP60 was not detected at the fragment scale under either control or heat stress, whereas isolated cells exhibited transient HSP60 expression during exposure to both control and heated temperatures (+5 degree Celsius above optimum), indicating that HSP60 regulation in this coral is strongly context-dependent and potentially suppressed at the tissue level. In contrast, E. diaphana and C. xamachana showed gradual, temperature-dependent accumulation of HSP60 over 24 h under heated conditions (+5 degree Celsius above its thermal optimum); however, C. xamachana also displayed constitutive basal expression under control conditions. These contrasting profiles highlight clear lineage-specific differences in HSP60 regulation among cnidarians. The consistent antibody cross-reactivity observed across all three species then prompted us to explore the evolutionary basis of this conservation. Phylogenetic analyses of HSP60 sequences confirmed that cnidarian proteins are orthologous to the canonical vertebrate HSP60 (human HSPD1), demonstrating deep structural and evolutionary conservation of this chaperonin across Metazoa. Collectively, these findings reveal that while HSP60 is evolutionarily ancient and conserved, its regulation under thermal stress varies across lineages and physiological contexts, reflecting complex modulation of mitochondrial proteostasis in early-diverging metazoans. This lineage- and context-dependent regulatory framework provides new insight into how chaperone plasticity contributes to cnidarian thermal tolerance and the differential susceptibility of reef taxa to bleaching under ocean warming.