Warm-blooded vertebrates such as birds and mammals are adapted to maintain their physiological body temperature around 36–42°C. The regulation of body temperature based on their surroundings helps them to adapt at diverse temperature zones (Ivanov et al., 2006). Maintaining temperature homeostasis is crucial for metabolism and various cellular pathways. Failure to sense temperature and respond to it can lead to either hypothermia (at low temperature), or hyperthermia (at high temperature). Thermal homeostasis is also relevant at the cellular and subcellular levels, though the exact mechanisms are not well understood. Studies suggest that thermal mapping of a cell can also give critical insight into the differentiation between a diseased and healthy state of the cell (Huang et al., 2021). In addition to the surrounding temperature, intracellular processes also cause thermal variations. At the cellular level, there is generation, dissipation and transfer of heat. Various metabolic reactions and organelle functions are involved in heat generation. Additionally, thermal status within a cell can also affect cellular functions e.g., DNA replication, gene expression, membrane fusion, and are also involved in cancer metabolism, etc. (Yang et al., 2022, Kamei et al., 2009, Vreugdenburg et al., 2013). Recent therapeutic approach to treat cancer conditions also involves changing the thermal state at microscale in specific conditions (Dutz and Hergt, 2013). In spite of potential applications in therapeutics and in diseases, there is very limited knowledge about the thermoregulation at cellular and subcellular level.
Recent studies based on novel probes and sensors suggest existence of thermal heterogeneity at subcellular level (Kucsko et al., 2013, Yang et al., 2011, Okabe et al., 2012). In general, few subcellular compartments (e.g. mitochondria and nucleus) remain hotter than the cytoplasm (Okabe et al., 2012; Chretien et al., 2017). An optimum thermal range of subcellular organelle is crucial for the maintenance of its proper dynamics, thermal-flux, metabolic-flux and functions. Reportedly, several functions of subcellular organelles such as mitochondria and lysosomes are temperature-dependent (Di et al., 2022). Deregulation of subcellular organelle temperatures not only expected to affect the functions of respective organelles but also can be associated with various disease conditions (Huang et al., 2021; Zaynab et al., 2022). In the same line, it has been reported heat stress helps in muscle atrophy by restoring the mitochondrial respiratory capacity (Hafen et al., 1985). It has also been observed that cold exposure and exercise also regulate functions of mitochondria-related genes in adipose tissue (Chung et al., 2017). Thermoregulation also plays a role in mitochondria-related diseases and pathophysiology. While thermoregulation can affect mitochondrial function, in reverse mitochondrial dysfunctions may also affect the temperature of other subcellular organelles, cells, and critical organs (such as brain and heart). In mitochondrial dysfunction condition, hypothermic brain condition was reported (Rango et al., 2014). Previous research has shown a direct relationship between mitochondrial temperature with metabolism, and hence mitochondrial temperature is also considered as an indicator to differentiate between healthy and dysfunctional cells (Tang et al., 2020; Jonckheere et al., 2012). Therefore, the “temperature” as well as the “change in temperature” of the mitochondria may be related to a large number of mitochondrial disorders, commonly known as “mitopathy”, which are complex and difficult to understand.
Dysfunction of other subcellular organelles are also known to cause various disease conditions. For example, lysosomal dysfunctions, commonly known as “lysopathy,” is involved in several rare genetic and complex disorders (Poswar et al., 2019). For example, mutations in TRPV3, a hot-sensitive ion channel induces a rare disorder known as “Olmsted syndrome” where lysosomal abnormalities are prominent (Yadav and Goswami, 2017, Jain et al., 2022). Relevant to thermal homeostasis, the subcellular organelles communicate with each other by direct transmission of materials (such as exocytosis, endocytosis, vesicle budding, etc.) as well as by through contact points (such as ER-mitochondrial contact points, mitochondrial-lysosomal contact points, etc.). However, it is not clear if the temperature of each organelle remains different or not under certain conditions. Though recent studies indicate the crucial role of different subcellular organelles in cell functions and diseases, till date, the thermoregulation of these different organelles and their thermal relationship with each other remain unexplored. Due to lack of sufficient tools and techniques, thermal regulation within cells, especially at the subcellular organelle levels, is still not well understood.
In year 2002, TRPM8, a member of TRP superfamily was identified that could be activated by cold stimuli in trigeminal sensory neurons, and thus tagged as cold responsive ion channel (McKemy et al., 2002). The same receptor was able to get activated by cooling agents such as by menthol or Icilin. This functional ion channel has been detected in both trigeminal sensory neurons and dorsal root ganglion neurons, which were also remain both menthol and cold-sensitive (Reid and Flonta, 2002; Thut et al., 2003). TRPM8 behaves similar to other ligand-gated ion channel, and thus shows rapid ligand-dependent channel opening, followed by quick elevation of cytosolic Ca2+-levels (Chuang et al., 2004). The in vivo studies further confirmed the behavioral defects against cold stimulus in TRPM8 knockout animals (Dhaka et al., 2007). Studies in 2018 and onwards confirmed the presence of TRPM8 in various brain regions e.g., hypothalamus, septum, thalamic reticular nucleus, certain cortices, and other limbic structures, which strongly indicate the involvement of TRPM8 in autonomic and behavioral thermoregulation (Ordás et al., 2021; Tsuneoka et al., 2023). At present TRPM8 has been well accepted cold-activated ion channel that can also get activated by various natural and synthetic modulators (Dhaka et al., 2007, Izquierdo et al., 2021). The expression and function of TRPM8 has been studied in a wide range of cell types, including various immune cells and neuronal cells (Yu et al., 2015; Khalil et al., 2016). Recently, we have reported the endogenous presence of TRPM8 in microglia and showed that modulation of TRPM8 regulates the functions of different organelles such as lysosomes and mitochondria (Shikha et al., 2023; Chakraborty and Goswami, 2022).
The subcellular organelle-specific thermo-regulation is also crucial for all cell types to maintain cellular homeostasis and also for the regulation of thermal diffusion of compounds and, thus, regulation of metabolic flux. Accordingly, recent studies also highlighted the significance of thermal-regulation of subcellular organelle in cell-functions, and change in thermal regulations are related to the development of disease conditions (Huang et al., 2021). Changes in relative temperature of various subcellular organelle have also been studied to get insight of thermo-regulation of various cell types with the help of well-established thermo-dyes (Acharya et al., 2022b; Acharya et al., 2023; Kumar et al., 2023). In this study, for the first time, we have elucidated the in vitro thermoregulation of microglial cells (BV2 cell) and especially the subcellular organelles as a factor of TRPM8 modulation for a deep understanding of thermal homeostasis at subcellular level. Our study describes a useful methodology to study spatio-temporal changes of the subcellular organelles, both qualitatively as well as quantitatively. Our study sheds light on the unexplored aspect of TRPM8 on the regulation of critical interplay between cellular homeostasis and temperature regulation.