Research Projects

Embryonic stem cells (ES) cells are pluripotent cells derived from the inner cell mass of the pre-implantation embryo. A number of pathways and processes are involved in regulating the pluripotency of stem cells, including Clathrin mediated endocytosis or CME (Mote et al., 2020; Narayana et al., 2019). The basic unit of the clathrin coat is a clathrin triskelia, which is formed by the polymerization of three molecules each of clathrin heavy chains (CHCs) and clathrin light chains (CLCs). While the clathrin heavy chain is essential for cell survival, the role of Clathrin light chains appears to be more regulatory. In higher vertebrates, two isoforms of CLCs are present, CLCa and CLCb (encoded by genes Clta and Cltb respectively) (Das et al., 2021). Clathrin light chains are associated with a plethora of cellular processes such as clathrin lattice assembly, clathrin coated vesicle (CCV) formation, neurotransmitter trafficking and synaptic vesicle recycling, actin assembly, cell division and migration. Our lab has generated and characterized clathrin light chain knockout mouse ESCs (mESCs) (Clta-/-; Cltb-/- and Clta_Cltb-/-), which display changes in expression of specific differentiation markers when compared to their wild type counterparts (Tiwari et al 2025). Our preliminary findings revealed that loss of CLCs resulted in 1) an altered mitochondrial morphology in stem cells through electron microscopy, accompanied by changes in the rate of proliferation. Unbiased proteomic analysis from the CLC knockout mESCs also revealed alterations in the abundance of proteins involved in mitochondrial respirosome and mitochondrial translation (preliminary data). Interestingly there was no change in the levels of transcripts encoding for these proteins through unbiased RNA-seq analysis (Tiwari et al, 2025), indicative of post-transcriptional regulation. Mitochondrial function is correlated to its shape and distribution in the cell, which requires a balance between mitochondrial fission, fusion, actin dynamics and mitophagy. The proper functioning of mitochondria is crucial for regulating ESC proliferation, controlling differentiation, and preventing the formation of tumor cells during the differentiation process. Mitochondria in pluripotent stem cells exhibit a small, fragmented morphology that is distinct from the elongated, tubular mitochondrial networks seen in differentiated cells. During differentiation, pluripotent stem cells undergo a drastic change in mitochondrial morphology, transitioning from small and fragmented to elongated, tubular networks, which is accompanied by a significant increase in the number of mitochondria in mouse and human embryonic stem cells. These changes in mitochondrial shape and number upon differentiation are also accompanied by an increase in oxidative phosphorylation-based metabolism. Moreover, the dynamic changes in mitochondrial morphology and number that occur during differentiation of mouse embryonic stem cells are tightly regulated by mitochondrial fission and fusion proteins, and are critical for proper cellular metabolism and differentiation. The connection between CME and mitochondria was revelealed some years ago when it was observed that lipophilic dyes were trafficked to the mitochondria from the plasma membrane via CME. Further, molecules such as Endosidin, function to inhibit CME while also acting as a mitochondrial uncoupler. Additionally, a number of membrane remodelling proteins are also involved at the mitochondria, similar to what is seen in endocytic processes. Our preliminary data revealed that loss of CLCs resulted in an altered mitochondrial morphology in stem cells. In light of these observations we hypothesized that CLCs may be involved in regulating mitochondrial function and thereby alter the pluripotency of stem cells. As the function of clathrin light chains in the context of mitochondria is completely unknown, we propose to address these lacunae through this proposal.