Protein oligomerization plays an essential role in many signaling pathways, ranging from light-controlled optogenetic systems to programmed cell death mechanisms such as necroptosis. However, measuring oligomerization states in living cells with temporal resolution remains challenging. Here, we apply photon counting histogram (PCH) analysis to quantify oligomerization states of HaloTag-labeled proteins in live HEK293 cells. Proteins of interest were fused to HaloTag and labeled with the far-red dye Janelia Fluro 646. As proof of concept, we examined different variants of optogenetic proteins AuLOV and CRY2, which undergo oligomerization upon blue light stimulation. PCH measurements revealed distinct light-dependent and concentration-dependent changes in oligomerization state, validating the sensitivity of the method in live-cell conditions. Importantly, this approach provides quantitative resolution of molecular assemblies beyond ensemble-averaged fluorescence intensity measurements. Building on these results, we aim to extend this methodology to investigate mixed lineage kinase domain-like protein (MLKL), the terminal executioner of necroptosis. MLKL oligomerization at the plasma membrane is a hallmark of necroptotic signaling, yet real-time quantification in living cells remains limited. Our approach offers a direct and quantitative means to measure MLKL oligomerization dynamics during necroptosis, providing new insight into the regulation of cell death execution. More broadly, this strategy establishes a versatile platform for dissecting the dynamic assembly of signaling proteins in living cells with high temporal and molecular resolution.
Li et al. (Sun,) studied this question.
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