The mixed lineage kinase domain-like (MLKL) pseudokinase is an essential molecular component of necroptosis, a form of programmed cell death with morphological characteristics of necrosis. Necroptosis results in plasma membrane perforation and the release of cellular contents, which elicit an immune response from neighbouring cells. It has an important function in many human inflammatory diseases. To cause cell death, MLKL needs to oligomerize and translocate to the cell membrane. Nanobodies fused with fluorescent proteins are an important tool in the visualization of almost any antigen within the living cell. Nanobodies against proteins involved in inflammation have been developed to modulate an immune response. In our study, we confirmed the binding of the fusion protein M33-mCherry by size-exclusion chromatography. Nanobody M33 recognizes the N-terminal region of MLKL (MLKLN-154). Detection of MLKL protein expressed in HEK293T cells with M33-mCherry fusion protein was unsuccessful. Further on, we determine whether M33-mCherry and MLKLN-154 colocalize in HEK293T cells after transfection with both constructs. Membrane translocation of MLKL is crucial for necroptosis, so colocalization might be observed on the plasma membrane. MLKLN-154 localizes in the cytoplasm and cannot induce necroptosis, but D144K mutant, which mimics activated MLKL, causes cell death. We determined the time point when MLKLN-154 D144K is expressed, but cells are still alive and also confirmed the expression of the M33-mCherry fusion protein. However, we could not observe their colocalization. There was no difference in cell death level between samples with nanobody and without it, so we concluded that in this case, the M33-mCherry nanobody does not decrease the level of cell death.
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