LP-1

Functional Effects of TBC1 Domain Containing Kinase Deletion in Immortalized B Cells and Plasma Cells

TBC1 domain containing kinase (TBCK) is highly expressed in neurons and glial cells, and its mutations cause intellectual disability and hypotonia. TBCK is part of the FERRY complex involved in mRNA transport and mTORC1 signaling. Here, Beck et al. generated TBCK knockout cell lines (Raji and LP-1 cells) to study its impact on B cells and plasma cells.

In Raji cells, lentiviral transduction with guide RNAs KO2, KO4, KO5, or KO6 significantly reduced TBCK expression (Fig. 1A-B). In LP-1 cells, initial TBCK knockdown was modest, with only KO4 and KO6 showing some reduction. To improve knockdown in LP-1 cells, Beck et al. generated single-cell clones, and some of these clones had satisfactory TBCK knockdown (Fig. 1C-D). They then used flow cytometry to see if the effects of TBCK knockdown seen in other cell models could be reproduced in Raji or LP-1 cells. They grew these cells for 24 hours in media with either 10% FBS or 0% FBS to mimic conditions with and without EGFR signaling. Surprisingly, neither Raji nor LP-1 cells showed any sensitivity to TBCK loss under normal or serum-free conditions. All modified cell lines had similar viability, cell size, and S6 phosphorylation levels. They also had similar side-scatter profiles, indicating no increase in cellular granularity or autophagosome accumulation in TBCK knockout cells. As expected, both cell lines had reduced S6 phosphorylation after serum withdrawal, especially in LP-1 cells, suggesting a greater reliance on EGF for homeostasis.