PNNL

Abstract

Single-cell proteomics can reveal the changing protein composition of differentiating cells. We used shotgun mass spectrometry to identify the abundant proteins present in single or small pools of cells from the embryonic day 15 (E15) utricle of the chicken inner ear, when many hair cells are differentiating from progenitor cells. In single hair cells, positive for labeling with FM1-43, we identified 40-70 proteins or protein groups. In each pool of 20 cells, we identified ~600 proteins. Cells that were negative for FM1-43 (presumptive progenitor/supporting cells) had 12-50 identifications in single cells and ~300 in each pool of 20 cells. Data are available via ProteomeXchange with identifier PXD014256. Comparison of relative abundance of identified proteins revealed that the actin monomer binding protein thymosin ß4 (TMSB4X) was present in E15 progenitor cells at nearly equimolar levels relative to actin, but dropped to one-tenth that value in hair cells, with little change in total actin. Single-cell RNA-seq analysis of E15 utricle cells showed that TMSB4X transcripts fell in abundance once hair-cell differentiation initiated. These results suggest that most actin is sequestered in progenitor cells, but upon differentiation to hair cells, actin is released, permitting assembly of the sensory hair bundle.