Background: Myo1e is a non-muscle motor protein enriched in podocytes, with mutations in MYO1E associated with steroid-resistant nephrotic syndrome (SRNS). Most of the MYO1E variants identified by genomic sequencing have not been functionally characterized. Here, we set out to analyze two mutations in the Myo1e motor domain, T119I and D388H, selected based on protein sequence conservation. Methods: EGFP-tagged human MYO1e constructs were delivered into the Myo1e-KO mouse podocyte-derived cells via adenoviral infection to analyze Myo1e protein stability, Myo1e localization, and clathrin-dependent endocytosis, which is known to involve Myo1e activity. Furthermore, truncated MYO1e constructs were expressed using the baculovirus expression system and used to measure Myo1e ATPase and motor activity in vitro. Results: Both mutants were expressed as full-length proteins in the Myo1e-KO cells. However, unlike wild-type (WT) Myo1e, the T119I variant was not enriched at the cell junctions or clathrin-coated vesicles (CCVs). In contrast, D388H variant localization was similar to WT. The rate of dissociation of the D388H variant from cell-cell junctions and CCVs was decreased, suggesting that this mutation affects Myo1e interactions with binding partners. ATPase activity and ability to translocate actin filaments were drastically reduced for the D388H mutant, supporting findings from cell-based experiments. Conclusions: T119I and D388H mutations are deleterious to Myo1e functions. The experimental approaches used in this study can be applied to future characterization of novel MYO1E variants associated with SRNS.
Expression of the unconventional myosin, Myosin-1e (Myo1e), has been shown to contribute to tumor progression in the MMTV-PyMT mouse model of mammary tumorigenesis and is associated with poor outcome in breast cancer patients. However, the specific effects of Myo1e expression on the mammary tumor cells remain unidentified. Here, we used Myo1e-KO and wild-type (WT) MMTV-PyMT mice on a pure genetic background to further investigate the molecular and cellular effects of Myo1e expression. Myo1e-WT tumors were characterized by an increased abundance of intra-epithelial macrophages and lower amounts of the extracellular matrix. Transcriptomic profiling of WT and Myo1e-KO tumors identified a pattern of differential expression of tumor suppressor and tumor-promoting genes that was consistent with the observed differences in tumor progression and morphology between the genotypes, and also revealed differential expression of genes associated with secretion and cell-cell adhesion. In agreement with the RNA-seq findings, Myo1e-expressing tumor cells exhibited increased proliferation and elevated nuclear enrichment of YAP1 transcriptional activator compared to Myo1e-KO tumor cells. To investigate tumor cell-autonomous effects of Myo1e expression, we used the epithelial cell line PY-230 derived from the MMTV-PyMT-induced mouse tumor to create Myo1e-depleted cells by Crispr-mediated genome editing. Cells deficient in Myo1e had increased expression of genes encoding milk components compared to the wild-type cells. Electric cell-substrate impedance sensing (ECIS) measurements showed that depletion of Myo1e in PY-230 cells resulted in increased resistance to electrical current indicating enhanced epithelial barrier function. Overall, we find that Myo1e expression biases tumors towards a less-differentiated, pro-tumorigenic state, and that depletion of Myo1e is associated with a pro-secretory, more differentiated state.
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