EFFECTS OF FOCAL SEGMENTAL GLOMERULOSCLEROSIS-ASSOCIATED MUTATIONS ON MYOSIN 1E LOCALIZATION AND ACTIVITY
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AuthorKarchin, Jing Bi
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AbstractOur lab has discovered that an actin-dependent molecular motor called Myosin 1e (Myo1e) is required for maintaining normal morphology and function in vivo of podocytes, a specialized epithelial cell in the kidney. We have found that Myo1e-null mice develop proteinuria, and mutations in the MYO1E gene, including missense mutations A159P and T119I, and nonsense mutation Y695X, have been identified in focal segmental glomerulosclerosis (FSGS), a primary kidney disease that often leads to end stage renal disease (ESRD). Based on these findings, we have proposed that Myo1e and especially its motor domain, plays a key role in regulating actin cytoskeleton organization in kidney podocytes. To study Myo1e activity at the junctions, we have used cell culture systems. We confirmed that Myo1e is a component of the podocyte slit diaphragm using glomerular fractionation assay and immune-gold labeling electron microscopy. Disruption of Myo1e motor activity by point mutation (A159P) completely disrupted Myo1e cellular localization and led to defective actin assembly at nascent cell-cell contacts. Domain mapping experiments in MDCK cells have suggested that the Myo1e TH2 domain is necessary, but not sufficient for its localization, but addition of the TH1 domain restores its localization to junctions. We have also found that the Myo1e SH3 domain interacts with ZO-1, a slit diaphragm and tight junction protein, in invitro pulldown assays, which might contribute to ZO-1 exchange activity at the junctions. Another FSGS-associated Myo1e motor domain mutation (T119I) also caused mis-localization of Myo1e in the cultured mouse podocytes, suggesting loss-of-function of the motor domain mutants. We have also shown that ZO-1 is not recruited to the nascent cell-cell contacts at the same time with the Myo1e T119I mutants. Finally, by using fission yeast as a model system, we have demonstrated that human kidney disease-associated mutations in fission yeast caused defects in yeast growth and endocytosis processes. Interestingly, after analyzing the colocalization patterns between the FSGS-associated Myo1 mutants and Chaperone Rng3, we have proposed that these two kidney disease-associated mutants likely possess different disease-causing mechanisms.Above all, we have concluded that Myo1e motor domain plays an important role in its localization and activity in podocyte actin cytoskeleton, which might be the link to the disease mechanism of FSGS at the molecular level.
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