Leveraging Microvillus Inclusion Disease Causing Patient Mutations to Investigate Myosin 5B Motor Function

Abstract

Microvillus Inclusion Disease (MVID) is a rare neonatal congenital diarrheal disorder. Mutations in the unconventional myosin, Myosin 5b (MYO5B) cause MVID. These MYO5B mutations, which cause loss of function, lead to the mis-trafficking of apical components in enterocytes, resulting in congenital diarrhea and the mislocalization of apical components. MVID can manifest in two phenotypes: in both the intestine and the liver or the liver alone. Many previous studies have utilized MYO5B knockout mouse models to help elucidate the pathology of MVID; however, many patients have missense mutations that lead to the expression of a dysfunctional MYO5B. The mechanisms by which these point mutations lead to a broad spectrum of disease severity and the development of two distinct disease phenotypes are still not fully understood. To investigate the effect of MVID patient mutations on the function of the MYO5B motor domain, I developed an in-cell assay to evaluate MYO5B motor function independent of cargo binding. This was done by tagging a truncation version of MYO5B containing only the motor head, neck, and a small portion of the coiled-coiled domain, with a triple tandem citrine repeat. This construct was then introduced into LLC-PK1-CL4 cells that produce microvilli, and I was able to use confocal imaging and fluorescence recovery after photobleaching (FRAP) to evaluate the effect of mutations on motor function. The patient mutations demonstrated a range of effects in these assays, from rigor-like behavior (P660L) to loss of actin-binding (I408F). Additionally, analysis of FRAP turnover kinetics suggests that some mutations impact the MYO5B actin binding kinetics. Collectively, the findings of my work indicate that patient mutations affect the MYO5B motor domain in diverse ways, consistent with the spectrum of phenotypes observed in patients.