Microvillus inclusion disease-causing MYO5B point mutations exert differential effects on motor function

Abstract

Microvillus inclusion disease (MVID) is a rare congenital diarrheal disorder typically caused by loss of function mutations in the unconventional myosin, myosin 5b (MYO5B), which leads to the mistrafficking of apical components in enterocytes. MVID can manifest in two phenotypes: in both the intestine and liver or the liver alone. Although previous studies seeking to understand MVID disease pathology used MYO5B KO models, many patients have point mutations and thus express a dysfunctional MYO5B. How these point mutations lead to a broad spectrum of disease severity and the development of two distinct disease phenotypes is still not known. Here, we investigate the effect of MVID patient mutations on the function of the MYO5B motor domain, independent of cargo binding, using confocal imaging and fluorescence recovery after photobleaching. Patient mutations demonstrated a range of effects in these assays, from rigor-like behavior to loss of actin binding. Additionally, analysis of fluorescence recovery after photobleaching turnover kinetics suggests that some mutations negatively impact the ability of MYO5B to stay bound to actin. Collectively, our findings indicate that patient mutations affect the MYO5B motor domain in diverse ways, consistent with the spectrum of phenotypes observed in patients.

Keywords: actin; confocal microscopy; congenital diarrhea; intestinal epithelium; intracellular trafficking; microvilli; myosin.

Figure 1

Point mutations in the MYO5B motor with a predicted outcome disrupt normal MYO5B motor localization. A, MYO5B dimer diagram (top left), boxed area shown below using an AlphaFold structure of the MYO5B motor head indicating positions of prediction point mutations (left) and important actin-binding domains (right). MYO5B-MD expression construct diagram. B, WT MYO5B-MD constructs are more localized to the tips of microvilli. C, predicted rigor mutation, N208A, localizes to the bases of microvilli. D, predicted actin-nonbinding mutation, E443A, localizes to the cytoplasm of the cells. E, predicted uncoupled mutation, I439A, localizes along the length of microvilli. F, the point along the normalized length of microvilli at which the intensity of the MYO5B-MD-CC-3x-Citrine signal was the highest plotted. ∗∗∗∗p value <0.0001. Each condition has an N ≥ 12 cells, where five or more microvilli have been measured from a replicate of three independent coverslips. Phalloidin is used to mark F-actin (magenta), and the MYO5B motor was transiently transfected into the cells (green). The scale bar represents 10 μm. MYO5B, myosin 5b.

Figure 2

Patient MYO5B motor mutations disrupt normal MYO5B localization.A, position of patient mutations shown on an AlphaFold structure of MYO5B motor head. B, patient mutation, P660L, localizes to the bases of microvilli. C, patient mutation, G519R, localizes along the length of microvilli. D, patient mutation, I408F, localizes intracellularly. E, patient mutation, C266R, localizes along the length of the microvilli. F, patient mutation, R824C, localizes more toward the bases of microvilli. G, patient mutation, R92C, localizes along the length of microvilli and more toward the tips of microvilli. H, the point along the normalized length of microvilli at which the intensity of the MYO5B-MD signal was the highest plotted for WT MYO5B-MD, MYO5B-MD(N208A), MYO5B-MD(P660L), and MYO5B-MD(G159R). Underlined mutants were shown in the previous figure. I, the point along the normalized length of microvilli at which the intensity of the MYO5B-MD signal was the highest plotted of the WT MYO5B-MD, MYO5B-MD(E443A), and MYO5B-MD(I408F). Underlined mutants were shown in the previous figure. J, the point along the normalized length of microvilli at which the intensity of the MYO5B-MD signal was the highest plotted of the WT MYO5B-MD, MYO5B-MD(C266R), MYO5B-MD(R824C), and MYO5B-MD(R92C). Underlined mutants were shown in the previous figure. ∗∗∗∗p value <0.0001. Each condition has an N ≥ 12 cells, where five or more microvilli have been measured from a replicate of three independent coverslips. Phalloidin is used to mark F-actin (magenta), and the MYO5B motor was transiently transfected into the cells (green). The scale bar represents 10 μm. MYOB, myosin 5b.

Figure 3

Point mutations in the MYO5B motor with a predicted outcome disrupt normal MYO5B motor localization.A, FRAP of the WT MYO5B motor that localizes to the tips of microvilli. B, FRAP of the I439A MYO5B motor, the predicted uncoupled mutation. C, FRAP of the patient mutation, G519R. D, FRAP of the liver-specific patient mutation, C266R. E, FRAP of the liver-specific patient mutation, R824C. F, FRAP of the liver-specific patient mutation, R92C. G, this graph depicts an overlay of the initial recovery for all MYO5B motors assayed with FRAP. This graph shows that mutant motors I439A, G519R, and C266R have a steeper initial recovery than the WT motor. H, a cartoon depicting that the MYO5B mutations may increase the on/off rate of actin-binding kinetics than the WT (figure prepared with Biorender). Each condition has an N ≥ 11 cells from a replicate of three independent MatTek dishes. The 3x-Citrine-Myo5b motor overexpression is shown in inverted grayscale. The movies are deconvolved and have been histogram-corrected for bleaching. The scale bar represents 5 μm. FRAP, fluorescence recovery after photobleaching; MYOB, myosin 5b.