Steroid-Resistant Nephrotic Syndrome–Associated MYO1E Mutations Have Differential Effects on Myosin 1e Localization, Dynamics, and Activity

Significance Statement

MYO1E is a gene linked to early onset steroid-resistant nephrotic syndrome (SRNS), which has a poor prognosis without kidney transplantation. Using live-cell imaging and myosin motor activity assays in mouse podocyte–derived cells using human constructs, we characterized two disease-associated mutations in the Myo1e motor domain, T119I and D388H, which are deleterious to Myo1e localization and functions. These findings can assist in interpreting genetic diagnosis of SRNS, lead to a more precise and efficient treatment, and improve understanding of Myo1e function in podocytes.

Abstract

Background

Myo1e is a nonmuscle motor protein enriched in podocytes. Mutations in MYO1E are 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, which were selected on the basis of 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 that of WT. The rate of dissociation of the D388H variant from cell-cell junctions and CCVs was decreased, suggesting 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.

Steroid-resistant nephrotic syndrome (SRNS) is often associated with mutations that undermine the selective permeability of the glomerular filtration barrier. Patients with familial SRNS are good candidates for kidney transplantation due to low risk of disease recurrence.^1–4 One of the genes linked to primary SRNS is MYO1E (encodes Myo1e), which exhibits an autosomal recessive pattern of inheritance of disease-associated variants.⁵ In this paper, we set out to characterize two mutations in MYO1E identified by genomic sequencing^6–8 to determine whether they disrupt Myo1e functions.

Myo1e is an actin-dependent ATPase^9,10 and a member of myosin class 1.¹¹ Class 1 myosins bind actin filaments via their motor domains and plasma membrane via their tail domains, thus serving as actin-membrane linkers. Myo1e is highly expressed in podocytes (terminally differentiated glomerular epithelial cells)^5,12,13 and is enriched in podocyte foot processes, which are elongated protrusions connected to each other by slit diaphragms (modified tight/adherens junctions). Podocyte foot processes and slit diaphragms are important components of the glomerular filter. Knockout (KO) of Myo1e (Myo1e-KO) in mice, either via germ line (complete) or podocyte-specific KO, leads to proteinuria, foot process effacement, and irregular thickening of the glomerular basement membrane.^12–14 Patients with MYO1E mutations also exhibit proteinuria along with podocyte foot process effacement and glomerular basement membrane defects.⁵

Myo1e localizes to cell-cell junctions^5,12,15 and clathrin-coated vesicles (CCVs)¹⁶ and binds the endocytic proteins dynamin and synaptojanin¹⁷ and junctional component ZO-1.¹⁵ Because KOs of the endocytic proteins Myo1e, dynamin, synaptojanin, and endophilin in podocytes lead to severe proteinuria in mice, clathrin-mediated endocytosis (CME) appears to play an important role in normal glomerular filtration.^12,18 These observations suggest Myo1e may contribute to maintaining slit diaphragm integrity or selective filtration via its roles in endocytosis and cell-cell contact assembly, and mutations that affect Myo1e localization to cell-cell junctions or CCVs in podocytes are likely to be pathogenic.

Several novel MYO1E variants were recently found in a Saudi Arabian cohort and a global cohort of families with SRNS.^6,7 Here, we have characterized protein expression/stability and intracellular localization of two of the novel Myo1e variants, T119I and D388H (Figure 1), using adenoviral expression of mutant constructs in the Myo1e-KO, podocyte-derived cell line. We found these mutations altered Myo1e localization and dynamics, and expression of these variants affected the density and lifetimes of CCVs. We also performed measurements of myosin motor activity using ATPase kinetics and filament sliding assays, with the latter providing the first characterization of the Myo1e-driven motility in vitro. The D388H mutation in the Myo1e motor domain led to a dramatic reduction of myosin ATPase activity and loss of motility, whereas the T119I variant was too unstable to be used for these measurements. Overall, our work provides functional evidence for the pathogenic effects of these mutations in MYO1E.

Methods

Podocyte Culture

Conditionally immortalized wild-type (WT) podocytes (mouse podocyte clones [MPCs]) were a generous gift from Dr. Peter Mundel.^19,20 Conditionally immortalized Myo1e-KO podocytes were derived from the Myo1e-KO mice as previously described.^12,15 Briefly, we derived Myo1e-KO cells from glomeruli of Myo1e-KO mice carrying the Immortomouse transgene (tsA58TAg).²¹ We isolated glomeruli using sequential sieving with 180-μm and 100-μm sieves, collecting glomeruli on a 71-μm sieve. We obtained individual podocyte clones by growing glomerular explant cells in culture and dilution cloning and initially characterized cells by immunoblotting and immunostaining to verify the lack of myo1e expression and the presence of the podocyte markers synaptopodin and nephrin. We have maintained podocyte-derived clones in our laboratory, and, as previously described for other podocyte cell lines,²² they have gradually lost nephrin expression but retained synaptopodin expression. Results of the RNAseq analysis of the WT and Myo1e-KO podocyte-derived cell lines, including expression of podocyte-specific markers, are reported in Supplemental Figure 1. Podocytes were grown on collagen I–coated (354236; Corning) culture dishes in RPMI-1640 with 10% FBS, 1% antibiotic-antimycotic, and 50 μg/ml IFN-γ (407303; EMD Millipore) at 33°C with 5% carbon dioxide. For differentiation, we cultured podocytes at 37°C without IFN-γ.^19,20 Podocytes were differentiated for 10 days before adenoviral transduction for protein analysis and live-cell imaging, following previously described transduction procedures.²³

Constructs

Point mutations were introduced into the WT human Myo1e in the pEGFP-C1 vector using the QuikChange Lightning Site-Directed Mutagenesis Kit (210519; Agilent Technologies).^5,17 We used mutant constructs for subcloning into the pAdEasy adenoviral vector and packaging into recombinant adenoviral particles, as described.^15,23 For the baculoviral expression, an EGFP-FLAG-AviTag–encoding linear DNA segment was prepared by gene synthesis (GENEWIZ) and inserted into the pFastBac vector using the In-Fusion kit (638910; Takara). Inserts encoding WT or mutant Myo1e fragments (motor domain plus IQ motif, amino acids 1–724) were PCR amplified from the pEGFP-C1-Myo1e constructs and subcloned by In-Fusion into the pFastBac/EGFP-FLAG-Avi vector. Primers used for cloning are listed in Supplemental Table 1.

Protein Expression in Podocytes and Drug Treatments

To measure protein expression, we collected cell lysates 24 hours after infection with adenoviral vectors encoding Myo1e. To measure the effects of stopping protein synthesis, we collected cell lysates after a 3-hour treatment of podocytes with 20 μg/ml cycloheximide (CHX; C7698; Sigma-Aldrich) in the complete RPMI medium at 37°C, whereas control lysates were collected from cells treated with DMSO. We performed proteasome inhibition using a 3-hour treatment of podocytes with 10 μM MG-132 (2194; Cell Signaling Technology) in the RPMI medium at 37°C. We analyzed cell lysates by Western blotting (WB) as described below.

Real-Time PCR

We used SYBR Green–based real-time quantitative PCR to detect adenoviral infection (the amount of adenoviral DNA introduced into cells) and expression of EGFP-Myo1e RNA (Supplemental Figure 2). Primer sequences are listed in the Supplemental Table 1. To verify the delivery of the Myo1e constructs into podocytes, podocytes were washed three times with warm 1× PBS at 24 hours postinfection, and the viral DNA was extracted using the DNeasy Blood and Tissue kit (69504; Qiagen).²⁴ We used the TRIzol method (15596026; Invitrogen) for RNA isolation and the iScript cDNA Synthesis Kit (1708890; Bio-Rad) for cDNA synthesis. We used the iTaq Universal SYBR Green Supermix (172-5121; Bio-Rad) for real-time PCR, which was performed on the Bio-Rad CFX 384 Real-Time PCR System.

Western Blotting and SDS-PAGE

Cells were harvested in CHAPS lysis buffer (20 mM Tris–hydrogen chloride [pH 7.5], 500 mM sodium chloride, 0.5% wt/vol CHAPS) with protease inhibitors (A32965; Thermo Scientific), pipetted up and down 30 times, and then left on ice for 30 minutes before boiling with sample buffer. Before performing WB, we separated each set of lysate samples by SDS-PAGE and stained them with Coomassie blue to verify that total protein amounts (determined for each gel lane using Fiji) were equal between the samples loaded onto each gel. The same amounts of cell lysates as those used for the Coomassie-stained gels were used for WB. Proteins were separated by SDS-PAGE, transferred to polyvinylidene difluoride membrane, and detected using rabbit anti-GFP antibody (A6455; RRID, AB_221570; Thermo Fisher Scientific) and goat anti-rabbit HRP-labeled secondary antibody (Jackson ImmunoResearch). We detected chemiluminescence using the WesternBright Quantum (Advansta) and a Bio-Rad ChemiDoc imaging system, using Image Lab software to quantify WB band intensity (Figure 2, Supplemental Figure 3). Gel images and full-size WB images are shown in Supplemental Appendix I.

Podocyte Culture for Live-Cell Imaging

A total of 5 × 10³ podocytes were plated onto collagen IV–coated (356233; Corning), 35-mm, glass-bottom MatTek dishes (P35G-15-14-C; MatTek) for differentiation for 10 days. At day 10, podocytes were infected with recombinant adenoviruses. After 24 hours of adenoviral transduction, podocytes were washed with warm RPMI media three times and observed using confocal or total internal reflection fluorescence (TIRF) microscopy.

Confocal Microscopy

For cell-cell junction and fluorescence recovery after photobleaching (FRAP) analysis, images were taken using a PerkinElmer UltraView VoX Spinning Disk Confocal system mounted on a Nikon Eclipse Ti-E microscope equipped with a Hamamatsu C9100-50 EMCCD camera and a Nikon Apo TIRF 60× (1.49 N.A.) oil objective, controlled by Volocity software. An environmental chamber was used to maintain cells at 37°C. During imaging, cells were maintained in RPMI supplemented with 10% FBS and 1% antibiotics-antimycotics.

Total Internal Reflection Flourescence Microscopy

For CCV analysis, we imaged cells using a TIRF illumination setup (Nikon) mounted on a Nikon Eclipse TE-2000E inverted microscope equipped with a perfect focus system (Nikon), a Nikon Apo TIRF 100× (1.49 N.A.) oil objective, and Prime 95B camera (Photometrics), controlled by NIS-Elements software. An environmental enclosure was used to maintain cells at 37°C. During imaging, we maintained cells in RPMI supplemented with 10% FBS and 1% antibiotic-antimycotic solution or in imaging buffer (136 mM sodium chloride, 2.5 mM potassium chloride [KCl], 2 mM calcium chloride, 1.3 mM magnesium chloride [MgCl₂], and 10 mM HEPES [pH 7.4]). Both 488-nm and 561-nm laser power were set at 60%. Images were acquired every 5 seconds for 6 minutes, with exposures of 200 or 800 ms for the 561-nm and 800 ms for the 488-nm wavelength.

Fluorescence Intensity and Kymographs

We used the “Measure” and “Multi kymograph” functions in Fiji to collect fluorescence intensity measurements for junctional enrichment (Figure 3, Supplemental Figure 4), FRAP (Figure 3, Supplemental Figure 5), region-of-interest (ROI) mean fluorescence intensity (Supplemental Figures 4, B and C, and 6, B and C), endocytic puncta intensity (Supplemental Figure 7), and endocytic vesicle kymographs (Figure 4D).^15,25–27

Figure 3.

Junctional localization of Myo1e^T119I is disrupted and junctional protein exchange of Myo1e^D388H is decreased in podocytes. (A) Single confocal sections of Myo1e-KO podocytes coexpressing ZO-1–mCherry and EGFP-Myo1e^WT, -Myo1e^T119I, or -Myo1e^D388H. Yellow arrowheads indicate cell-cell junctions. Scale bar, 20 μm. (B) Bar graph of Myo1e enrichment at the cell-cell junctions (mean±SD). A total of 44 Myo1e^WT-, 51 Myo1e^T119I-, and 47 Myo1e^D388H-expressing cells from two to three independent experiments were quantified. (C) Merged single confocal sections of KO podocytes coexpressing EGFP-Myo1e^T119I and ZO-1–mCherry after 10 μM MG-132 treatment for 3 hours. Dotted white boxes indicate the zoomed-in regions shown in the lower panels. Yellow arrowheads point to podocyte junctions. Scale bar, 20 μm. (D) Quantification of junctional protein fluorescence intensity in (C) by line scan. Line scans of junctions without (D, left) and with (D, right) MG-132 treatment. Hollow circles indicate mean fluorescence intensity of ZO-1–mCherry and solid circles indicate mean fluorescence intensity of EFGP-Myo1e^T119I (mean±SD). A total of 40 MG-132 (−) cells and 26 MG-132 (+) cells from one independent experiment were quantified. (E) Graphs of mobile fraction and recovery t_1/2 of EGFP-Myo1e^T119I at cell-cell junctions (E, two left panels) and in the cytosol (E, two right panels) with DMSO or 10 μM MG-132 treatment for 3 hours. DMSO-treated junctional FRAP was quantified from seven Myo1e^WT- and six Myo1e^T119I-expressing cells. MG-132–treated junctional FRAP was quantified from 15 Myo1e^WT- and 11 Myo1e^T119I-expressing cells. DMSO-treated cytosolic FRAP was quantified from four Myo1e^WT- and four Myo1e^T119I-expressing cells. MG-132–treated cytosolic FRAP was quantified from five Myo1e^WT- and seven Myo1e^T119I-expressing cells. Data from one to two independent experiments. (F) Graphs of mobile fraction and recovery t_1/2 of EGFP-Myo1e^WT, Myo1e^D388H, and Myo1e^N164A at cell-cell junctions at steady state. A total of 26 Myo1e^WT-, 33 Myo1e^D388H-, and 33 Myo1e^N164A-expressing cells from two to three independent experiments were quantified. (E–F) Box-and-whisker plots indicate the median value, interquartile range, and full range of data points. Asterisks indicate significant difference from the Myo1e^WT control as determined by one-way ANOVA. *P≤0.05, ***P≤0.001, ****P≤0.0001.

Figure 4.

Myo1e^T119I and Myo1e^D388H expression in place of Myo1e^WT leads to changes in CCV density, peak intensity, and lifetimes in podocytes. (A) Percent (mean±SD) of CCVs that contain Myo1e puncta in KO podocytes expressing Myo1e mutant constructs in a single time point image. (B) Density (mean±SD) of CCV puncta/μm²) in KO podocytes expressing Myo1e mutant constructs. (A–B) Nine KO (clathrin light chain alone, no Myo1e expression), 35 Myo1e^WT-, 13 Myo1e^T119I-, and 26 Myo1e^D388H-expressing cells from one to three independent experiments were quantified. (C) Peak fluorescence intensity (mean±SD) of the CCV tracks that contain Myo1e, as measured by Imaris after local background subtraction. Mean value of each experimental group is shown. A total of 28 Myo1e^WT-, 13 Myo1e^T119I-, and 26 Myo1e^D388H-expressing cells from two to three independent experiments were quantified. (D) Kymographs showing CCV density, dynamics, and Myo1e recruitment in Myo1e^WT-, Myo1e^T119I-, and Myo1e^D388H-expressing KO podocytes. (E) Distribution of CCV lifetimes. (F) Distribution of the lifetimes of CCVs that contain Myo1e. (E–F) Lifetimes of CCVs were measured and categorized into abortive (≤20 seconds), productive (>20 but ≤90 seconds), long-lived (>90 but ≤300 seconds), and stable (>300 seconds). The total number of puncta analyzed is indicated below each construct. (G) Duration of Myo1e association with CCVs expressed as a fraction of the CCV lifetime. Only subpopulations with the lifetimes >20 seconds were analyzed. (H) Duration of Myo1e association with CCVs for the CCVs that exhibited only a single Myo1e association event and CCV lifetimes >20 seconds. (G–H) Duration of Myo1e association was measured and categorized into quartiles (≤25% of CCV track duration, >25% but ≤50% of CCV track duration, >50% but ≤75% of CCV track duration, >75% of CCV track duration). (E–H) Percent values of events are indicated on the bar plots. A total of 28 Myo1e^WT-, 13 Myo1e^T119I-, and 26 Myo1e^D388H-expressing cells from two to three independent experiments were quantified. Asterisks indicate a significant difference from Myo1e^WT, as determined by one-way ANOVA. ***P≤0.001, ****P≤0.0001.

Fluorescence Recovery after Photobleaching Imaging and Analysis

For photobleaching, a 488-nm argon laser with full power was used to bleach a 30 × 75 pixel ROI at the podocyte junctions. We collected five frames of prebleaching images at one frame per second. Postbleaching images were acquired using the 488-nm laser at 25% of laser power; the acquisition rate for the first 40 seconds was set to two frames per second, and to one frame every 2 seconds for the subsequent 120 seconds. Fluorescence intensity of the bleached area was measured over time and normalized relative to the background, a control ROI (to correct for acquisition bleaching), and the prebleaching images. Lastly, the fluorescence intensity of the ROI at the first time point after bleaching was set to zero. The best-fit curve for fluorescence recovery was obtained using the exponential one-phase association model in Prism 8. We used the following equation: y=a(1−e^−bx), where x is time in seconds.²⁶ The t_1/2 of recovery was determined using b from the previous equation, where t_1/2=ln 0.5/−b. We performed analysis on 16-bit images. Occasionally, cells contracted or expanded slightly during imaging, which, in some cases, resulted in mobile fraction values above one. Although we excluded from analysis the cells that exhibited a dramatic retraction or change in position, we did not exclude the cells that exhibited subtle motion during the recovery period to avoid arbitrary exclusion of datasets.

Endocytic Vesicle Detection, Tracking, and Analysis

We used Imaris software (version 9.7.1; Bitplane Inc.) to perform quantification of the CCVs and Myo1e colocalization (Figure 4A), density (Figure 4B), peak intensity (Figure 4C), and lifetimes (Figure 4, E and F). CCVs were automatically detected using the spot module and particle tracking, following previously described procedures with additional modifications.²⁸ We first subjected TIRF microscopy images to ROI segmentation (to obtain images with an area of about 900 μm²) and background subtraction. We then detected CCVs using the spot module, with an estimated spot diameter of 0.5 μm. The detected vesicles were filtered with the Quality function in Imaris (defined as the intensity at the center of the spot, Gaussian filtered by three quarters of the spot radius). The threshold values of Quality were set to minimize the noise (as determined by visual inspection). The Brownian motion particle-tracking algorithm was applied to trace objects through sequential frames of the time-lapse movies. If the distance between the candidate spot position and the predicted future position exceeded the maximum distance of 0.35 μm, the connections between a spot and a future position match were rejected. To connect tracks that were segmented due to the object being temporarily out of focus, the maximum permissible gap length was set to one frame. Track outputs were then edited to correct for tracking errors by visual inspection. Only tracks that appeared from the first to the last frame (stable CCVs) and those that appeared and disappeared (dynamic CCVs) during the image acquisition (72 or 73 frames, 6 minutes) were subjected to lifetime analysis.^28,29

After the ROI segmentation for CCV tracking, Myo1e puncta were detected using the surface module (without creating tracks for the Myo1e particles). The puncta were first background subtracted and then the surface grain size was set to 0.1 μm and the diameter of the largest sphere was set to 0.28 μm. In addition, split touching objects (region growing) was enabled, and seed point diameter was set to 0.4 μm. The thresholds for background subtraction, quality, and number of voxels were set on the basis of visual inspection to minimize noise.

To collect the CCV-Myo1e colocalization information, object-object statistics in the CCV module was applied and CCV tracks were temporarily unlinked. Next, the shortest distance of CCV spot to Myo1e surface was filtered by setting the threshold to the maximum of 0.25 μm. Percentages of CCVs containing Myo1e (CCV-Myo1e colocalization rate; Figure 4A) were counted in a selected frame (the frame that is equivalent to the 90 second time point in a 6-minute movie). Further, the selected (Myo1e-colocalized) CCVs were tagged, and the data for mean intensity (Figure 4C), track duration (Figure 4, E and F), and shortest distance to the Myo1e surface (Figure 4, G and H) were extracted from the statistics section. Additionally, Gaussian filter (threshold=0.111) and local background subtraction (threshold=0.5 μm) were applied before collecting CCV peak intensity.

CCV intensity (Figure 4C), lifetimes (Figure 4, E and F), and association duration (Figure 4, G and H) data collected from Imaris were first processed manually for statistical analysis. To reduce mistakes during repeated manual data processing, the data were then rearranged using Python code (https://github.com/fomightez/pjl), relying mainly on the Pandas module; the scripts were generously implemented by Wayne A. Decatur and provided within active Jupyter-based sessions served via MyBinder.org. For data rearrangement for CCV intensity analysis (Figure 4C), maximum intensity value in each track was extracted and rearranged in a new list, with each list corresponding to a different experimental group for statistical analysis. For CCV lifetimes (Figure 4, E and F), in each experimental group, the values of track duration were moved to a new list for statistical analysis. For the association duration (Figure 4G), the total frames of a track and the frames with the CCV-Myo1e distance of ≤0.25 μm were counted. In the resulting list, if the total number of frames was five or less (abortive CCVs), the track was removed from further analysis. Lastly, to calculate the duration of the Myo1e-CCV contact, the number of frames where Myo1e was associated with the CCV was divided by the total number of frames. The values of each experimental group were moved to a list for statistical analysis. Furthermore, identification of the CCVs that were only associated with Myo1e for a single frame was performed using C# code (https://github.com/opidopi/DataFilter), which was kindly created by Mr. Sean M. Lantry.

Expression and Purification of Myo1e for Motor Function Assays

We produced the recombinant baculoviruses expressing Myo1e constructs containing the motor and IQ domain and the carboxy-terminal (C-terminal) GFP, Avi, and FLAG tags as described previously.^30–32 The C-terminal tags were built into the Myo1e constructs to facilitate protein purification and motility assays. WT and D388H Myo1e were coexpressed with calmodulin in SF9 cells and purified with anti-FLAG affinity chromatography.³³ The purified Myo1e constructs were examined by Coomassie blue staining of SDS-PAGE gels, and concentrations were determined by GFP absorbance (ε_{488 nm}=55,000 M⁻¹·cm⁻¹) and Bradford assays using BSA as a standard. Actin was purified from acetone powder derived from rabbit skeletal muscle (Pell-freeze), using the method of Pardee and Spudich.³⁴

Steady-State ATPase Measurements

We examined the actin-activated ATPase activity of purified Myo1e using the NADH-coupled assay³⁵ in KMg50 buffer (10 mM imidazole [pH 7.0], 50 mM KCl, 1 mM EGTA, 2 mM MgCl₂, and 1 mM dithiothreitol) at 25°C. The absorbance of NADH was examined over a 200-second period in an Applied Photophysics Stopped-Flow apparatus. We used a standard curve of known ADP concentrations to determine the absorbance units per concentration of ADP. The ATPase rates (V) were plotted as a function of actin concentration, and the data were fit to a Michaelis–Menten equation (V=V₀+[k_cat×[actin]])/(K_ATPase+[actin]), which allowed determination of the maximum rate of ATPase activity, reported as the addition of the ATPase in the absence of actin (V₀) and the actin-activated ATPase (k_cat) (V_Max=V₀+k_cat), and actin concentration ([actin]) at which ATPase activity is one-half maximal (K_ATPase). To remove nonfunctional Myo1e motors, we first bound Myo1e to actin, pelleted the actomyosin using ultracentrifugation (10 minutes at 95,000 rpm in a TLA.120.2 rotor at 4°C), and subsequently released the Myo1e from actin with a second ultracentrifugation step in the presence of 2 mM ATP and additional salt (final 150 mM KCl). The Myo1e released from actin in the presence of ATP was used directly in the ATPase and motility assays.

Single Nucleotide Turnover

We used mant-labeled ATP (mantATP) to perform single ATP turnover experiments with FLAG-purified WT and D388H Myo1e in the absence of actin. A substoichiometric concentration of mantATP was mixed with Myo1e in the stopped-flow apparatus, and mant fluorescence was examined over a 500-second period. The mant fluorescence, which increases when bound to myosin, was excited at 290 nm, and the emission measured with a 395-nm, long-pass filter. The fluorescence decays were fit to the exponential equation that best fit the data.

In Vitro Motility

The in vitro actin-gliding assay was performed as previously described.^36,37 Although, in our design, either the anti-GFP antibody or biotin-streptavidin bonds could be used to attach Myo1e to the coverslip, we found that attachment of the EGFP-tagged Myo1e^WT to the coverslip via an anti-GFP antibody supported robust motor activity in vitro and that biotinylation was not needed to provide strong adhesion to the motility chamber. Myo1e was adhered to 1% nitrocellulose-coated coverslips that contained anti-GFP antibody (0.1 mg/ml), and the surface was blocked with BSA (2 mg/ml). Sheared unlabeled actin (2 µM) followed by 2 mM ATP were added to block the dead myosin heads on the surface. The activation buffer, containing KMg50 buffer, 0.35% methylcellulose, 0.45 mM phosphoenolpyruvate, 45 U/ml pyruvate kinase, 0.1 mg/ml glucose oxidase, 5 mg/ml glucose, 0.018 mg/ml catalase, and 2 mM ATP was added right before video acquisition. Alexa 555 phalloidin-labeled actin was visualized with a Nikon TE2000 fluorescence microscope equipped with a 60×/1.4 N.A. lens and a Coolsnap HQ2 cooled CCD camera controlled using Nikon Elements 3.0. Videos were collected for up to 10 minutes at a 10-second frame rate. The velocities were manually analyzed by tracking actin filaments using MTrackJ in ImageJ.³⁸

Protein Sequence Alignment and Structure Modeling

Protein sequence alignments were performed using the ClustalW alignment function in MacVector (version 17.0.10). The sequence accession numbers are listed in Supplemental Table 2. Protein structure modeling and rotamer predictions were performed using UCSF Chimera.³⁹

Statistical Analysis

For multiple comparisons of the WT and mutants, data were analyzed using a one-way ANOVA with Tukey post hoc test, with statistical significance set at P≤0.05. All statistical analyses and graphing were performed using GraphPad Prism software.

Results

MYO1E Variants Found in Patients with Nephrotic Syndrome and Their Predicted Effects on Protein Structure

Genomic sequencing has identified a number of disease-associated variants in MYO1E.^5–8 Patients homozygous for MYO1E^T119I or MYO1E^D388H presented with FSGS on their renal biopsy specimens and were resistant to steroid treatment (Supplemental Table 3).^6,7 T119 and D388 are highly conserved residues in the myosin motor domain (Figure 1, A–C). T119 is located within the P-loop, which, along with the switch-1 region, constitutes the structural elements essential for nucleotide binding⁴⁰ and sensing whether the bound nucleotide is ATP or ADP.⁴¹ D388 is in the switch-2 region, which connects with the relay helix to convey the conformational changes to the converter domain during the ATPase cycle.^42,43 To determine whether these mutations affect the key elements that are involved in myosin activity, we selected a class 1 myosin for which the crystal structure has been determined, namely, the short-tailed Dictyostelium myosin myo1E.⁴⁴ By introducing the corresponding mutations into the myo1E structure and modeling possible side-chain orientations, we found several possible clashes between the mutated residues and other amino acid residues in the motor domain (Figure 1, D–F). On the basis of this modeling, the T119I and D388H mutations may affect the ATPase activity of Myo1e. We then set out to experimentally test the effects of these mutations.

Figure 1.

Amino acid residues T119 and D388 in the Myo1e motor domain are highly conserved. (A) Domain map of human Myo1e indicating the locations of the SRNS-associated Myo1e mutations T119I and D388H. (B and C) Alignment of myosin protein sequences for the regions containing (B) T119 and (C) D388 residues. Both amino acid residues are conserved among class 1 myosins, including long-tailed class-1 myosins from Caenorhabditis elegans (worm), Dictyostelium discoideum (slime mold), S. pombe (fission yeast), and S. cerevisiae (budding yeast); the short-tailed class 1 myosin myo1E of D. discoideum that we used for the rotamer analysis; and in myosin 1e of Mus musculus (mouse), Gallus gallus (chicken), Xenopus laevis (frog), and Danio rerio (zebrafish). These residues are also conserved in other myosin classes, including human nonmuscle myosin 2a–c, 5a, and 6. The conserved residues corresponding to the mutation site are highlighted in blue boxes. (D) Ribbon diagram representation of the ADP-bound D. discoideum myo1E motor domain (PDB: 1LKX) showing the residues T108 and D386, which are equivalent to the human Myo1e residues T119 and D388. Their locations are shown relative to the key structural elements of the magnesium-binding site. The T108 residue in myo1E binds the magnesium ion associated with the ATP. The backbone of the myo1E structure is shown as a blue ribbon. P-loop is highlighted as a green ribbon. Switch-1 is highlighted as an orange ribbon. Switch-2 is highlighted as a cyan ribbon. Magnesium is highlighted in pink. D. discoideum myo1E residue numbers are shown. (E) A more detailed view of the T108 residue (green). Upon changing the threonine at position 108 to isoleucine to model the most likely effects of the T118I mutation, the isoleucine side chain clashed with the magnesium ion and switch-1 residue S158. (F) A more detailed view of the D386 residue (cyan). Replacing D386 with a histidine, we modeled a number of possible histidine side-chain orientations. In rotamer 1 (probability score, 0.18), the histidine clashed with S111 on the α-helix that connects to the P-loop (F, upper panel). In rotamer 2 (probability score, 0.13), this histidine clashed with the T108 on the P-loop, which is the above-mentioned residue binding to the magnesium ion (F, lower panel). In both cases, the D386H mutation may disrupt the P-loop interaction with magnesium-ATP.

Expression of the Steroid-Resistant Nephrotic Syndrome-Associated Myo1e Variants as Recombinant Proteins in Podocyte-Derived Cell Lines

To investigate whether the T119I and D388H mutations affect Myo1e protein stability, we expressed amino-terminally EGFP-tagged human Myo1e constructs in several cell lines, including previously described, conditionally immortalized, podocyte-derived cells obtained from Myo1e-KO mice (KO podocytes).^12,15 These cells can be differentiated in culture and used in conjunction with adenoviral vectors for protein expression.²³ Because many cell lines, including podocyte-derived immortalized cells, may undergo changes in protein expression and morphology in culture, we performed transcriptomic characterization of the Myo1e-KO cell line used in this study and compared it with the mouse podocyte–derived cell line obtained from WT mice (MPCs).¹⁹ Our transcriptomic analysis revealed that both Myo1e-KO cells and MPCs expressed many podocyte markers, although expression levels of some markers differed between the two cell lines, and some transcripts, such as nephrin and podocin, were not detectable in either cell line, in agreement with previous observations (Supplemental Figure 1).²² Due to the subtle differences between the Myo1e-KO podocyte–derived cells (KO podocytes) and MPCs (WT podocytes), we used both cell lines for initial testing of Myo1e stability, although primarily KO podocytes were used to examine Myo1e localization and activity.

Full-length proteins corresponding to the EGFP-myo1e fusions were detected by WB in the Myo1e^WT-, Myo1e^T119I-, and Myo1e^D388H-transduced cells at approximately equal levels (Figure 2, A and B, Supplemental Figure 2, A and B). Additionally, average RNA expression for the mutants was 50 times higher than for Myo1e^WT (Supplemental Figure 2C). The increased mRNA expression/stability could potentially compensate for any changes in protein stability. To examine the rate of turnover of mutant versus WT proteins, we treated Myo1e-expressing podocytes with CHX, a ribosome inhibitor, to stop protein expression and measure Myo1e degradation over time. The decrease in the mutant Myo1e protein level was not significantly different from that in the Myo1e^WT (Figure 2, C and D, Supplemental Figure 2D). In addition, we inhibited proteasomal protein degradation in the Myo1e-KO podocytes using MG-132 and found that accumulation of Myo1e^T119I or Myo1e^D388H was not significantly different from that of Myo1e^WT (Figure 2, E and F, Supplemental Figure 2E). On the other hand, when protein expression level and protein turnover were examined using expression of EGFP-Myo1e in WT mouse podocytes or in HEK-293 human cells, Myo1e^T119I was found to be expressed at a lower level than Myo1e^WT and was rapidly degraded (Supplemental Figure 3). Similarly, our attempts to express and purify Myo1e^T119I in the baculovirus system resulted in very low protein yield and significant protein degradation. Myo1e^D388H was less stable than Myo1e^WT when expressed in the baculovirus system, but did not exhibit increased degradation in the WT podocytes or HEK-293 cells. Overall, although the Myo1e^T119I and Myo1e^D388H variants exhibit no evidence of increased degradation in Myo1e-KO podocytes, these mutations may affect Myo1e stability in other cell types or in the presence of the WT Myo1e.

Figure 2.

Myo1e^T119I and Myo1e^D388H are expressed as full-length proteins in Myo1e-KO podocytes. (A) WB analysis of total cell lysates of Myo1e-KO podocytes expressing Myo1e^WT, Myo1e^T119I, and Myo1e^D388H at steady state. (B) Quantification of band intensity in (A), normalized to the WT (mean±SD). (C) WB analysis of total cell lysates of Myo1e-KO podocytes expressing Myo1e^WT, Myo1e^T119I, and Myo1e^D388H treated with 20 μg/ml CHX or DMSO for 3 hours. (D) Measurement of Myo1e degradation (band intensity in the DMSO-treated sample divided by the band intensity in the CHX-treated sample for the same construct) after CHX treatment in KO podocytes (mean±SD). (E) WB analysis of total cell lysates of Myo1e-KO podocytes expressing Myo1e^WT, Myo1e^T119I, and Myo1e^D388H treated with DMSO or 10 μM MG-132 for 3 hours. (F) Measurement of Myo1e accumulation (band intensity in the MG-132–treated lysates divided by band intensity in the DMSO control) after the MG-132 treatment (mean±SD). (A, C, and E) Blots were probed with the anti-GFP antibody. Equal protein loading verified by Coomassie blue staining. (B, D, and F) Data collected from three independent experiments. There is no significant difference (P>0.05) among groups as determined by one-way ANOVA.

Myo1e^T119I Localization to Podocyte Cell-Cell Junctions Is Disrupted

Because Myo1e localizes to cell-cell junctions in podocytes,^5,15 we set out to determine whether the SRNS-associated mutations affect Myo1e localization to cell-cell contacts. Whereas EGFP-Myo1e^WT and Myo1e^D388H colocalized with the junctional marker mCherry-ZO-1, Myo1e^T119I was mostly absent from cell-cell contacts (Figure 3, A and B, Supplemental Figure 4A). Myo1e junctional enrichment did not correlate with the level of Myo1e or ZO-1 expression in each cell (Supplemental Figure 4, B and C), indicating differences in expression between cells likely do not affect the outcome of this analysis.

Because Myo1e^T119I protein level was elevated after proteasomal inhibition (Supplemental Figure 2E), we tested if increasing mutant protein availability would enhance its localization to the cell-cell junctions. After MG-132 treatment, Myo1e^T119I (but not Myo1e^WT) formed patchy clumps in the cytosol that, in some cases, aligned with cell-cell contacts (Figure 3C, Supplemental Figure 4D), but were not precisely colocalized with ZO-1 (Figure 3D). To test whether Myo1e^T119I patches in MG-132–treated cells may represent misfolded/immobilized protein aggregates, we measured EGFP-Myo1e^T119I dynamics using FRAP. Compared with the Myo1e^WT or DMSO-treated Myo1e^T119I, Myo1e^T119I puncta at the junctions and the cytosol exhibited reduced fluorescence recovery after MG-132 treatment (Figure 3E). In addition, in the MG-132–treated cells, Myo1e^T119I and Myo1e^D388H had lower solubility than Myo1e^WT, with the majority of the protein remaining in the pellet fraction after cell lysis (Supplemental Figure 4, E and F). Thus, when the misfolded Myo1e^T119I protein is prevented from being degraded by proteasomes, it forms immobile protein aggregates.

Myo1e^D388H Localizes to Cell-Cell Junctions but Exhibits Decreased Dissociation from the Junctions

Because Myo1e^D388H localized to cell-cell junctions (Figure 3, A and B), we examined the dissociation of Myo1e^D388H from cell-cell contacts using FRAP analysis (Figure 3F, Supplemental Figure 5). Myo1e^N164A, designed to be a rigor mutant with strong actin binding,⁴⁵ was used as a positive control for FRAP experiments. Both Myo1e^D388H and Myo1e^N164A exhibited decreased dissociation from cell-cell junctions (Figure 3F, Supplemental Figure 5, B and C, Supplemental Video 1). Thus, Myo1e^D388H mutation changes Myo1e-actin binding or other protein-protein interactions at cell-cell junctions, resulting in slower protein exchange.

Expression of Myo1e^T119I or Myo1e^D388H Affects Clathrin-Coated Vesicle Dynamics

To test whether mutations affect Myo1e localization to CCVs, we coexpressed mCherry-tagged clathrin light chain and EGFP-Myo1e in KO podocytes and used TIRF microscopy to image CCVs (Supplemental Figure 6A). The extent of CCV/Myo1e colocalization was significantly lower for the Myo1e^T119I than for Myo1e^WT and Myo1e^D388H (Figure 4A).

The density of CCVs was significantly reduced in cells expressing Myo1e^T119I and significantly increased in cells expressing Myo1e^D388H compared with the KO podocytes expressing Myo1e^WT (Figure 4B). The CCV density in the KO podocytes expressing Myo1e^T119I was similar to the Myo1e-uninfected KO podocytes (Figure 4B). Peak clathrin intensity in the Myo1e-containing CCVs was significantly increased in the Myo1e^D388H-expressing KO podocytes, but no change was found in Myo1e^T119I-expressing cells compared with the Myo1e^WT-expressing cells (Figure 4C, Supplemental Figure 7). These findings suggest Myo1e not only influences vesicle internalization or scission, as previously suggested, but may also affect clathrin recruitment or CCV stabilization during the earlier steps in endocytosis, affecting the number and size of CCVs.

Myo1e is recruited to endocytic invaginations immediately before the internalization of CCVs.¹⁶ Whereas Myo1e^WT was transiently recruited to CCVs immediately before their disappearance from the TIRF microscopy image, Myo1e^T119I localization was diffuse, and very few instances of its transient recruitment to CCVs were observed (Figure 4D). However, Myo1e^D388H formed longer-lived and numerous puncta, some of which were colocalized with CCVs (Figure 4D). By tracking mCherry-tagged clathrin light chain in time-lapse series, we classified CCV lifetimes into abortive (≤20 seconds), productive (>20 but ≤90 seconds), long-lived (>90 but ≤300 seconds), and stable (>300 seconds).^25,46 Both Myo1e^T119I- and Myo1e^D388H-expressing cells contained a higher fraction of abortive CCVs than Myo1e^WT-expressing cells (Figure 4E). We then examined separately the lifetimes of CCVs that contained Myo1e; they were characterized by a lower proportion of abortive CCVs than the general CCV population (Figure 4F). There was a further decrease in the abortive CCV population, and an increase in the stable CCV population among Myo1e^T119I-positive CCVs, compared with the Myo1e^WT-positive CCVs (Figure 4F), although this analysis may be complicated by the low number of the Myo1e^T119I-positive CCVs (Figure 4A). In contrast, in the Myo1e^D388H-expressing cells, the proportion of abortive CCVs was elevated and that of the stable population was reduced (Figure 4F). We next measured the duration of Myo1e association with the CCVs as a fraction of each CCV’s lifetime. In the Myo1e^T119I-expressing cells, 91% of the Myo1e-containing CCVs were associated with Myo1e for ≤25% of the track lifetime, whereas this short-duration fraction constituted only 43.61% of the CCVs in the Myo1e^WT-expressing cells (Figure 4G). In some cases, Myo1e was recruited to a single CCV track multiple times. Because the nature of these repeated Myo1e-CCV interactions is unclear, we excluded these CCVs from our analysis and reanalyzed Myo1e association for those CCVs that exhibited only a single event of Myo1e recruitment (Figure 4H). With this analysis, the Myo1e^D388H was characterized by a higher proportion of the CCVs that exhibited prolonged association with Myo1e compared with the Myo1e^WT (Figure 4H). Overall, our analysis indicates Myo1e^T119I is not recruited to CCVs to the same extent as Myo1e^WT, and expression of this variant affects CCV density and internalization, whereas Myo1e^D388H is characterized by the prolonged retention at the sites of CME.

Steroid-Resistant Nephrotic Syndrome-Associated Mutations Affect Myo1e Motor Activity

We used the baculovirus system to express and purify a Myo1e construct for enzymatic and biophysical characterization.¹⁰ In an earlier study, a truncated Myo1e protein, lacking the C-terminal tail but containing the motor domain and calmodulin-binding neck region, was used for enzymatic studies. We expressed a similar version but added C-terminal tags for motility assays: an EGFP tag that could elevate the myosin motor domain above the coverslip to avoid steric interference with the motor activity, and an Avi-tag that could be used to couple the motor to the coverslip via biotin-streptavidin bonds (Figure 5A).

Figure 5.

D388H mutation in Myo1e disrupts myosin ATPase activity. (A) Myo1e construct design for baculovirus expression. The star indicates the point mutation of D388H (c.1162G>C) on Myo1e motor domain. (B) Actin-dependent ATPase activity of the FLAG- and actin-cosedimentation–purified Myo1e constructs was measured using the NADH-coupled ATPase assay. The data points represent the mean±SD from three independent experiments for both WT and D388H. The solid lines are best fit to a Michaelis–Menten model. (C) Single ATP turnover kinetics using FLAG-purified Myo1e in the absence of actin. Substoichiometric amount of mantATP was mixed with Myo1e. We observed a fluorescence increase that was followed by an exponential decrease, which was used to estimate the single turnover rate constant. (D) The fluorescence decay of Myo1e^WT was fit to a three-exponential equation, (E) whereas the decay of Myo1e^D388H was single exponential (Supplemental Table 2). The single turnover data were collected from a single experiment for both WT and D388H.

Due to the susceptibility of Myo1e^T119I to proteolysis, we were not able to purify enough of this protein to perform in vitro assays. Myo1e^D388H was more stable, but still exhibited elevated degradation compared with Myo1e^WT (Supplemental Figure 8A). To further purify Myo1e before performing the motor characterization assays, we performed actin cosedimentation and ATP release, which isolates the enzymatically “active” myosin (e.g., myosin that binds actin in an ATP-dependent manner). The proteolytic bands remained even after this additional purification in both Myo1e^WT and Myo1e^D388H, albeit the latter showed more proteolysis than the former (Supplemental Figure 8A). Thus, the proteolytic cleavage may occur on a surface loop instead of the key structural elements of the motor domain. Therefore, we used both actin-purified and standard myosin preparations in our experiments.

We measured Myo1e ATPase activity in the presence of varying actin concentrations (Figure 5B, Supplemental Figure 8B). In the absence of actin, Myo1e^WT exhibited relatively high basal ATPase activity (V₀=0.82±0.05 seconds⁻¹), which was observed in a previous study¹⁰ and is unusual for myosins. In the presence of actin, ATPase was maximally increased approximately 30% (V_Max=1.05±0.08 seconds⁻¹). The basal ATPase activity of Myo1e^D388H was substantially reduced (V₀=0.09±0.04 seconds⁻¹) compared with that of Myo1e^WT. However, the ATPase activity was activated approximately two-fold in the presence of actin (V_Max=0.21±0.07 seconds⁻¹). Overall, the maximum ATPase rate (V_Max) of Myo1e^WT was five times higher than that of Myo1e^D388H. The actin concentration needed to achieve half-maximum ATPase activity (K_ATPase [µM]) was similar in WT and mutant Myo1e (Figure 5B, Supplemental Table 1). The measurements of Myo1e ATPase activity were also examined without the extra step of purifying by actin cosedimentation (FLAG purified), which demonstrated the actin cosedimentation step slightly increased the maximum ATPase activity, but did not change the relative differences observed between Myo1e^WT and Myo1e^D388H (Supplemental Figure 8, B and C). To further examine the ATPase kinetics in the absence of actin, single ATP turnover experiments were performed on FLAG-purified Myo1e. By mixing Myo1e with fluorescently labeled ATP (mantATP) we observed a fluorescence increase, associated with mantATP binding to myosin, followed by a fluorescence decay, which represents the period of time required for ATP hydrolysis, phosphate release, and then mantADP release (single turnover). We found the fluorescence decay was best fit by a single exponential function for Myo1e^D388H but was triphasic for Myo1e^WT (Figure 5, C–E). The results showed the fast phase of the fluorescence decay in Myo1e^WT is most similar to the basal ATPase rate constant, whereas the slower phases may represent a different conformation of Myo1e that releases products slowly. Myo1e^D388H was able to bind and hydrolyze ATP, and the single turnover rate constant was relatively similar to the basal ATPase rate measured in the steady-state ATPase assay (Figure 5, C–E, Supplemental Table 2).

To investigate the functional consequences of the reduced ATPase activity in Myo1e^D388H, we performed in vitro motility measurements with the Myo1e motor-IQ constructs (WT and D388H) (Figure 6). Using comparable motor densities, Myo1e^WT supported robust F-actin sliding (V_avg=117.9±49.4 nm/s, at 1 µM Myo1e surface density) (Figure 6A, Supplemental Table 3, Supplemental Video 2), whereas no F-actin translocation was detected in the assays with the Myo1e^D388H preparations at any density (Supplemental Table 3, Supplemental Video 2). When analyzing the motor density dependence of the Myo1e^WT-mediated motility, we observed some variability but overall similar velocities at varying motor densities (Figure 6B). Finally, by mixing 1 μM Myo1e^WT with varying amounts of Myo1e^D388H, we found the observed velocity of F-actin decreased with the increasing amount of Myo1e^D388H. This indicates Myo1e^D388H can bind to F-actin and slow down Myo1e^WT motility (Figure 6C, Supplemental Video 2).

Figure 6.

Myo1e^D388H lacks motor activity. (A) Actin filament translocation in in vitro motility assays using FLAG- and actin-cosedimentation–purified Myo1e constructs (1 μM surface density). The average sliding velocity (actin translocation speed) of Myo1e was determined by pooling the filaments from four independent experiments, each with a separate protein preparation (n=121 filaments, approximately 30 filaments from each preparation) and fitting the data to a Gaussian function. (B) The sliding velocity of Myo1e^WT was examined at a series of loading concentrations. The data are the summary of two independent experiments from two separate protein preparations. (C) Myo1e^WT was slowed by the presence of Myo1e^D388H in a dose-dependent manner in the mixed motility assay. The average sliding velocity is plotted as the function of Myo1e^D388H present (concentration of WT=1 µM and varying concentration of D388H) collected from one independent experiment. [D388H Myo1e], D388H Myo1e concentration; [Myo1e], Myo1e concentration.

Discussion

The assignment of gene variants as pathogenic or benign can be challenging, and direct functional testing of the effects of the mutations represents one of the key lines of evidence for variant annotation.^47–49 In this study, we used a combination of cell biology and biochemistry approaches to evaluate the effects of two SRNS-associated MYO1E mutations. In addition to identifying sensitive readouts for Myo1e activity, we also revealed the effects of the loss of Myo1e activity on specific podocyte functions, such as endocytosis. Thus, this work not only demonstrates that the two missense variants in Myo1e lead to functional defects but also sheds new light on the roles of Myo1e in podocytes and lays foundation for developing functional assays that can be used for MYO1E variant annotation in the future.

We hypothesized that mutations could affect Myo1e folding and degradation. Unexpectedly, we found the stability of the Myo1e variants varied depending on the cell type used for their expression, with the stability of Myo1e^T119I drastically decreased in cells containing endogenous Myo1e (WT podocytes and HEK-293 cells), but not in the Myo1e-KO podocytes. When we previously introduced a Myo1e^T119I equivalent mutation into the genomic copy of the fission yeast Schizosaccharomyces pombe class 1 myosin, Myo1, the mutant protein was stably expressed and did not exhibit any evidence of degradation or misfolding.⁵⁰ Our findings indicate Myo1e^T119I and even Myo1e^D388H may exhibit decreased stability in some cells, although the mechanism resulting in differences in protein degradation between cell types is unknown. Whether this loss of stability may be sufficient to completely disrupt Myo1e activity and lead to SRNS is unclear because the mutant proteins are still expressed in Myo1e-null podocytes. Overall, due to the high variability of the mutant protein expression in different cell types, we conclude WB should be used primarily as an adjunct to the microscopy-based assays (to verify that recombinant Myo1e used in these assays is present as a full-length protein), but not as the main readout for protein stability.

Junctional localization analysis revealed Myo1e^T119I was deficient in the ability to localize to podocyte cell-cell contacts, whereas Myo1e^D388H had a lower junctional dissociation rate than Myo1e^WT. The inability of Myo1e to serve as a dynamic membrane-actin linker at the cell-cell junctions may result in the disruption of the organization or dynamics of the slit diaphragm complexes, which are critical for glomerular integrity.^51,52

Analysis of CME provides another sensitive and quantitative readout for Myo1e activity. The involvement of class 1 myosins, such as Myo1e in mammalian cells and its yeast homologs, Myo3/5 (budding yeast) and Myo1 (fission yeast), in endocytosis is highly conserved.⁵³ Overexpression of the Myo1e-tail construct (lacking the motor domain) inhibits transferrin uptake in HeLa cells,¹⁷ and introduction of motor domain mutations into the single genomic copy of Myo1 in fission yeast inhibits endocytic internalization.⁵⁰ Whereas internalization of endocytic cargo has been traditionally used to measure the efficiency of endocytosis, either bulk cargo internalization measurements or measurements of CCV movement away from the plasma membrane may not accurately reflect changes in the early steps of clathrin coat assembly, such as initiation or stabilization of clathrin coats.^54,55 In this study, detailed tracking of CCV intensities and lifetimes and Myo1e localization to CCVs enabled identification of the subtle differences between the WT and mutant Myo1e. Specifically, the extent of colocalization between Myo1e and CCVs, the duration of Myo1e association with CCVs, the density of CCVs at the plasma membrane, and the distribution of CCV lifetimes were all affected by the expression of the Myo1e variants.

Our analysis of the CCV dynamics in the Myo1e-KO cells expressing Myo1e constructs revealed differences between the two mutations and unexpected contributions of Myo1e to CME. Previously, Myo1e was thought to act solely as a component of the vesicle internalization and scission machinery on the basis of the timing of Myo1e recruitment¹⁶ and its interactions with dynamin and synaptojanin¹⁷ and with actin assembly regulators.^25,56 Indeed, deletion of Myo1e homologs in yeast slows down the endocytic pit invagination and actin assembly.^57,58 Thus, in cells lacking active Myo1e, we expected to observe prolonged CCV lifetimes, representing less efficient scission, and the presence of a large fraction of stable (noninternalizing) CCVs. Indeed, we found the lifetimes of Myo1e-containing CCVs were increased in the Myo1e^T119I-expressing cells. Because Myo1e^T119I exhibits weak colocalization with the CCVs, it may not be able to contribute to scission. However, we also made some unexpected findings: the absence of Myo1e or the expression of Myo1e^T119I were associated with decreased density of CCVs on the plasma membrane, suggesting Myo1e may contribute to the initial steps in clathrin coat assembly or stabilization. Indeed, a recent survey of the functional effects of the knockdowns of endocytic accessory proteins, which relied on the automated CCV analysis, placed Myo1e into the group of accessory proteins that regulate CCV stabilization,⁵⁴ suggesting sensitive approaches to the analysis of CCV dynamics can reveal novel roles for some endocytic proteins, such as Myo1e. Unlike expression of Myo1e^T119I, Myo1e^D388H expression was associated with increased CCV density and intensity. Thus, overall, decreased interactions between Myo1e and CCVs result in a decrease in both CCV assembly (reduced density) and internalization (extended lifetimes), whereas prolonged interactions lead to the increased density and intensity of the CCVs. In a model of Drosophila nephrocytes, dynamin-dependent CME was recently shown to internalize nephrin proteins that are not in the lipid raft.⁵⁹ Thus, the interaction of Myo1e and dynamin may assist in maintaining a functional slit diaphragm and prevent filter clogging by removing defective nephrin.^17,18,59 Additional insight into Myo1e functions in CME may be gained in the future by using specific endocytic cargos in podocytes⁶⁰ to develop new endocytosis assays.

ATPase activity measurements and in vitro motor activity assays represent the most direct tests of motor domain function. Previously, Myo1e ATPase activity was characterized using a purified protein consisting of the motor and neck domains,¹⁰ although in vitro motility was not studied. We were able to measure Myo1e-driven actin motility for the first time, resulting in an estimate of the actin sliding speed of approximately 120 nm/s, which is faster than some of the short-tailed class 1 myosins, Myo1a, with the actin sliding speed of 28 nm/s,⁶¹ and Myo1b, with the speed of 56 nm/s,⁶² and comparable with the short-tailed Myo1c at 83 nm/s.⁶³ The high basal ATPase activity and maximal ATPase activity of Myo1e^WT is consistent with the previous kinetics study,¹⁰ but the concentration of actin required to activate ATPase activity is ten times higher in this study than that of the previous study (K_ATPase=10.7±7.5 μM in this study versus 1.2±0.36 μM in that of El Mezgueldi et al.¹⁰). The buffer in this study contained a two-fold higher MgCl₂ concentration, which can have an effect on myosin ATPase kinetics.³² The dramatic reduction in both basal and actin-activated ATPase activity of Myo1e^D388H indicates a severe defect in the ATPase cycle of this mutant. The results of single ATP turnover measurement are consistent with the steady-state ATPase assay for Myo1e^WT and Myo1e^D388H, supporting the conclusion of the deficient, but not completely abolished, ATPase activity of Myo1e^D388H. We demonstrate Myo1e^D388H does not support actin gliding in the motility assay, which is consistent with previous work on the corresponding mutation in Dictyostelium myosin II (D454 being an equivalent residue to D388 in Myo1e).⁶⁴ Our work suggests this aspartate residue in the switch-2 region is essential for myosin motor activity in both class 1 and class 2 myosins.

Overall, our analysis identified differences between the two mutants tested and between the mutants and the WT Myo1e, including reduced association of Myo1e^T119I with CCVs and cell-cell junctions and prolonged association of Myo1e^D388H with these structures (Figure 7). One possible explanation for these differences is that actin binding via the motor domain or actin-dependent transport are required for Myo1e recruitment to its sites of action, and that Myo1e^T119I lacks the ability to bind actin, which we were not able to test directly due to its proteolysis during purification. However, our previous studies show a Myo1e tail construct (lacking the motor and neck domains) is enriched in the cell-cell junctions and CCVs,^15,17 arguing against the direct contribution of the motor domain to localization. Second, the T119I mutation could lead to complete misfolding, including misfolding of the tail domain, preventing the tail from performing its normal function in Myo1e localization. Third, although it is not known whether Myo1e motor activity is regulated via intramolecular interactions, other myosins have been shown to be autoinhibited via tail-motor binding.^9,65 If this mode of regulation is also applicable to Myo1e, the mutant motor domain may not be able to undergo the conformational change required to relieve the autoinhibition. The prolonged association of Myo1e^D388H with cell junctions and CCVs could potentially be explained by a reduced rate of ADP dissociation or by slower dissociation from actin in the presence of ATP, which could prolong actin binding. However, both Myo1e^WT and Myo1e^D388H were able to dissociate from actin in the presence of ATP and have a similar K_ATPase, suggesting Myo1e^D388H is not a true rigor mutant. Alternatively, if the autoinhibition model for regulation of Myo1e activity is correct, perhaps the D388H mutation allows Myo1e to remain in an open (activated) conformation longer, thereby prolonging tail domain interactions with its binding partners. Because Myo1e^D388H expression increases CCV density, we hypothesize prolonged tail domain interactions with the plasma membrane phospholipids or endocytic accessory proteins may promote assembly and maturation of CCVs (Figure 7).

Figure 7.

Model describing how expression of Myo1e variants may affect cell junctions and clathrin-dependent endocytosis. Normal Myo1e regulation and activity: Myo1e^WT transiently localizes to the boundary between plasma membrane and branched actin at the (A) cell-cell junctions and (B) clathrin-coated pits, where it interacts with its binding partners (ZO-1, dynamin, and synaptojanin). Myo1e motor/head domain binds branched actin (shown in beige in [B]), whereas tail domain interacts with plasma membrane. Myo1e tail domain interactions promote clustering or activation of its binding partners, including phospholipids and endocytic accessory proteins (as shown in [B] in blue), thus facilitating CCV maturation or scission and slit diaphragm assembly. Interactions of the Myo1e motor/head domain with actin are transient, as indicated by the two-headed arrows in (A) and (B). We hypothesize that, as Myo1e head domain dissociates from actin, it folds into an autoinhibited conformation, preventing the tail domain from interacting with proteins or lipids. Effects of motor domain mutations: Myo1e^T119I is a misfolded/inactive mutant with diffuse localization. In the absence of Myo1e recruitment to the CCVs, the lifetimes of the CCVs are extended, and the number of CCVs is reduced due to the failure of the mislocalized Myo1e^T119I to support vesicle maturation, invagination, and scission. Myo1e^D388H is a motor activity–deficient mutant characterized by the prolonged retention at the cell-cell junctions and CCVs. Because its tail domain is still functional and capable of interacting with its binding partners, expression of this mutant results in the increased accumulation of endocytic accessory proteins, leading to an increase in the CCVs’ density and intensity.

In summary, in this study, we have provided direct functional evidence for the deleterious effects of the T119I and D388H mutations in Myo1e. In the future, we hope to apply a similar experimental design to examine other MYO1E variants. In particular, an important next step would be applying the assays developed in this study to the more common MYO1E variants, whose pathogenicity cannot be decisively established on the basis of their population frequency.

Disclosures

F. Hildebrandt reports having consultancy agreements with, having ownership interest in, serving in an advisory or leadership role for, being the cofounder of, and serving as a scientific advisory board member of Goldfinch-Bio; receiving research funding from National Institutes of Health (NIH); receiving honoraria from Sanofi; and having patents or royalties involving NPHP1. All remaining authors have nothing to disclose.

Funding

This work was supported by an American Society of Nephrology predoctoral fellowship to P.-J. Liu, American Heart Association fellowship 14PRE20380534 to J. Bi-Karchin, and the National Institute of Diabetes and Digestive and Kidney Diseases awards R01DK083345 to M. Krendel and RC2 DK122397-03 to F. Hildebrandt.

Author Contributions

J. Bi-Karchin, S.E. Chase, M.E. Garone, L.K. Gunther, M. Krendel, P.-J. Liu, C.E. Martin, C.D. Pellenz, D. Perez, E.L. Plante, M.F. Presti, and C.M. Yengo were responsible for investigation; M.E. Garone, M. Krendel, P.-J. Liu, S. Lovric, C.M. Yengo, and C. Zhang were responsible for methodology; L.K. Gunther, M. Krendel, P.-J. Liu, S. Lovric, and C. Zhang were responsible for formal analysis; L.K. Gunther, P.-J. Liu, and C.M. Yengo were responsible for visualization; F. Hildebrandt and M. Krendel were responsible for funding acquisition; F. Hildebrandt, M. Krendel, P.-J. Liu, S. Lovric, and C.M. Yengo conceptualized the study; F. Hildebrandt, M. Krendel, P.-J. Liu, and C.M. Yengo reviewed and edited the manuscript; M. Krendel was responsible for project administration; M. Krendel, P.-J. Liu, and C.M. Yengo wrote the original draft; M. Krendel and C.M. Yengo provided supervision; and C. Zhang was responsible for data curation.

Data Sharing Statement

The original experimental data reported in this paper have been deposited to Gene Expression Omnibus, accession no. GSE202684.

Supplemental Material

This article contains the following supplemental material online at http://jasn.asnjournals.org/lookup/suppl/doi:10.1681/ASN.2021111505/-/DCSupplemental.

Supplemental Table 1. List of primer sequences used in this paper.

Supplemental Table 2. Accession numbers for the sequences used for the Myo1e alignments.

Supplemental Table 3. Summary of the recently identified MYO1E variants.

Supplemental Figure 1. RNA sequencing identifies expression of podocyte-specific markers in both WT and Myo1e-KO podocyte-derived cell lines used in the current study.

Supplemental Figure 2. Expression of recombinant Myo1e constructs in Myo1e-KO podocytes using adenoviral vectors (related to Figure 2).

Supplemental Figure 3. Myo1eT119I is unstable in the cell lines with endogenous Myo1e.

Supplemental Figure 4. Analysis of EGFP-Myo1e localization at cell-cell junctions in the Myo1e-KO podocytes (related to Figure 3).

Supplemental Figure 5. Myo1eD388H exhibits slower protein exchange at the podocyte junctions (related to Figure 3).

Supplemental Figure 6. Analysis of EGFP-Myo1e co-localization with CCVs by TIRF microscopy (related to Figure 4).

Supplemental Figure 7. Analysis of the fluorescence intensity of CCVs co-localized with Myo1e (related to Figure 4).

Supplemental Figure 8. Purification of Myo1eWT and Myo1eD388H constructs for in vitro motor activity experiments (related to Figure 5).

Supplemental Video 1. Representative movies of fluorescence recovery after photobleaching (FRAP) at the Myo1e-KO podocyte junctions (related to Figure 3, E and F, Supplemental Figure 5, B and C).

Supplemental Video 2. Representative movies of F-actin translocation by Myo1e motor+IQ (related to Figure 6).

Supplemental Appendix 1. Source data for all figures (Excel file).