Molecular Control of Non-Muscle Myosin II-A Aggregation and Intracellular Dynamics by motor- or tail-specific MYH9 Mutations

Figures

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  Figure 1.. N93K is a bona fide molecular motor. (A) Left, model of an assembly-competent (6S) NM2-A, highlighting the functional domains of the heavy chain as indicated. This conformation is induced by phosphorylation of the RLC in Ser19 when NM2-A is in the compact, filament assembly-incompetent (10S) conformation (top right). Extended NM2-A rapidly form bipolar filaments (bottom right). Dephosphorylation of filamentous NM2-A triggers filament disassembly and induces a rapid return to the 10S conformation. (B) Actin-activated steady-state ATPase activity of NM2-A WT (grey) and NM2-A N93K (orange). The measured k100 of NM2-A N93K and NM2-A WT are 0.07±0.008 s−1 and 0.09±0.098 s−1, respectively. The kcat and Kapp lie outside the assessable F-actin concentration range and thus could not be determined. (C) Inhibition of the actin-activated steady-state ATPase (100 μM F-actin) of NM2-A WT (grey) and NM2-A N93K (orange) by blebbistatin. Blebbistatin inhibits the enzymatic activity of NM2-A N93K and NM2-A WT with an IC50 of 1.33±0.25 μM and 2.7±0.85 μM, respectively. (D) The in vitro gliding speed of actin filaments propelled by NM2-A N93K (103.5±6.36 nm·s−1) is reduced comparted to NM2-A WT…

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  Figure 2.. A molecular chimera of MHCII-B and -A but devoid of ATPase activity does not restore cellular contractility but accumulates at the rear of polarized cells. (A) Representative COS7 cells transfected, adhered and stained as in (A), illustrating the relative position of the GFP-tagged protein. Outline is drawn from F-actin staining (not shown). Note the proximity to the leading edge in wild type (WT), N93K and the BH-AT chimera, and the posterior accumulation of the BHR709C-AT chimera. Scale bar=20 μm. (B-D) Quantification of the percentage of adhesive area (B), number of adhesions per mm2 (C), and area per individual adhesion (D). Data was calculated as indicated in Material and Methods. n>30 cells from two independent experiments. Significance was calculated using non-parametric Mann Whitney test. * p<0.05; ** p<0.01; *** p<0.001; **** p<10−4; n.s., not significant. (E) Quantification of the average distance of the most anterior fluorescent signal for each construct. The edge was determined by paxillin staining as shown in (A). n>30 cells from two independent experiments. Significance was calculated using non-parametric Mann Whitney test. * p<0.05; **** p<10−4.

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  Figure 3.. Mutations localized in different positions of MHCII-A display a similar subcellular distribution and display comparable filament kinetics. (A) COS7 cells were transfected with Halo-MHCII-A wild type (WT) the GFP-MHCII-A carrying the indicated mutation. After 24h, cells were allowed to spread on fibronectin (2 μg/mL)-coated dishes overnight, fixed and stained for endogenous MHCII-B (magenta in overlay). In the II-A WT composite images, Halo-MHCII-A WT is magenta (top row), and in each horizontal row the myosin bearing the indicated mutation is green. In the II-B composite images, the myosin bearing the indicated mutation is green and endogenous MHCII-B is blue. Representative cells are shown. Scale bar=10 μm. (B-D) NM2-A N93K and NM2-A E1841K, but not NM2-A R705H, display increased stability within actomyosin filaments. (B) FRAP curves of EGFP-MHCII-A decorated actomyosin filaments. COS-7 cells, transfected with either EGFP-MHCII-A WT or EGFP-MHCII-A bearing the indicated mutations were allowed to adhere on fibronectin-coated (8 μg/mL) coverslips for 2h and then imaged. FRAP was performed always in comparable regions across mutants in well-delineated fibers. Data is the average of…

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  Figure 4.. UNC45a displays an increased association with the mutant variant NM2A N93K in cells. (A) COS-7 cells were co-transfected with pEGFP-N1 or UNC45-A-GFP plasmids and FLAG-MHCIIA WT or N93K as indicated. After 48h, cells were lysed and incubated with GFP-trap beads and separated using SDS-PAGE. Top panels represent input; bottom panels represent the immunoprecipitated fraction. (B) Densitometric quantification of experiments as in (A), corrected by the amount of UNC45a-GFP detected in the immunoprecipitated fraction. Data represents nine independent experiments. * p < 0.05. (C) COS7 cells were co-transfected with UNC45a-GFP and the indicated FLAG-tagged MHCII-A construct. After 48h, cells were lysed and incubated with anti-FLAG-conjugated magnetic beads and separated using SDS-PAGE. Top panels represent input; bottom panels represent the immunoprecipitated fraction. (D) Densitometric quantification of experiments as in (D), corrected by the amount of FLAG-MHCII-A detected in the immunoprecipitated fraction. Data represents ten independent experiments. ** p < 0.01. (E) IBAQ-based quantification of the interaction of UNC45a with WT and N93K MHCII-A. HEK-293 cells were transfected with…

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  Figure 5.. UNC45 depletion differentially affects the ability of WT and N93K NM2-A to localize in filaments. (A) COS7 cells were co-transfected with pLKO.1 control or a sequence targeting UNC45a. After 96h, cells were allowed to spread on fibronectin (2 μg/mL)-coated dishes overnight, fixed and stained for endogenous UNC45a (right column). Representative cells are shown. Scale bar=10 μm. (B) Quantification of the percentage of cells displaying GFP-positive filaments in experiments as in (A). Each dot represents a count of at least 50 cells in 5-10 fields across four independent experiments. Non-parametric Mann-Whitney U test was used to evaluate statistical significance. ** p < 0.01; *** p < 0.001, **** p < 0.0001, n.s., not significant.

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  Figure 6.. In silico modeling predicts weakening of the autoinhibited state in N93K. Homology model of NM2-A in the autoinhibited state suggests weakened interactions between N93K in the blocked head and R1417 in segment 2 of the folded tail that may compromise the stability of 10S. Notably, the respective mutation in the free head has no effect on interactions with the tail or other portions of the protein.

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  Figure 7.. N93K forms aggregates at high concentration in MEG-01 cells (A) FCS gating strategy for sorting. MEG-01 cells were co-transfected with mNeonGreen-MHCII-A WT and Halo-MHCII-A WT, N93K, R705H or E1841K. After 48h, cells were incubated with JF635, sorted and allowed to adhere to fibronectin (25 μg/mL)-coated coverslips, fixed and imaged using STED. X axis represents mNeonGreen levels; Y axis represents Halotag-JF635 levels. Low expressors were collected in the red box; High expressors were collected in the green box. (B) Representative STED images of MEG-01 cells transfected as indicated in (A) and collected from the red box (low expressors). Scale bar= 5 μm. (C) Representative STED images of MEG-01 cells transfected as indicated in (A) and collected from the green box (high expressors). Scale bar= 5 μm. In the composite images, magenta represents the Halo-tagged mutant and green is WT. Yellow boxes represent the magnified region shown in the zoom panels. Scale bar in the zoom panels= 1 μm. (D) Quantification of the percentage of cells displaying aggregates in experiments as in (B, C). Each dot represents a count of at least 50 cells in 5-10 fields across two independent…

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  Figure 8.. Analysis of particle intensity, density and degree of aggregation by STED-ICS in MEG-01 cells expressing Halo-MHCII-A mutants and mNeonGreen-MHCII-A wild type. Images similar to those shown in Fig. 7C were analyzed by ICS as indicated in Material and Methods. Data is the intensity (A-D), particle density (E-H) and degree of aggregation (I-L) of Halo-MHCII-A WT (A, E, I), N93K (B, F, J), R705H (C, G, K) and E1841 (D, H, L) against mNeonGreen-MHCII-A WT in the same region. (M) represents the comparison of the degree of aggregation of the Halo-tagged mutants from one condition to another, whereas (N) represents mNeonGreen-MHCII-A WT in cells expressing the indicated Halo-tagged mutant. Analysis was performed on 70-120 120x120 pixels ROIs of 20-30 MEG-01 STED images per condition from two independent experiments. Statistical significance was determined with the nonparametric Mann-Whitney U test. ** p < 0.01; *** p < 0.001, **** p < 0.0001, n.s., not significant. In (M), * significance vs. WT; # significance vs. E1841K.

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  Figure 9.. UNC45A co-localizes with N93K aggregates but is mostly excluded from E1841K aggregates. (A) Representative images of MEG-01 cells transfected as indicated in Fig. 7, collected from the green box in Fig. 7A (high expressors) and stained for endogenous UNC45A. Scale bar= 5 μm. In composite images, red represents the Halo-mutant, green is WT and blue is endogenous UNC45a. Colored boxes on right represent the zoomed regions indicated in the composite images. Note the color pattern, which is similar in wild type (first row) and R705H (third row), with white dominating N93K (second row), and major segregation of red (E1841K) and blue (UNC45A) in the fourth row. (B) Pearson correlation was calculated using the Coloc2 algorithm in Fiji in images as in (A). Data represents 4-5 non-overlapping regions from 4-5 cells (high level of expression, as described in Fig. 7A) per condition. **** p<0.0001; n.s., not significant.