Fig 1: Cellular morphology and motility are regulated by the subcellular localization of wildtype MLC1. a Representative image of COS-7 cells expressing MLC1-GFP either in the intracellular organelles (*) or on the plasma membrane (***). COS-7 cell showing intermediate distribution pattern of MLC1 is located on the top of the image (**). Plasma membrane-targeted MLC1 was stained using anti-Myc antibody (red, surface MLC1, before permeabilization). Total MLC1 expression was visualized using GFP (green, total MLC1, after permeabilization), while nuclear expression was examined using DAPI (blue). Scale bar: 20 μm (upper), 5 μm (bottom). b The polarized distribution of surface MLC1-GFP and filopodia. The histogram for each signal was analyzed using ImageJ. Scale bar: 20 μm. c Kymographs of COS-7 cells expressing PM-MLC1 and ER-MLC1. Scale bar: 20 μm. Motility of cells expressing PM-MLC1 (n = 6) or ER-MLC1 (n = 6), as determined via live cell imaging. Mean velocity (d) and directionality (e) were analyzed. f Time-lapse imaging of the subcellular localization of MLC1-GFP in moving COS-7 cells (yellow, merged with mCherry). mCherry (red) was used as a marker of transfection, allowing us to determine cellular morphology using a fluorescence microscope. Scale bar: 25 μm
Fig 2: Reduced single-cell motility in an MLC1-expressing cell. a Representative images from a wound healing assay of COS-7 cells expressing wildtype MLC1 and patient-derived mutants (P92S- and S280 L-GFP). COS-7 cells expressing GFP-tagged proteins were visualized by 488 nm filter set. Scale bar: 200 µm. b Quantification of wound healing activity ((width of initial wound–width of final wound)/24 h) of cells expressing GFP (n = 19), MLC1-GFP (n = 22), and patient-derived mutants (P92S-GFP (n = 16) and S280 L-GFP (n = 17)). c Representative snapshots and trajectories of single-cell level live cell imaging. Scale bar: 50 µm. Live cell imaging was used to analyze single-cell motility in COS-7 cells expressing GFP (n = 27), MLC1-GFP (n = 40), and patient-derived mutants (P92S-GFP, n = 19 and S280 L-GFP, n = 10). Trajectory (d), mean velocity (e), and directionality (f) were analyzed as described in the Methods section. Lower directionality scores are indicative of more random movement
Fig 3: MLC1 induces morphological changes. COS-7 cells were transiently transfected with MLC1-GFP (a) or GFP (b), followed by immunofluorescence staining with anti-GFP (green, transfection marker) antibody and phalloidin (magenta, fibrous actin). Empty and filled arrowheads (magnified image) indicate fibrous actin bundles and branched actin networks, respectively. Scale bar: 20 µm (upper) and 5 µm (bottom). c Expression of MLC1-GFP in COS-7 cells was confirmed via Western blotting with anti-GFP antibody
Fig 4: Interaction between MLC1 and ARP2/3 complex. a and b FLAG-MLC1-GFP, ARP3-V5, and ARP3-Myc were co-expressed in COS-7 cells and used for co-immunoprecipitation. MLC1 and ARP2 or 3 were immunoprecipitated with anti-FLAG, V5, Myc antibodies, respectively. MLC1 was detected in V5- and Myc-precipitants, additionally, ARP3 and ARP2 were detected in FLAG-precipitant. Actin was used as loading control. c Primary astrocytes were infected with rAAV-GFP-shScr or -shMlc1. Endogenous Arp3 and Cortactin were immunoprecipitated with specific antibodies (Input = 10% of IP)
Fig 5: Effect of patient-derived MLC1 mutants on cellular morphology. a Heterologously expressed GFP, MLC1-GFP, and patient-derived mutants (P92S- and S280 L-GFP) in COS-7 cells were stained with anti-GFP antibody (green, MLC1), GRP78 (blue, ER), and phalloidin (magenta, fibrous actin). filled and empty arrowheads indicate branched actin networks and fibrous actin bundles, respectively. Scale bar: 25 µm (upper) and 5 µm (bottom). The number of filopodia per cell (b) and average length of filopodia (c) in cells transfected with GFP (n = 27 cells and 763 filopodia), MLC1-GFP (n = 24 cells and 1788 filopodia), P92S-GFP (n = 13 cells and 425 filopodia), and S280 L-GFP (n = 17 cells and 281 filopodia) were analyzed by FlioQuant. d Surface expression of wildtype and mutant MLC1 in COS-7 cells was analyzed via surface biotinylation (total = 10% of surface). Tubulin was used as a loading control and a cytosol-specific marker. Transferrin receptor (TfR) was used as a surface-specific marker. e Densitometric analysis of surface MLC1 expression for wildtype and mutant constructs. Levels of fractional surface expression (surface expression/total protein expression) of tested MLC1 constructs (n = 3) were normalized to that of wildtype value (n = 3)
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