yoU-Net follows a U-Net architecture to binarize immunofluorescence images.

A) Representative images of α-actinin-2 and actin filaments (phalloidin) in a hiCM. The α-actinin-2 binary was thresholded in FIJI using Otsu’s method. Orange arrowhead denotes Z-Bodies. B) yoU-Net architecture from input image to output binary. Details can be found in the Supplement and in Figure S1. C) yoU-Net-generated binary of α-actinin-2, predicted using the trained U-Net described in Figure 1B. Orange arrowhead: Z-Bodies. D) Model of muscle stress fibers (MSFs) and myofibrils during sarcomere formation. Black arrow denotes direction of MSF translocation as α-actinin-2-positive Z-Bodies elongate and coalesce to form Z-Lines.

Quantifying sarcomere and myofibril organization using α-actinin-2 binaries.

A) Z-Lines and myofibrils identified by sarcApp. Each line denotes a Z-Line, and each different color represents a different myofibril. B) Quantification scheme for myofibrils and Z-Lines. Details can be found in Figure S2. C) Z-Bodies identified by sarcApp. Each red circle denotes a Z-Body. D) Quantification scheme for MSFs and Z-Bodies. Details can be found in Figure S2. E) Distribution of myofibrils per hiCM plated for 24 hours (N=188 cells; 4 biological replicates). F) Distributions of Z-Lines per hiCMs from Figure 2E. G) Distribution of average Z-Line length per cell from Figure 2F (N=104 cells). H) Distribution of average myofibril persistence lengths per cell from 2G (N=104 cells; quantification details can be found in Figure S2). I) Distribution of MSFs per cell from Figure 2E. J) Distribution of Z-Bodies per cell from Figure 2E. K) Distribution of average Z-Body length per cell from Figure 2E. L) Distribution of average MSF persistence lengths per cell from Figure 2E. M) Myofibril long axes identified by sarcApp. N) Quantification scheme for myofibril angle relative to edge. Briefly, the closest edge segment to the myofibril long axis (perpendicularly) is used as the reference angle, and the numerical output is the difference between the myofibril long axis angle and the reference edge angle. O) Distribution of myofibril orientation relative to cell edge in the same cells as Figure 2E. (N=188 cells, 1217 myofibrils). Note that most myofibrils are relatively parallel to the edge in hiCMs plated for 24 hours.

Blebbistatin treatment ablates Z-Line formation in hiCMs

A) Representative images and insets of α-actinin-2 and F-actin in hiCMs treated with DMSO, 50 µM Blebbistatin, or 100 µM Blebbistatin. B) Representative platinum replica EM image of a control hiCM and a 50 µM Blebbistatin-treated hiCM. Arrows indicate an elongated Z-Line in the DMSO-treated hiCM and a Z-Body in the Blebbistatin-treated hiCM. C) Myofibrils per cell in hiCMs (N=118 DMSO cells, 108 50 µM Blebbistatin cells, and 93 100 µM Blebbistatin cells; 4 biological replicates). D) Z-Lines per cell in hiCMs from Figure 3C. E) Average myofibril persistence length per cell in hiCMs from Figure 3C (N=104 DMSO control cells, 45 50 µM Blebbistatin cells, and 45 100 µM Blebbistatin cells). F) Average Z-Line length per cell from Figure 3E. G) Average size of all α-actinin-2-positive puncta in hiCMs from Figure 3C. H) Myofibril orientation relative to the cell edge segment closest to the myofibril center, perpendicularly. N=4 biological replicates, 1217 DMSO control myofibrils, 385 50 µM Blebbistatin myofibrils, 220 100 µM Blebbistatin myofibrils.

sarcApp uses titin binaries to identify myofibrils and precursor ring structures

A) Representative image of titin and α-actinin-2 in a control hiCM. Arrow: an α-actinin-2-positive Z-Body with titin localized in a ring around it. B) Model of titin localization during sarcomere formation. C) Titin doublets identified by sarcApp. Each line denotes a doublet with titin localized, and each color is a myofibril. E) Quantification scheme for myofibrils and titin doublets. Details can be found in Figure S2 and the Supplemental Methods. E) Titin precursor rings identified by sarcApp (red). F) Quantification scheme for titin precursor rings. Details can be found in Figure S2 and the Supplemental Methods.

Blebbistatin affects myofibril orientation and the morphology of titin structures

A)Representative images of titin and F-actin in hiCMs treated with DMSO, 50 µM Blebbistatin, and 100 µM Blebbistatin. B)Myofibrils per cell in hiCMs (N=3 biological replicates, 107 DMSO cells, 95 50 µM Blebbistatin cells, and 84 100 µM Blebbistatin cells). C) Doublets per cell in hiCMs from Figure 5B. D) Doublet length per cell in hiCMs from Figure 5B: (N= 58 DMSO cells, 32 50 µM Blebbistatin cells, and 21 100 µM Blebbistatin cells. E) Rings per cell in hiCMs from Figure 5B. F) Average ring aspect ratio per cell in hiCMs from Figure 5B (N= 105 DMSO control cells, 95 50 µM Blebbistatin cells, and 78 100 µM Blebbistatin cells. G) Myofibril orientation (N=292 DMSO control myofibrils, 151 50 µM Blebbistatin myofibrils, and 69 100 µM Blebbistatin myofibrils

sarcApp uses myomesin binaries to identify myofibrils and M-Lines in hiCMs

A) Schematic showing myomesin localization at the M-Line. B) Representative image of myomesin and F-actin in a hiCM. The myomesin binary was predicted using yoU-net as described in the Supplemental Methods. C) Myofibrils and M-Lines identified by sarcApp. Each line denotes an M-Line, and each color represents a myofibril. D) Quantification scheme for myofibrils and M-Lines. Details can be found in Figure S2. E) Representative images of myomesin and F-actin in hiCMs treated with DMSO, 50 µM Blebbistatin, and 100 µM Blebbistatin. F) Myofibrils per cell in hiCMs (N=3 biological replicates; 112 DMSO cells, 90 50 µM Blebbistatin cells, and 89 100 µM Blebbistatin cells). G) M-Lines per cell in hiCMs from Figure 6F. H) Average M-Line length per cell in hiCMs from Figure 6F (N=97 DMSO control cells, 16 50 µM Blebbistatin cells, and 13 100 µM Blebbistatin cells). I) Myofibril orientation (N=752 DMSO control myofibrils, 49 50 µM Blebbistatin myofibrils, and 60 100 µM Blebbistatin myofibrils).

Knockdown of α or β cardiac myosin II reduces but does not eliminate sarcomeres

A) Schematic showing cardiac myosin localization in a sarcomere. B) Representative western blot showing α cardiac myosin (MYH6) knockdown in hiCMs. C) Representative images of α-actinin-2, titin, and myomesin in siControl hiCMs and α cardiac myosin (MYH6) knockdown hiCMs. D) Number of Z-Lines per cell in hiCMs in two independent groups of siControl (scramble)-treated hiCMs and two separate MYH6 siRNA-treated hiCMs (sequences 1 and 2) (N=3 biological replicates, 81 siCon cells and 78 siMYH6 (1) cells, and 88 siCon cells and 63 siMYH6 (2) cells). E) Average Z-Line length per cell in hiCMs from Figure 7D (N=68 siCon cells and 75 siMYH6 (1) cells, and 83 siCon cells and 62 siMYH6 (2) cells). F) Average doublet length per cell in hiCMs in siCon (scramble)-treated hiCMs and two MYH6 siRNA sequences (1 and 2) (71 siCon cells, 49 siMYH6 (1) cells, and 50 siMYH6 (2) cells. G) Average M-Line length per hiCMs (85 siCon cells, 78 siMYH6 (1) cells, and 64 siMYH6 (2) cells). H) Representative western blot showing β cardiac myosin (MYH7) knockdown in hiCMs. I) Representative images of α-actinin-2, titin, and myomesin in β cardiac myosin (MYH7) knockdown hiCMs. J) Number of Z-Lines per cell in hiCMs in two independent groups of siCon (scramble)-treated hiCMs and two separate MYH7 siRNA-treated hiCMs (pools 1 and 2) (N= 86 siCon cells and 66 siMYH7 (1) cells, and 97 siCon cells and 62 siMYH7 (2) cells). L) Average Z-Line length per cell in hiCMs from Figure 7J (81 siCon cells and 63 siMYH7 (1) cells, and 81 siCon cells and 59 siMYH7 (2) cells). M) Number of rings per hiCM (N=3 biological replicates, 94 siCon cells, 72 siMYH7 (1) cells, and 66 siMYH7 (2) cells). N) Average M-Line length per cell in hiCMs (N= 87 siCon cells, 65 siMYH7 (1) cells, and 62 siMYH7 (2) cells).

Myomesin knockdown alters titin and cardiac myosin II localization, but not α-actinin-2

A) Representative images of sarcomeric proteins α-actinin-2 and titin in myomesin (MYOM) knockdown hiCMs. B) Representative western blot and quantification showing MYOM knockdown in hiCMs, N=3 biological replicates. C) Number of Z-Lines per cell in siCon (scramble)-treated hiCMs and two separate MYOM siRNA-treated hiCMs (sequences 1 and 2). N=3 biological replicates, 132 siCon cells, 105 siMYOM (1) cells, and 103 siMYOM (2) cells. D) Average Z-Line length per cell in hiCMs from Figure 8C: N=4 biological replicates, 117 siCon cells, 104 siMYOM (1) cells, and 92 siMYOM (2) cells (only cells with myofibrils were quantified for Figure 8D). E) Titin doublets per cell in hiCMs using experimental treatments in Figure 8C. N=3 biological replicates, 117 siCon cells, 90 siMYOM (1) cells, and 100 siMYOM (2) cells. F) Average doublet length per cell in hiCMs from Figure 8E: N=3 biological replicates, 74 siCon cells, 68 siMYOM (1) cells, and 70 siMYOM (2) cells. (Only cells with myofibrils were quantified for Figure 8F). G) Rings per cell in hiCMs from Figure 8E. H) Representative images of MYH7 and F-actin in siCon, siMYOM (1), and siMYOM (2) hiCMs. I) Length of β cardiac myosin stacks in siCon, siMYOM (1), and siMYOM (2) hiCMs

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