Myoblast Choreographic Alignment

Abstract

The alignment of spindle-shaped cells is crucial for physiological processes, such as muscle regeneration and tissue engineering. Despite their importance, the underlying mechanisms that govern this alignment remain poorly understood. In this study, we explore the collective motion and alignment of C2C12 myoblasts, cells essential for muscle regeneration, across a range of cell densities and substrate stiffness. Contrary to the conventional view that C2C12 cells exhibit weak cell-cell adhesion, leading to uncoordinated motion, our study shows that these cells demonstrate anisotropic adhesion properties. Specifically, cells connected longitudinally by nanoscale focal adherens junctions (FAJs) exhibit strong adhesion, whereas those connected laterally by nanoscale linear adherens junctions (LAJs) display weak adhesion. The interplay between these molecular junctions facilitates spontaneous alignment, even beyond confluence, contributing to the formation of multinucleated myotubes. Moreover, we show that softer substrates reduce the level of C2C12 cell alignment, emphasizing the role of extracellular environments in regulating cell behavior. These insights into the alignment dynamics of C2C12 myoblasts advance our understanding of contractile cell behavior and offer valuable contributions to the fields of muscle regeneration, tissue engineering, and cellular biomechanics.

Keywords: adherens junctions; cell morphogenesis; correlation length; extracellular interactions; soft matter.

1

Dynamics of nematic order and spontaneous alignment of C2C12 cells. (a) Schematic colormaps, and (b–d) representative in vitro orientation colormaps (top) and corresponding cell angle distribution polar plots (bottom), showing the two distinctive C2C12 patterns: low nematic order (b) versus high nematic order (c, d), wherein the colors represent the values of angles. (e) The vector fields of C2C12 velocity (red) and director fields of C2C12 orientation (blue) at t = T ₀ (light color) and t = T ₀ + 60 h (dark color). (f, g) Interpreted correlation functions of C2C12 velocity and orientation at t = T ₀ and t = T ₀ + 60 h. Dash lines represent the slope of the exponential fitting. (Scale bar: 200 μm.).

2

Characterization of nematic order of C2C12 cells during temporal evolution. (a) Orientation colormaps of C2C12 cells during evolution. (Scale bar: 500 μm.) (b) Time series depicting C2C12 orientation correlation functions, color-coded for different incubation time points. (c) Evolutionary profile of C2C12 orientation ξ (black open squares), juxtaposed with cell density dynamics (blue open circles). Temporal orientations ξ are derived from the correlation function curves in (b). Three distinct regimes emerge upon comparison: visible extension of ξ with rising cell density (pink region), ξ independent of cell density (yellow region), and renewed cell proliferation without changes in ξ (blue region). The black dashed line marks confluence attainment (t = T ₀), and the blue dashed line indicates the baseline for zero cell density. (d) Cell density dependence of nucleus area fraction Φ_N by the ratio between total projection area occupied by cell nuclei and the total projection area occupied by cells (shown by the inset). Colors are aligned with time points in (b). (e) Time series of cell nuclei, color-coded for nucleus area (top row) and nucleus aspect ratio (second row), respectively. (Scale bar: 100 μm.) (f) Mean value of the single nucleus area over time, color-coded as in (b). (g) Mean value of single-cell aspect ratio, the ratio between major axis and minor axis of the cell nucleus (inset), over time. Analysis conducted on n > 200 nuclei. (N = 3 samples, n = 9 FOVs, error bars indicate 1 SD).

3

Role of cell–cell adherens junctions in C2C12 correlation build-up. (a) Cell density for samples before MMC treatment (blue), incubated with MMC for 1 day (orange), and incubated normally for 1 day (green). (b) Correlation comparison of the cell with varying MMC treatment conditions and corresponding (c) correlation lengths. (d) Illustration displaying the interference caused by N-cadherin antibodies (red) binding to N-cadherin’s extracellular domains (yellow), hindering effective cell interaction. Scenarios of (i) normal and (ii) adherens junction blocking groups are shown. (e) Colormaps depicting cell orientation for cases (i) and (ii), where color region size indicates the extent of cell alignment. (f) Correlation function curves, and corresponding (g) correlation lengths of untreated cells (red) and cells subjected to antibody treatment (blue) (N = 3 samples, n = 9 FOVs, error bars indicate 1 SD, **P-value <0.01, ***P-value <0.005). (Scale bar: 200 μm.).

4

Individual cell migration among cell groups. (a) Time-lapse tracking of the initially parallel cells (i) and the respective trajectories (ii) when transiting into separation with different orientation and velocity (iii). (b) The same sets of figures as (a) but with different modes of motion: encountering cells forming contact and separating in opposite velocity direction and aligned cell orientation, and (c) initially perpendicular cells transiting into alignment in both orientation and velocity, eventually by end-to-end contact and entrained motion. Full videos can be found in Videos S1–S3. The orientation of cells was identified as the orientation of the major axis of cells. (Scale bar: 100 μm.)

5

Roles of differentiated adherens junctions and actin activity in C2C12 cell correlation. (a) Fluorescence images capturing changes in adherens junction structures with varying cell density. Merged images of actin (green, immuno-stained by Phalloidin), nucleus (blue, immuno-stained by DAPI), and β-catenin (red, immuno-stained by anti-β-catenin) at low, intermediate, and high cell density (I–III), with zoomed-in images (i–iii) highlighting FAJs and LAJs marked by yellow and green arrows, respectively. Cell density states are differentiated by fixing time after cell seeding (t _seed = 12, 24, and 36 h, respectively). (b) Structure of coexisting LAJs and FAJs. (c) The schematic of LAJs and FAJs between neighboring cells. (d) The quantification of LAJs (green) and FAJs (yellow) at low cell density (ρ = 280 cells/mm^–2) and high density (ρ = 867 cells/mm^–2), including the mean area of each type of AJs per cell and proportion of each type of AJs. (e) Impact of blebbistatin-induced actomyosin contractility inhibition on C2C12 correlation function at t _seed = 12 h. The inset shows the order parameter Q, computed based on the orientation of nuclei. (n = 9 FOVs, error bars represent 1 SD, ***P-value <0.005). (f) Upon treatment with blebbistatin, C2C12 cells exhibited a reduced mean square displacement (MSD) accompanied by a lower scaling exponent α. A linear fit was applied to the log–log MSD curve within the highlighted gray window (Δt from 60 to 600 min). Tracking of cell motion was initiated after 3 days of incubation. The full video is found in Video S5. MSD curves were averaged across n = 5 FOVs. (Scale bar: 100 μm.).

6

Role of cell–matrix interaction in C2C12 cell correlation build-up. (a) Organization of focal adhesions along the aligned cells cultured on a coverslip. (Scale bar: 50 μm.) (b) Subcellular structure of a migrating C2C12 cell, highlighting various key structures of the cytoskeleton. (c) Schematic of PDMS substrate fabrication. Ultrathin PDMS as a control group (thickness <20 μm) and thick PDMS as a experimental group (thickness >1 mm) are made by spin coating and normal deposition, respectively. (d) The correspondence between the mixing ratio (base reagent vs cross-linker) and Young’s modulus of thick PDMS measured by rheology test. (e) Morphology of sparsely distributed C2C12 cells on different substrates. (Scale bar: 100 μm.) (f–h) Quantification of average area of each cell (f), average of cell shape index (g), and orientation correlation function for cells on different substrates (h). Experimental group (red); Control group (blue). Error bars represent 1 SD (n = 9 FOVs). (i) Color-coded orientational fields and fluorescently stained C2C12 monolayers on stiff substrate (E = 406 kPa) and soft substrate (E = 114 kPa) in experimental groups. (Scale bar: 1000 μm.).