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Abstract
Contractile actomyosin bundles, stress fibers, contribute to morphogenesis, migration, and mechanosensing of non-muscle cells. In addition to actin and non-muscle myosin II (NMII), stress fibers contain a large array of proteins that control their assembly, turnover, and contractility. Calponin-3 (Cnn3) is an actin-binding protein that associates with stress fibers. However, whether Cnn3 promotes stress fiber assembly, or serves as either a positive or negative regulator of their contractility has remained obscure. Here, we applied U2OS osteosarcoma cells as a model system to study the function of Cnn3. We show that Cnn3 localizes to both NMII-containing contractile ventral stress fibers and transverse arcs, as well as to non-contractile dorsal stress fibers that do not contain NMII. Fluorescence-recovery-after-photobleaching experiments revealed that Cnn3 is a dynamic component of stress fibers. Importantly, CRISPR/Cas9 knockout and RNAi knockdown studies demonstrated that Cnn3 is not essential for stress fiber assembly. However, Cnn3 depletion resulted in increased and uncoordinated contractility of stress fibers that often led to breakage of individual actomyosin bundles within the stress fiber network. Collectively these results provide evidence that Cnn3 is dispensable for the assembly of actomyosin bundles, but that it is required for controlling proper contractility of the stress fiber network.
Highlights
Contractile actomyosin bundles, stress fibers, are important for morphogenesis, migration and mechanosensing of non-muscle cells
Calponin-3 is a dynamic component of contractile stress fibers
The conventional immunofluorescence microscopy and 3D structural-illumination microscopy (3D-SIM) experiments demonstrated that both endogenous Cnn[3] (Fig. 1A,B) and exogenously expressed Cnn3-mCherry (Fig. 1C) localize to all three stress fibers categories; dorsal stress fibers, transverse arcs, and ventral stress fibers. 3D-SIM experiments further revealed that in U2OS cells Cnn[3] is not uniformly distributed along stress fibers, but it is enriched in α-actinin-1 foci in transverse arcs and ventral stress fibers (Fig. 1C,D)
Summary
Contractile actomyosin bundles, stress fibers, are important for morphogenesis, migration and mechanosensing of non-muscle cells. Other studies provided evidence that Cnn[1] may affect the mechanical properties of actin filaments and regulate organization of the actin cytoskeleton in smooth muscle cells[14,15]. The precise functions of Cnn[2] and Cnn[3] in non-muscle cells remain somewhat controversial Both proteins localize to contractile actomyosin bundles, such as stress fibers[16,21,27]. We show that Cnn[3] is a dynamic component of stress fibers, and that its depletion results in increased and uncontrolled contractility of the stress fiber network These results provide evidence that, like Cnn[1], Cnn[3] functions as a negative regulator of actomyosin contractility in cells
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