Cardiac protein homeostasis, sarcomere assembly, and integration of titin as the sarcomeric backbone are tightly regulated to facilitate adaptation and repair. Very little is known on how the >3-MDa titin protein is synthesized, moved, inserted into sarcomeres, detached, and degraded. Here, we generated a bifluorescently labeled knockin mouse to simultaneously visualize both ends of the molecule and follow titin’s life cycle in vivo. We find titin mRNA, protein synthesis and degradation compartmentalized toward the Z-disk in adult, but not embryonic cardiomyocytes. Originating at the Z-disk, titin contributes to a soluble protein pool (>15% of total titin) before it is integrated into the sarcomere lattice. Titin integration, disintegration, and reintegration are stochastic and do not proceed sequentially from Z-disk to M-band, as suggested previously. Exchange between soluble and integrated titin depends on titin protein composition and differs between individual cardiomyocytes. Thus, titin dynamics facilitate embryonic vs. adult sarcomere remodeling with implications for cardiac development and disease.

Cellular signaling systems precisely transmit information in the presence of molecular noise while retaining flexibility to accommodate the needs of individual cells. To understand design principles underlying such versatile signaling, we analyzed the response of the tumor suppressor p53 to varying levels of DNA damage in hundreds of individual cells and observed a switch between distinct signaling modes characterized by isolated pulses and sustained oscillations of p53 accumulation. Guided by dynamic systems theory we show that this requires an excitable network structure comprising positive feedback and provide experimental evidence for its molecular identity. The resulting data-driven model reproduced all features of measured signaling responses and is sufficient to explain their heterogeneity in individual cells. We present evidence that heterogeneity in the levels of the feedback regulator Wip1 sets cell-specific thresholds for p53 activation, providing means to modulate its response through interacting signaling pathways. Our results demonstrate how excitable signaling networks can provide high specificity, sensitivity and robustness while retaining unique possibilities to adjust their function to the physiology of individual cells.

Highlights d Single-cell and computational analyses show how signaling adapts to internal states d The p53 response to damage is posttranscriptionally adjusted to cell cycle states d PCNA-CRL4 cdt2-mediated degradation leads to delayed p21 dynamics during S phase d Accumulation of non-degradable p21 during S phase induces genome instability

Three states in one blow: a FRET relay system distinguishes between (i) the absence of RNA target, (ii) the presence of C → U edited RNA or (iii) unedited RNA target by means of ratio measurements.