The giant muscle protein titin is a major contributor to passive force; however, its role in active force generation is unresolved. Here, we use a novel titin-cleavage (TC) mouse model that allows specific and rapid cutting of elastic titin to quantify how titin-based forces define myocyte ultrastructure and mechanics. We show that under mechanical strain, as titin cleavage doubles from heterozygous to homozygous TC muscles, Z-disks become increasingly out of register while passive and active forces are reduced. Interactions of elastic titin with sarcomeric actin filaments are revealed. Strikingly, when titin-cleaved muscles contract, myosin-containing A-bands become split and adjacent myosin filaments move in opposite directions while also shedding myosins. This establishes intact titin filaments as critical force-transmission networks, buffering the forces observed by myosin filaments during contraction. To perform this function, elastic titin must change stiffness or extensible length, unveiling its fundamental role as an activation-dependent spring in contracting muscle.
All data generated or analysed during this study are included in the manuscript and supporting files. Source data files have been provided for Figures 1 to 7.
- Wolfgang A Linke
- Wolfgang A Linke
- Wolfgang A Linke
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Animal experimentation: Laboratory mice were bred and housed at the University Clinic Muenster, in strict accordance with, and approval from the local authorities (LANUV NRW, Germany, 81-02.04.2019.A472). All procedures were performed according to the guidelines of the animal care and use committee of the University Clinic Muenster. Adult mice were euthanized by an isoflurane gas overdose and cervical dislocation, as recommended by the local animal care and use committee, and every effort was made to minimize suffering.
- Alphee Michelot, Institut de Biologie du Développement, France
© 2020, Li et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
β-Arrestins are master regulators of cellular signaling that operate by desensitizing ligand-activated G-protein-coupled receptors (GPCRs) at the plasma membrane and promoting their subsequent endocytosis. The endocytic activity of β-arrestins is ligand dependent, triggered by GPCR binding, and increasingly recognized to have a multitude of downstream signaling and trafficking consequences that are specifically programmed by the bound GPCR. However, only one biochemical ‘mode’ for GPCR-mediated triggering of the endocytic activity is presently known – displacement of the β-arrestin C-terminus (CT) to expose clathrin-coated pit-binding determinants that are masked in the inactive state. Here, we revise this view by uncovering a second mode of GPCR-triggered endocytic activity that is independent of the β-arrestin CT and, instead, requires the cytosolic base of the β-arrestin C-lobe (CLB). We further show each of the discrete endocytic modes is triggered in a receptor-specific manner, with GPCRs that bind β-arrestin transiently (‘class A’) primarily triggering the CLB-dependent mode and GPCRs that bind more stably (‘class B’) triggering both the CT and CLB-dependent modes in combination. Moreover, we show that different modes have opposing effects on the net signaling output of receptors – with the CLB-dependent mode promoting rapid signal desensitization and the CT-dependent mode enabling prolonged signaling. Together, these results fundamentally revise understanding of how β-arrestins operate as efficient endocytic adaptors while facilitating diversity and flexibility in the control of cell signaling.
The insulin receptor (IR) and insulin-like growth factor 1 receptor (IGF1R) control metabolic homeostasis and cell growth and proliferation. The IR and IGF1R form similar disulfide bonds linked homodimers in the apo-state; however, their ligand binding properties and the structures in the active state differ substantially. It has been proposed that the disulfide-linked C-terminal segment of α-chain (αCTs) of the IR and IGF1R control the cooperativity of ligand binding and regulate the receptor activation. Nevertheless, the molecular basis for the roles of disulfide-linked αCTs in IR and IGF1R activation are still unclear. Here, we report the cryo-EM structures of full-length mouse IGF1R/IGF1 and IR/insulin complexes with modified αCTs that have increased flexibility. Unlike the Γ-shaped asymmetric IGF1R dimer with a single IGF1 bound, the IGF1R with the enhanced flexibility of αCTs can form a T-shaped symmetric dimer with two IGF1s bound. Meanwhile, the IR with non-covalently linked αCTs predominantly adopts an asymmetric conformation with four insulins bound, which is distinct from the T-shaped symmetric IR. Using cell-based experiments, we further showed that both IGF1R and IR with the modified αCTs cannot activate the downstream signaling potently. Collectively, our studies demonstrate that the certain structural rigidity of disulfide-linked αCTs is critical for optimal IR and IGF1R signaling activation.