Luca Fusi

Regulation of the SUMO pathway sensitizes differentiating human endometrial stromal cells to progesterone

cAMP is required for differentiation of human endometrial stromal cells (HESCs) into decidual cells in response to progesterone, although the underlying mechanism is not well understood. We now demonstrate that cAMP signaling attenuates ligand-dependent sumoylation of the progesterone receptor (PR) in HESCs. In fact, decidualization is associated with global hyposumoylation and redistribution of small ubiquitin-like modifier (SUMO)-1 conjugates into distinct nuclear foci. This altered pattern of global sumoylation was not attributable to impaired maturation of SUMO-1 precursor or altered expression of E1 (SAE1/SEA2) or E2 (Ubc9) enzymes but coincided with profound changes in the expression of E3 ligases and SUMO-specific proteases. Down-regulation of several members of the protein inhibitors of activated STAT (PIAS) family upon decidualization pointed toward a role of these E3 ligases in PR sumoylation. We demonstrate that PIAS1 interacts with the PR and serves as its E3 SUMO ligase upon activation of the receptor. Furthermore, we show that silencing of PIAS1 not only enhances PR-dependent transcription but also induces expression of prolactin, a decidual marker gene, in progestin-treated HESCs without the need of simultaneous activation of the cAMP pathway. Our findings demonstrate how dynamic changes in the SUMO pathway mediated by cAMP signaling determine the endometrial response to progesterone.

Probing myosin structural conformation in vivo by second-harmonic generation microscopy

Understanding of complex biological processes requires knowledge of molecular structures and measurement of their dynamics in vivo. The collective chemomechanical action of myosin molecules (the molecular motors) in the muscle sarcomere represents a paradigmatic example in this respect. Here, we describe a label-free imaging method sensitive to protein conformation in vivo. We employed the order-based contrast enhancement by second-harmonic generation (SHG) for the functional imaging of muscle cells. We found that SHG polarization anisotropy (SPA) measurements report on the structural state of the actomyosin motors, with significant sensitivity to the conformation of myosin. In fact, each physiological/biochemical state we probed (relaxed, rigor, isometric contraction) produced a distinct value of polarization anisotropy. Employing a full reconstruction of the contributing elementary SHG emitters in the actomyosin motor array at atomic scale, we provide a molecular interpretation of the SPA measurements in terms of myosin conformations. We applied this method to the discrimination between attached and detached myosin heads in an isometrically contracting intact fiber. Our observations indicate that isometrically contracting muscle sustains its tetanic force by steady-state commitment of 30% of myosin heads. Applying SPA and molecular structure modeling to the imaging of unstained living tissues provides the basis for a generation of imaging and diagnostic tools capable of probing molecular structures and dynamics in vivo.

Force generation by skeletal muscle is controlled by mechanosensing in myosin filaments

Contraction of both skeletal muscle and the heart is thought to be controlled by a calcium-dependent structural change in the actin-containing thin filaments, which permits the binding of myosin motors from the neighbouring thick filaments to drive filament sliding. Here we show by synchrotron small-angle X-ray diffraction of frog (Rana temporaria) single skeletal muscle cells that, although the well-known thin-filament mechanism is sufficient for regulation of muscle shortening against low load, force generation against high load requires a second permissive step linked to a change in the structure of the thick filament. The resting (switched ‘OFF’) structure of the thick filament is characterized by helical tracks of myosin motors on the filament surface and a short backbone periodicity. This OFF structure is almost completely preserved during low-load shortening, which is driven by a small fraction of constitutively active (switched ‘ON’) myosin motors outside thick-filament control. At higher load, these motors generate sufficient thick-filament stress to trigger the transition to its long-periodicity ON structure, unlocking the major population of motors required for high-load contraction. This concept of the thick filament as a regulatory mechanosensor provides a novel explanation for the dynamic and energetic properties of skeletal muscle. A similar mechanism probably operates in the heart.

The mechanism of the resistance to stretch of isometrically contracting single muscle fibres.

Rapid attachment to actin of the detached motor domain of myosin dimers with one motor domain already attached has been hypothesized to explain the stretch‐induced changes in X‐ray interference and stiffness of active muscle. Here, using half‐sarcomere mechanics in single frog muscle fibres (2.15 μm sarcomere length and 4°C), we show that: (1) an increase in stiffness of the half‐sarcomere under stretch is specific to isometric contraction and does not occur in rigor, indicating that the mechanism of stiffness increase is an increase in the number of attached motors; (2) 2 ms after 100 μs stretches (amplitude 2–8 nm per half‐sarcomere) imposed during an isometric tetanus, the stiffness of the array of myosin motors in each half‐sarcomere (em) increases above the isometric value (em0); (3) em has a sigmoidal dependence on the distortion of the motor domains (Δz) attached in isometric contraction, with a maximum ∼2 em0 for a distortion of ∼6 nm; em is influenced by detachment of motors at Δz > 6 nm; (4) at the end of the 100 μs stretch the relation between em/em0 and Δz lies slightly but not significantly above that at 2 ms. These results support the idea that stretch‐induced sliding of the actin filament distorts the actin‐attached motor domain of the myosin dimers away from the centre of the sarcomere, providing the steric conditions for rapid attachment of the second motor domain. The rate of new motor attachment must be as high as 7.5 × 104 s−1 and explains the rapid and efficient increase of the resistance of active muscle to stretch.

Sarcomere‐length dependence of myosin filament structure in skeletal muscle fibres of the frog

Contraction of skeletal muscle is thought to be regulated by a structural change in the actin‐containing thin filaments of the sarcomere, but recent results have suggested that a structural change in the myosin‐containing thick filaments may also be involved. We show that thick filament structure in resting muscle depends on the overlap with the thin filaments of the region of the thick filament containing myosin binding protein C (MyBP‐C). During isometric contraction, the regions of the thick filaments that do not overlap with thin filaments are highly disordered, in contrast to their helical order in resting muscle. The results provide strong support for the role of a structural transition in the thick filaments, mediated by an interaction between MyBP‐C and the thin filaments, in the physiological regulation of contraction in skeletal muscle.

Thick filament mechano-sensing is a calcium-independent regulatory mechanism in skeletal muscle

Recent X-ray diffraction studies on actively contracting fibres from skeletal muscle showed that the number of myosin motors available to interact with actin-containing thin filaments is controlled by the stress in the myosin-containing thick filaments. Those results suggested that thick filament mechano-sensing might constitute a novel regulatory mechanism in striated muscles that acts independently of the well-known thin filament-mediated calcium signalling pathway. Here we test that hypothesis using probes attached to the myosin regulatory light chain in demembranated muscle fibres. We show that both the extent and kinetics of thick filament activation depend on thick filament stress but are independent of intracellular calcium concentration in the physiological range. These results establish direct control of myosin motors by thick filament mechano-sensing as a general regulatory mechanism in skeletal muscle that is independent of the canonical calcium signalling pathway.

The Conformation of Myosin Heads in Relaxed Skeletal Muscle: Implications for Myosin-Based Regulation

In isolated thick filaments from many types of muscle, the two head domains of each myosin molecule are folded back against the filament backbone in a conformation called the interacting heads motif (IHM) in which actin interaction is inhibited. This conformation is present in resting skeletal muscle, but it is not known how exit from the IHM state is achieved during muscle activation. Here, we investigated this by measuring the in situ conformation of the light chain domain of the myosin heads in relaxed demembranated fibers from rabbit psoas muscle using fluorescence polarization from bifunctional rhodamine probes at four sites on the C-terminal lobe of the myosin regulatory light chain (RLC). The order parameter 〈P2〉 describing probe orientation with respect to the filament axis had a roughly sigmoidal dependence on temperature in relaxing conditions, with a half-maximal change at ∼19°C. Either lattice compression by 5% dextran T500 or addition of 25 μM blebbistatin decreased the transition temperature to ∼14°C. Maximum entropy analysis revealed three preferred orientations of the myosin RLC region at 25°C and above, two with its long axis roughly parallel to the filament axis and one roughly perpendicular. The parallel orientations are similar to those of the so-called blocked and free heads in the IHM and are stabilized by either lattice compression or blebbistatin. In relaxed skeletal muscle at near-physiological temperature and myofilament lattice spacing, the majority of the myosin heads have their light chain domains in IHM-like conformations, with a minority in a distinct conformation with their RLC regions roughly perpendicular to the filament axis. None of these three orientation populations were present during active contraction. These results are consistent with a regulatory transition of the thick filament in skeletal muscle associated with a conformational equilibrium of the myosin heads.

Structural changes in myosin motors and filaments during relaxation of skeletal muscle

Structural changes in myosin motors and filaments during relaxation from short tetanic contractions of intact single fibres of frog tibialis anterior muscles at sarcomere length 2.14 μm, 4°C were investigated by X‐ray diffraction. Force declined at a steady rate for several hundred milliseconds after the last stimulus, while sarcomere lengths remained almost constant. During this isometric phase of relaxation the intensities of the equatorial and meridional M3 X‐ray reflections associated with the radial and axial distributions of myosin motors also recovered at a steady rate towards their resting values, consistent with progressive net detachment of myosin motors from actin filaments. Stiffness measurements confirmed that the fraction of motors attached to actin declined at a constant rate, but also revealed a progressive increase in force per motor. The interference fine structure of the M3 reflection suggested that actin‐attached myosin motors are displaced towards the start of their working stroke during isometric relaxation. There was negligible recovery of the intensities of the meridional and layer‐line reflections associated with the quasi‐helical distribution of myosin motors in resting muscle during isometric relaxation, and the 1.5% increase in the axial periodicity of the myosin filament associated with muscle activation was not reversed. When force had decreased to roughly half its tetanus plateau value, the isometric phase of relaxation abruptly ended, and the ensuing chaotic relaxation had an exponential half‐time of ca 60 ms. Recovery of the equatorial X‐ray intensities was largely complete during chaotic relaxation, but the other X‐ray signals recovered more slowly than force.

Structural dynamics of troponin during activation of skeletal muscle

Significance Muscle contraction is controlled by structural changes in the thin filament of the muscle sarcomere triggered by calcium binding to troponin. It has long been suspected that myosin binding has an additional effect in switching on the thin filament, but the biological function of this effect was unknown. We have elucidated the in situ sequence of calcium-induced structural changes of troponin and identified a kinetic component tracking myosin binding to the thin filament. We propose a model of muscle regulation with kinetics determined by coordinated changes in the structures of both thick and thin filaments in response to mechanical conditions rather than, as in the conventional view, solely by the calcium transient and structural changes in the thin filament. Time-resolved changes in the conformation of troponin in the thin filaments of skeletal muscle were followed during activation in situ by photolysis of caged calcium using bifunctional fluorescent probes in the regulatory and the coiled-coil (IT arm) domains of troponin. Three sequential steps in the activation mechanism were identified. The fastest step (1,100 s−1) matches the rate of Ca2+ binding to the regulatory domain but also dominates the motion of the IT arm. The second step (120 s−1) coincides with the azimuthal motion of tropomyosin around the thin filament. The third step (15 s−1) was shown by three independent approaches to track myosin head binding to the thin filament, but is absent in the regulatory head. The results lead to a four-state structural kinetic model that describes the molecular mechanism of muscle activation in the thin filament–myosin head complex under physiological conditions.

The non‐linear elasticity of the muscle sarcomere and the compliance of myosin motors

The force in the half‐sarcomere (hs), the functional unit of muscle, is due to the contributions of individual myosin motors arranged in parallel in the half‐myosin filament and pulling on the opposing actin filament. According to a linear hs model, during an isometric contraction the force rises to its maximal steady value (T0) in proportion to the number of actin‐attached motors, while the hs strain rises with a slope that depends on the compliance of the myofilaments. We measured the hs stiffness, superimposing small 4 kHz length oscillations on the development of isometric contraction, and found an elastic element in parallel to the myosin motors with a constant stiffness ∼1/20th that of the motor array at T0. The results support a structural model in which myosin motors are distributed in multiple substates, of which only the first ones are occupied during isometric force generation, causing a motor strain of ∼1.7 nm.