Biology subjects

Reconditi, M.

Publications and source records attributed to Reconditi, M..

5 recordsLinked to original sources

bioRxiv2026

The distinct role of actin isoforms on mechanosensing-based maturation of hiPSC-derived neurons unveiled by isoform-specific mutations

Neuronal maturation is governed by the integration of intrinsic programs, such as gene regulation and cellular metabolism, with external mechanical cues. The conversion of mechanical forces into developmental responses (mechanotransduction) requires mechanical coupling of neurons to the extracellular matrix and neighboring cells. This coupling is mediated by the actin cytoskeleton and associated transmembrane protein complexes. However, the specific role of the {beta}- and {gamma}-actin isoforms in mechanotransduction, and the functional consequences of their pathogenic mutations, remain poorly understood. To address this question, we combined optical tweezers mechanics with immunofluorescence in human iPSC-derived neural progenitor cells (NPCs) and postmitotic neurons (NCs), both wild-type (WT) and carrying pathogenic mutations in {beta}-actin (R196H) or {gamma}-actin (T203M) associated with Baraitser-Winter cerebrofrontofacial (BWCFF) syndrome. Using membrane tether elongation as a proxy for early protrusion formation, we found that {gamma}-T203M NPCs require a fourfold lower force than WT NPCs and, upon differentiation, fail to develop the neurite-filopodia architecture retaining abundant immature protrusions In contrast, {beta}-R196H NPCs exhibit only reduction of cell surface tension associated with increased neuron fragility, without pronounced protrusion abnormalities. These results reveal non-redundant roles of {beta}- and {gamma}-actin isoforms in neuronal mechanotransduction and provide mechanistic insight into the pathogenesis of BWCFF syndrome.

neuroscience↗

bioRxiv2025

The Baraitser-Winter Cerebrofrontofacial Syndrome recurrent R196H variant in cytoplasmic β-actin impairs its cellular polymerization and stability

Variants in cytoskeletal actin encoding genes are associated with a broad spectrum of disorders, called non-muscle actinopathies. Among them, Baraitser-Winter cerebrofrontofacial syndrome (BWCFF) displays the most severe symptoms, such as intellectual disability and epilepsy. The exact consequences of the mutation on actins properties, however, are not fully understood. Here we explored the cellular effects of the R196H mutation in patient-derived fibroblasts. We show that the heterozygous mutation causes an actin polymerization defect in cells, leading to a fifty percent decrease in filamentous (F-) actin content. This effect can be rescued by the addition of the actin-polymerizing and stabilizing drug, jasplakinolide. We observed no significant defects either in the organization of the cellular actin cytoskeleton, analyzed by superresolution (STED) microscopy, or in the structure of purified filaments stabilized with phalloidin, explored with atomic force microscopy (AFM). The reduced F-actin content correlated with an approximately fourfold reduction in the stiffness of patient-derived cells probed with AFM. Manipulating the cells by mechanical forces through the application of the Dual Laser Optical Tweezers (DLOT) technique suggests that the mutation weakens the attachment of cytoskeletal actin to the plasma membrane. Inducing dynamical reorganization of actin by uniaxial stretching revealed that the interaction of cofilin with actin is also weakened by the mutation. Based on the existing cofilin-actin structures, the binding of cofilin may weaken the interaction of the neighboring residue E195 with K113, one of the lateral contacts stabilizing the filament. Thus, the mutation possibly exerts its effect through the destabilization of the interfilament interactions, potentially interfering allosterically with cofilin binding during actin depolymerization.

biophysics↗

bioRxiv2024

An integrated picture of the structural pathways controlling the heart performance

Regulation of heart function is attributed to a dual filament mechanism: (i) the Ca2+-dependent structural changes in the regulatory proteins of the thin, actin-containing filament making actin available for myosin motor attachment1, and (ii) the release of motors from their folded (OFF) state on the surface of the thick filament2 allowing them to attach and pull the actin filament. Thick filament mechanosensing is thought to control the number of motors switching ON in relation to the systolic performance3, but its molecular basis is still unknown. Here high spatial resolution X-ray diffraction data from electrically paced rat trabeculae and papillary muscles call for a revision of the mechanosensing hypothesis and provide a molecular explanation of the modulation of heart performance also in light of the recent cryo-EM thick filament structure4, 5. We find that upon stimulation titin activation6 triggers structural changes in the thick filament that switch motors ON throughout the filament within [~][1/2] the maximum systolic force. These structural changes also drive MyBP-C N-terminus to bind actin7 promoting first motor attachments from the central 1/3 of the half-thick filament. Progression of attachments towards the periphery of half-thick filament with increase in systolic force is carried on by near-neighbour cooperative thin filament activation by attached motors8. The identification of the roles of MyBP-C, titin, thin and thick filaments in heart regulation enables their targeting for potential therapeutic interventions.

physiology↗

bioRxiv2021

Titin switches from an extensible spring to a mechanical rectifier upon muscle activation

In contracting striated muscle titin acts as a spring in parallel with the array of myosin motors in each half-sarcomere and could prevent the intrinsic instability of thousands of serially linked half-sarcomeres, if its stiffness, at physiological sarcomere lengths (SL), were ten times larger than reported. Here we define titin mechanical properties during tetanic stimulation of single fibres of frog muscle by suppressing myosin motor responses with Para-Nitro-Blebbistatin, which is able to freeze thick filament in the resting state. We discover that thin filament activation switches I-band titin spring from the large SL-dependent extensibility of the OFF-state to an ON-state in which titin acts as a SL-independent mechanical rectifier, allowing free shortening while opposing stretch with an effective stiffness 4 pN nm-1 per half-thick filament. In this way during contraction titin limits weak half-sarcomere elongation to a few % and, also, provides an efficient link for mechanosensing-based thick filament activation.

physiology↗

bioRxiv2018

A mechanical model of the half-sarcomere which includes the contribution of titin

The evidence, in both resting and active muscle, for the presence of an I-band spring element like titin that anchors the Z line to the end of the thick filament did not yet produce a proper theoretical treatment in a complete model of the half-sarcomere. The textbook model developed by A.F. Huxley and his collaborators in 1981, which provides that the half-sarcomere compliance is due to the contribution of the compliances of the thin and thick filaments and actin-attached myosin motors, predicts that at any sarcomere length the absence of attached motors results in an infinite half-sarcomere compliance, in contrast with the observations. Growing evidence for the presence of a titin-like I-band spring urges the 1981 model to be implemented to include the contribution of this element in the mechanical model of the half-sarcomere. The model described here represents a tool for the interpretation of measurements of half-sarcomere compliance at long sarcomere lengths and for investigations of the possible role of titin as the mechano-sensor in thick filament regulation.

biophysics↗