Biology subjects

Hinits, Y.

Publications and source records attributed to Hinits, Y..

3 recordsLinked to original sources

bioRxiv2026

The generation and validation of a de novo-designed titin binder for use in fluorescence microscopy

Advances in the generation of proteins in silico has enabled the efficient design of such that can bind to a specified target. Here, we demonstrate the use of a fluorescently-labelled de novo-designed protein to bind its target in situ and be imaged using fluorescence microscopy, a widely used experimental technique that typically relies on antibodies or similar evolutionary derived binders to identify the presence and location of targets in their native environment. Our de novo-designed protein binds the C-terminal domain M10 (Ig-169) of the giant muscle protein titin, which spans half a sarcomere, the basic contractile unit of striated muscle. M10 antibodies suitable for fluorescence microscopy are unavailable. Confocal microscopy of muscle sections shows the binder localises to the M-band of the sarcomere - where M10 is found - and fails to label muscle in competition experiments and in mutant muscle where M10 is absent. These results demonstrate the utility of de novo-designed proteins in immunostaining-like experiments and suggest a future where targets can be routinely identified in complex biological samples by in silico-generated binders. Such an approach avoids the need to generate antibodies or similar binders either in vivo or in vitro, which can have technical, financial and ethical challenges.

bioengineering↗

bioRxiv2024

Location-Dependent Differences in Cardiac and Skeletal Muscle Dysfunction Associated With Truncating Titin (ttn.2) Variants

BackgroundTruncating variants (TTNtv) in the TTN gene, encoding the giant sarcomeric protein titin, cause a range of human cardiac and skeletal muscle disorders of varying penetrance and severity. The effects of variant location on clinical manifestations are incompletely understood. MethodsWe generated six zebrafish lines carrying tv in the ttn.2 gene at the Z-disk, I-band, A-band, and M-band titin regions. Expression of titin transcripts was evaluated using qPCR. Phenotype analysis was performed during embryonic development and in adult hearts. ResultsUsing location-specific primers, we found a significant reduction of Z-disk and I-band ttn.2 transcripts in all homozygous embryos but levels of A-band and M-band transcripts were reduced only in lines with truncations distal to the cronos promoter. Homozygous embryos uniformly died by 7-10 days post fertilization with marked impairment of cardiac morphology and function. Skeletal muscle motility and sarcomere organization were more disrupted in mutants with truncations distal to the cronos promoter compared to those proximal. The most C-terminal line (e232), which lacked only the titin kinase and M-band regions, differed from other lines, with homozygous embryos showing incorporation of truncated Ttn.2/Cronos protein and normal sarcomere assembly, but selective degradation of fast skeletal muscle sarcomeres. Heterozygous embryos were phenotypically indistinguishable from wild-type. High-frequency echocardiography in adult heterozygous fish showed reduced ventricular contraction under resting conditions in A-band mutants. Heterozygous Z-disk and I-band mutants had no significant baseline impairment but were unable to augment ventricular contraction in response to acute adrenaline exposure, indicating a lack of cardiac reserve. ConclusionsOur data suggest that cardiac and skeletal muscle dysfunction associated with truncating ttn.2 variants is influenced by age, variant location, and the amount of functional titin protein. The distinctive phenotype associated with distal C-terminal truncations may reflect different requirement for C-terminal titin for maintenance of fast, slow and cardiac muscle sarcomeres.

cell biology↗

bioRxiv2019

Fgf-driven Tbx protein activities directly induce myf5 and myod to initiate zebrafish myogenesis

Skeletal muscle derives from dorsal mesoderm that is formed during vertebrate gastrulation. Fibroblast growth factor (Fgf) signalling is known to cooperate with transcription factors of the Tbx family to promote dorsal mesoderm formation, but the role of these proteins in skeletal myogenesis has been unclear. Using the zebrafish, we show that dorsally-derived Fgf signals act through Tbx16 and Tbxta to induce two populations of slow and fast trunk muscle precursors at distinct dorsoventral positions. Tbx16 binds to and directly activates the myf5 and myod genes that are required for commitment to skeletal myogenesis. Tbx16 activity depends on Fgf signalling from the organiser. In contrast, Tbxta is not required for myf5 expression. However, Tbxta binds to a specific site upstream of myod not bound by Tbx16, driving myod expression in the adaxial slow precursors dependent upon Fgf signals, thereby initiating muscle differentiation in the trunk. After gastrulation, when similar muscle cell populations in the post-anal tail are generated from the tailbud, declining Fgf signalling is less effective at initiating adaxial myogenesis, which is instead initiated by Hedgehog signalling from the notochord. Our findings provide insight into the ancestral vertebrate trunk myogenic pattern and how it was co-opted during tail evolution to generate similar muscle by new mechanisms.

developmental biology↗