Biology subjects

Manstein, D. J.

Publications and source records attributed to Manstein, D. J..

6 recordsLinked to original sources

bioRxiv2025

Sarcomeric Remodelling in Human Heart Failure unraveled by single molecule long read sequencing

Dysregulation of alternative splicing - mediated by factors such as RBM20 or SLM2 - can affect proper gene isoform control disrupts gene isoform homeostasis and underpins severe cardiomyopathy in both animal models and patients. Although innovative therapies target various sarcomeric components, the impact of isoform switching in cardiac disease remains poorly understood. Here, we applied nanopore long-read sequencing to map the full-length transcriptome of left ventricular tissue from thirteen nonfailing controls, ten patients with dilated cardiomyopathy (DCM), and ten with ischemic cardiomyopathy (ICM). Our analysis identified 78,520 transcripts, 31% of which represent novel isoforms of known genes. Notably, the transcriptomes of DCM and ICM were largely indistinguishable, indicating that end-stage heart failure is characterized by a convergent isoform landscape, irrespective of disease etiology. Among 11 prototypical sarcomere genes, 10 displayed highly significant isoform shifts (p= 5.23x10-45 - 2.89x10-200). Focusing on tropomyosin, we observed that while the predominant cardiac gene TPM1 showed moderate up-regulation of its transcript isoforms, transcripts derived from TPM3--typically expressed at lower levels in the healthy heart--were markedly increased in heart failure.

genetics↗

bioRxiv2024

Baraitser-Winter Syndrome Hotspot Mutation R196H in Cytoskeletal β-actin Reduces F-actin Stability and Perturbs Interaction with the Arp2/3 Complex

Baraitser-Winter cerebrofrontofacial syndrome (BWCFF) is the most common and best-defined clinical entity associated with heterozygous single-point missense mutations in cytoskeletal {beta}-actin. Patients present with distinct craniofacial anomalies and neurodevelopmental disabilities of variable severity. To date, the most frequently observed variants affect residue R196 of cytoskeletal {beta}-actin, with the variant p.R196H being the most common. Patients carrying the p.R196H variant are likely to suffer from pachygyria, probably due to neuronal migration defects contributing to the development of abnormally thick convolutions of the cerebral cortex. Here, we describe the recombinant production, purification and biochemical characterization of the BWCFF hotspot variant p.R196H. The stability and nucleotide interaction of monomeric p.R196H are unaffected, indicating a disease mechanism involving incorporation of p.R196H protomers into actin filaments. Incorporation of the variant strongly affects F-actin stability and polymerization dynamics, consistent with the position of residue R196 close to the helical axis of the actin filament and an important interstrand contact. The changes observed include an increased critical concentration of polymerization, a reduced elongation rate and an increase in the rate of filament depolymerization. In the Arp2/3-generated branch junction complex, which is essential for cell migration and endocytosis, R196 is located at the interface between the first protomer of the nucleated daughter filament and the Arp2 subunit of the Arp2/3 complex. Assays probing the interaction of p.R196H filaments with the Arp2/3 complex show a reduced efficiency of branch generation. Branch stability is impaired, as evidenced by a reduction in the number of branches and spontaneous debranching events. Furthermore, in their interaction with different types of cytoskeletal myosin motors, p.R196H filaments show isoform-specific differences. While p.R196H filaments move WT-like on lawns of surface-immobilized non-muscle myosin-2A, motility on myosin-5A is 30 % faster.

biochemistry↗

bioRxiv2023

The non-muscle actinopathy-associated mutation E334Q in cytoskeletal gamma-actin perturbs interaction of actin filaments with myosin and ADF/cofilin family proteins

Various heterozygous cytoskeletal {gamma}-actin mutations have been shown to cause Baraitser-Winter cerebrofrontofacial syndrome, non-syndromic hearing loss, or isolated eye coloboma. Here, we report the biochemical characterization of human cytoskeletal {gamma}-actin carrying mutation E334Q, a mutation that leads to a hitherto unspecified non-muscle actinopathy. Following expression, purification, and removal of linker and thymosin {beta}4 tag sequences, the p.E334Q monomers show normal integration into linear and branched actin filaments. The mutation does not affect thermal stability, actin filament nucleation, elongation and turnover. Model building and normal mode analysis predict significant differences in the interaction of p.E334Q-filaments with myosin motors and members of the ADF/cofilin family of actin-binding proteins. Assays probing the interactions of p.E334Q-filaments with human class 2 and class 5 myosin motor constructs show significant reductions in sliding velocity and actin-affinity. E334Q differentially affects cofilin-mediated actin dynamics by increasing the rate of cofilin-mediated de novo nucleation of actin filaments and decreasing the efficiency of cofilin-mediated filament severing. Thus, it is likely that p.E334Q-mediated changes in myosin motor activity, as well as filament turnover contribute to the observed disease phenotype.

biochemistry↗

bioRxiv2020

Enhanced super-resolution microscopy by combined Airyscan and Quantum-Dot-Triexciton Imaging

Super-resolution fluorescence imaging provides critically improved information about the composition, organization and dynamics of sub-cellular structures. Quantum-Dot-Triexciton Imaging (QDTI) has been introduced as an easy-to-use sub-diffraction imaging method that achieves an almost 2-fold improvement in resolution when used with conventional confocal microscopes. Here we report an overall 3-fold increase in lateral and axial resolution compared to standard confocal microscopes by combining QDTI with the Airyscan approach.

biophysics↗

bioRxiv2018

Thrombocytopenia Microcephaly Syndrome - a novel phenotype associated with ACTB mutations

Introductory paragraphUntil recently missense germ-line mutations in ACTB, encoding the ubiquitously expressed {beta}-cytoplasmic actin (CYA), were exclusively associated with Baraitser-Winter Cerebrofrontofacial syndrome (BWCFF), a complex developmental disorder1,2. Here, we report six patients with previously undescribed heterozygous variants clustered in the 3-coding region of ACTB. These patients present with clinical features different from BWCFF, including thrombocytopenia, microcephaly, and mild developmental disability. Patient derived cells are morphologically and functionally distinct from controls. Assessment of cytoskeletal constituents identified a discrete filament population altered in these cells, which comprises force generating and transmitting actin binding proteins (ABP) known to be associated with thrombocytopenia3-8. In silico modelling and molecular dynamics (MD)-simulations support altered interactions between these ABP and mutant {beta}-CYA. Our results describe a new clinical syndrome associated with ACTB mutations with a distinct genotype-phenotype correlation, identify a cytoskeletal protein interaction network crucial for thrombopoiesis, and provide support for the hypomorphic nature of these actinopathy mutations.

genetics↗

bioRxiv2017

Mechanistic Insights Into The Active Site And Allosteric Communication Pathways In Human Nonmuscle Myosin-2C

The cyclical interaction of myosin with F-actin and nucleotides is the basis for contractility of the actin cytoskeleton. Despite a generic, highly conserved motor domain, ATP turnover kinetics and their activation by F-actin vary greatly between myosins-2 isoforms. Here, we present a 2.25 [A] crystal structure of the human nonmuscle myosin-2C motor domain, one of the slowest myosins characterized. In combination with integrated mutagenesis, ensemble-solution kinetics, and molecular dynamics simulations approaches, this study reveals an allosteric communication pathway that connects the distal end of the motor domain with the active site. Genetic disruption of this pathways reduces nucleotide binding and release kinetics up to 85-fold and abolishes nonmuscle myosin-2 specific kinetic signatures. These results provide insights into structural changes in the myosin motor domain that are triggered upon F-actin binding and contribute critically to the mechanochemical behavior of stress fibers, actin arcs, and cortical actin-based structures.

biochemistry↗