Biology subjects

Schultheis, D.

Publications and source records attributed to Schultheis, D..

6 recordsLinked to original sources

bioRxiv2026

Desmin Mutations Disrupt Filament Elongation and Drive Polymorphic Aggregation

In muscle cells, desmin intermediate filaments form a cytoskeletal network that maintains the structural integrity and mechanical coupling of myofibrils. Dominant missense mutations in desmin cause myopathies characterized by intracellular protein aggregation. To explore the process of aggregate formation, we investigate the assembly kinetics of wild-type desmin and four disease-associated variants (N342D, L345P, R350P, and R406W) using dual-wavelength stopped-flow spectroscopy, complemented by atomic force microscopy and molecular dynamics simulations. At low ionic strength, all proteins form uniform tetramers. Increasing the ionic strength initiates assembly byrapid lateral association of tetramers into unit-length filaments (ULFs), followed by longitudinal elongation and radial filament compaction. R406W forms ULFs with wild-type-like kinetics, whereas N342D, L345P, and R350P exhibit delayed lateral assembly. After short filaments have formed, all four mutants diverge from productive filament maturation, but through distinct pathways. Quantitative analysis of atomic force microscopy images together with kinetic modelling of the spectroscopic data shows that wild-type filaments elongate continuously, whereas R406W filaments progressively associate into fibrillar clusters and cease elongating. By contrast, N342D, L345P, and R350P rapidly collapse into globular complexes that subsequently coalesce into larger aggregates. Molecular dynamics simulations indicate that the mutations differentially destabilize coil 2, which leads to local structural perturbations and mutation-specific assembly defects. Together, these findings identify early filament maturation - when elongating ULF-derived filaments would normally undergo radial compaction to form stable, mature filaments - as a critical time point in desmin assembly. At this stage, pathogenic mutations redirect the internal reorganization of the filament from productive stabilization toward mutation-specific structural collapse and aggregation. SignificanceDominant mutations in the intermediate filament protein desmin cause myofibrillar myopathies characterized by protein aggregation and progressive muscle degeneration. Using in vitro filament-assembly experiments, we identify intrinsic assembly defects of disease-associated desmin mutants. Mutant proteins initially enter the normal assembly pathway, albeit with delayed kinetics. They subsequently fail at an early maturation step in which short filaments would normally reorganize to sustain productive elongation. Instead, mutant proteins form morphologically distinct aggregates. Similar structural abnormalities occur when mutant proteins co-assemble with wild-type desmin. Our findings identify the radial compaction phase during early longitudinal assembly as a critical checkpoint that determines whether desmin assembly yields functional intermediate filaments or is redirected into pathogenic aggregation pathways.

biophysics↗

bioRxiv2024

Integrated data from R405W desmin knock-in mice highlight alterations of mitochondrial function, protein quality control, and myofibrillar structure in the initial stages of myofibrillar myopathy

BackgroundMutations in the desmin gene cause skeletal myopathies and cardiomyopathies. The objective of this study was to elucidate the molecular pathology induced by the expression of R405W mutant desmin in murine skeletal muscle tissue. MethodsA comprehensive characterization of the skeletal muscle pathology in hetero- and homozygous R405W desmin knock-in mice was performed, employing grip strength, blood acylcarnitine and amino acid, histological, ultrastructural, immunofluorescence, immunoblot, ribosomal stalling, RNA sequencing and proteomic analyses. ResultsBoth hetero- and homozygous R405W desmin knock-in mice showed classical myopathological features of a myofibrillar myopathy with desmin-positive protein aggregation, degenerative changes of the myofibrillar apparatus, increased autophagic build-up, and mitochondrial alterations. Muscle weakness and increased blood concentrations of acylcarnitines and amino acids were only present in homozygous animals. During its translation, mutant desmin does not induce terminal ribosomal stalling. Analyses of RNA sequencing and proteomic data from soleus muscle of 3-month-old mice depicted 59 up- and 2 down-regulated mRNAs and 101 up- and 18 down-regulated proteins that were shared between the heterozygous and homozygous genotypes in the respective omics datasets compared to the wild-type genotype. Combined analysis of the omics data demonstrated 187 significantly dysregulated candidates distributed across four groups of regulation. A down-regulation on the mRNA and protein levels was observed for a multitude of mitochondrial proteins including essential proton gradient-dependent carriers. Up-regulation on both omics levels was present for the transcription factor Mlf1, which is a binding partner of protein quality control related Dnajb6. Down-regulated on mRNA but up-regulated on the protein level was the sarcomeric lesion marker Xirp2 (xin actin-binding repeat-containing protein 2), whereas Ces2c (acylcarnitine hydrolase) was regulated in the opposite way. ConclusionsThe present study demonstrates that the expression of mutant desmin results in a myofibrillar myopathy in hetero- and homozygous R405W desmin knock-in mice. Combined morphological, transcriptomic and proteomic analyses helped to decipher the complex pattern of early pathological changes induced by the expression of mutant desmin. Our findings highlight the importance of major mitochondrial alterations, including essential proton gradient-dependent carriers as well as Dnajb6-related protein quality control and Xin-related myofibrillar damage, in the molecular pathogenesis of desminopathies.

neuroscience↗

bioRxiv2022

Viscoelastic properties of suspended cells measured with shear flow deformation cytometry

Numerous cell functions are accompanied by phenotypic changes in viscoelastic properties, and measuring them can help elucidate higher-level cellular functions in health and disease. We present a high-throughput, simple and low-cost microfluidic method for quantitatively measuring the elastic (storage) and viscous (loss) modulus of individual cells. Cells are suspended in a high-viscosity fluid and are pumped with high pressure through a 5.8 cm long and 200 {micro}m wide microfluidic channel. The fluid shear stress induces large, near ellipsoidal cell deformations. In addition, the flow profile in the channel causes the cells to rotate in a tank-treading manner. From the cell deformation and tank treading frequency, we extract the frequency-dependent viscoelastic cell properties based on a theoretical framework developed by R. Roscoe1 that describes the deformation of a viscoelastic sphere in a viscous fluid under steady laminar flow. We confirm the accuracy of the method using atomic force microscopy-calibrated polyacrylamide beads and cells. Our measurements demonstrate that suspended cells exhibit power-law, soft glassy rheological behavior that is cell cycle-dependent and mediated by the physical interplay between the actin filament and intermediate filament networks.

biophysics↗

bioRxiv2021

Large portion of essential genes is missed by screening either fly or beetle indicating unexpected diversity of insect gene function

Most gene functions were detected by screens in very few model organisms but it has remained unclear how comprehensive these data are. Here, we expanded our RNAi screen in the red flour beetle Tribolium castaneum to cover more than half of the protein-coding genes and we compared the gene sets involved in several processes between beetle and fly. We find that around 50 % of the gene functions are detected in both species while the rest was found only in fly (~10%) or beetle (~40%) reflecting both technical and biological differences. We conclude that work in complementary model systems is required to gain a comprehensive picture on gene functions documented by the annotation of novel GO terms for 96 genes studied here. The RNAi screening resources developed in this project, the expanding transgenic tool-kit and our large-scale functional data make T. castaneum an excellent model system in that endeavor.

genetics↗

bioRxiv2018

A large-scale systemic RNAi screen in the red flour beetle Tribolium castaneum identifies novel genes involved in arthropod muscle development

Although muscle development has been widely studied in Drosophila melanogaster there are still many gaps in our knowledge, and it is not known to which extent this knowledge can be transferred to other insects. To help in closing these gaps we participated in a large-scale RNAi screen that used the red flour beetle, Tribolium castaneum, as a screening platform. The effects of systemic RNAi were screened upon double-stranded RNA injections into appropriate muscle-EGFP tester strains. Injections into pupae were followed by the analysis of the late embryonic/early larval muscle patterns, and injections into larvae by the analysis of the adult thoracic muscle patterns. Herein we describe the results of the first-pass screens with pupal and larval injections, which covered ~8,500 and ~5,000 genes, respectively, of a total of ~16,500 genes of the Tribolium genome. Apart from many genes known from Drosophila as regulators of muscle development, a collection of genes previously unconnected to muscle development yielded phenotypes in larval body wall and leg muscles as well as in indirect flight muscles. We then present the main candidates from the pupal injection screen that remained after being processed through a series of verification and selection steps. Further, we discuss why distinct though overlapping sets of genes are revealed by the Drosophila and Tribolium screening approaches.

developmental biology↗

bioRxiv2018

RNAi screen in Tribolium reveals involvement of F-BAR proteins in myoblast fusion and visceral muscle morphogenesis in arthropods

In a large-scale RNAi screen in Tribolium castaneum for genes with knock-down phenotypes in the larval somatic musculature, one recurring phenotype was the appearance of larval muscle fibers that were significantly thinner than those in control animals. Several of the genes producing this knock-down phenotype corresponded to orthologs of Drosophila genes that are known to participate in myoblast fusion, particularly via their effects on actin polymerization. A new gene previously not implicated in myoblast fusion but displaying a similar thin-muscle knock-down phenotype was the Tribolium ortholog of Nostrin, which encodes an F-BAR and SH3 domain protein. Our genetic studies of Nostrin and Cip4, a gene encoding a structurally related protein, in Drosophila show that the encoded F-BAR proteins jointly contribute to efficient myoblast fusion during larval muscle development. Together with the F-Bar protein Syndapin they are also required for normal embryonic midgut morphogenesis. In addition, Cip4 is required together with Nostrin during the profound remodeling of the midgut visceral musculature during metamorphosis. We propose that these F-Bar proteins help govern proper morphogenesis particularly of the longitudinal midgut muscles during metamorphosis.

developmental biology↗