Proteomic Analysis of the Dysferlin Protein Complex Unveils Its Importance for Sarcolemmal Maintenance and Integrity

Dysferlin is critical for repair of muscle membranes after damage. Mutations in dysferlin lead to a progressive muscular dystrophy. Recent studies suggest additional roles for dysferlin. We set out to study dysferlin's protein-protein interactions to obtain comprehensive knowledge of dysferlin functionalities in a myogenic context. We developed a robust and reproducible method to isolate dysferlin protein complexes from cells and tissue. We analyzed the composition of these complexes in cultured myoblasts, myotubes and skeletal muscle tissue by mass spectrometry and subsequently inferred potential protein functions through bioinformatics analyses. Our data confirm previously reported interactions and support a function for dysferlin as a vesicle trafficking protein. In addition novel potential functionalities were uncovered, including phagocytosis and focal adhesion. Our data reveal that the dysferlin protein complex has a dynamic composition as a function of myogenic differentiation. We provide additional experimental evidence and show dysferlin localization to, and interaction with the focal adhesion protein vinculin at the sarcolemma. Finally, our studies reveal evidence for cross-talk between dysferlin and its protein family member myoferlin. Together our analyses show that dysferlin is not only a membrane repair protein but also important for muscle membrane maintenance and integrity

Genetic modifiers ameliorate endocytic and neuromuscular defects in a model of spinal muscular atrophy

© 2020 The Author(s). This article is licensed under a Creative Commons Attribution 4.0 International License (https://creativecommons.org/licenses/by/4.0/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made.Background: Understanding the genetic modifiers of neurodegenerative diseases can provide insight into the mechanisms underlying these disorders. Here, we examine the relationship between the motor neuron disease spinal muscular atrophy (SMA), which is caused by reduced levels of the survival of motor neuron (SMN) protein, and the actin-bundling protein Plastin 3 (PLS3). Increased PLS3 levels suppress symptoms in a subset of SMA patients and ameliorate defects in SMA disease models, but the functional connection between PLS3 and SMN is poorly understood.Results: We provide immunohistochemical and biochemical evidence for large protein complexes localized in vertebrate motor neuron processes that contain PLS3, SMN and members of the hnRNP F/H family of proteins. Using a Caenorhabditis elegans (C. elegans) SMA model, we determine that overexpression of PLS3 or loss of the C. elegans hnRNP F/H ortholog SYM-2 enhances endocytic function and ameliorates neuromuscular defects caused by decreased SMN-1 levels. Furthermore, either increasing PLS3 or decreasing SYM-2 levels suppresses defects in a C. elegans ALS model.Conclusions: We propose that hnRNP F/H act in the same protein complex as PLS3 and SMN and that the function of this complex is critical for endocytic pathways, suggesting that hnRNP F/H proteins could be potential targets for therapy development.Peer reviewe

Macrolides rapidly inhibit red blood cell invasion by the human malaria parasite, Plasmodium falciparum

BACKGROUND Malaria invasion of red blood cells involves multiple parasite-specific targets that are easily accessible to inhibitory compounds, making it an attractive target for antimalarial development. However, no current antimalarial agents act against host cell invasion. RESULTS Here, we demonstrate that the clinically used macrolide antibiotic azithromycin, which is known to kill human malaria asexual blood-stage parasites by blocking protein synthesis in their apicoplast, is also a rapid inhibitor of red blood cell invasion in human (Plasmodium falciparum) and rodent (P. berghei) malarias. Multiple lines of evidence demonstrate that the action of azithromycin in inhibiting parasite invasion of red blood cells is independent of its inhibition of protein synthesis in the parasite apicoplast, opening up a new strategy to develop a single drug with multiple parasite targets. We identified derivatives of azithromycin and erythromycin that are better invasion inhibitors than parent compounds, offering promise for development of this novel antimalarial strategy. CONCLUSIONS Safe and effective macrolide antibiotics with dual modalities could be developed to combat malaria and reduce the parasite’s options for resistance.Danny W Wilson, Christopher D Goodman, Brad E Sleebs, Greta E Weiss, Nienke WM de Jong, Fiona Angrisano, Christine Langer, Jake Baum, Brendan S Crabb, Paul R Gilson, Geoffrey I McFadden, and James G Beeso

East Adriatic: a reservoir region of severe Citrus tristeza virus strains

Citrus tristeza virus (CTV) represents one of the major threats to citrus production worldwide. In the East Adriatic region, CTV symptoms are mostly absent due to traditional citrus grafting on trifoliate orange (Poncirus trifoliata), a CTV-tolerant rootstock. Therefore, the virus has been continuously spreading by the propagation of infected material. The genetic variability of CTV was studied on nineteen citrus samples, collected from orchards in the coastal region of Croatia, Montenegro and Albania, that previously tested positive by ELISA and immunocapture RT-PCR. Single-strand conformation polymorphism of the amplified coat protein gene demonstrated the presence of different CTV variants in each amplicon, while sequence analysis of cloned CP gene variants confirmed their clustering into six out of the seven phylogenetic groups so far delineated. Four of these groups include sequences of severe quick decline, seedling yellows and stem-pitting (SP) isolates, thought to be found only rarely in the Mediterranean region. Regardless of the lack of symptoms in the field, CTV isolates from the East Adriatic displayed high genetic variability and pathogenic potential, additionally confirmed by biological characterisation. The high percentage of mixed infections suggest the potential for further diversification and a greater risk of severe variants spreading into new areas

Material Properties of Caenorhabditis elegans Swimming at Low Reynolds Number

Undulatory locomotion, as seen in the nematode Caenorhabditis elegans, is a common swimming gait of organisms in the low Reynolds number regime, where viscous forces are dominant. Although the nematode's motility is expected to be a strong function of its material properties, measurements remain scarce. Here, the swimming behavior of C. elegans is investigated in experiments and in a simple model. Experiments reveal that nematodes swim in a periodic fashion and generate traveling waves that decay from head to tail. The model is able to capture the experiments' main features and is used to estimate the nematode's Young's modulus E and tissue viscosity η. For wild-type C. elegans, we find E ≈ 3.77 kPa and η ≈ −860 Pa·s; values of η for live C. elegans are negative because the tissue is generating rather than dissipating energy. Results show that material properties are sensitive to changes in muscle functional properties, and are useful quantitative tools with which to more accurately describe new and existing muscle mutants