Remmelzwaal, Sanne et al. (2021) International Worm Meeting "BBLN-1 is essential for intermediate filament organization and apical membrane morphology"

Jarosinska, Olga, Leube, Rudolf, van der Horst, Suzanne, Boxem, Mike, Altelaar, Maarten, Ramalho, Joao, Pasolli, Milena, Akhmanova, Anna, Stucchi, Riccardo, Remmelzwaal, Sanne, Richardson, Christine, Geisler, Florian, Kroll, Jason

[International Worm Meeting, 2021]

Epithelial tubes are essential components of metazoan organ systems that control the flow of fluids and the exchange of materials between body compartments and the outside environment. The size and shape of the central lumen confer important characteristics to tubular organs and need to be carefully controlled. Here, we identify the small coiled-coil protein BBLN-1 as a regulator of lumen morphology in the C. elegans intestine. Loss of BBLN-1 causes the formation of bubble-shaped invaginations of the apical membrane into the cytoplasm of intestinal cells, and abnormal aggregation of the subapical intermediate filament (IF) network. BBLN-1 interacts with IF proteins and localizes to the IF network in an IF-dependent manner. The appearance of invaginations is a result of the abnormal IF aggregation, indicating a direct role for the IF network in maintaining lumen homeostasis. Finally, we identify bublin (BBLN) as the mammalian ortholog of BBLN-1. When expressed in the C. elegans intestine, bublin recapitulates the localization pattern of BBLN-1 and can compensate for the loss of BBLN-1. In mouse intestinal organoids, bublin localizes subapically, together with the IF protein keratin 8. Our results therefore may have implications for understanding the role of IFs in regulating epithelial tube morphology in mammals.

Kroll, Jason et al. (2021) International Worm Meeting "Light-induced protein clustering to study protein-protein interactions in C. elegans"

Kroll, Jason, Boxem, Mike, Remmelzwaal, Sanne

[International Worm Meeting, 2021]

Interactions among proteins are fundamental for life, and misregulation of them can lead to diseases such as cancer. We adapted the light-activated reversible inhibition by assembled trap (LARIAT) system and the light-induced co-clustering assay for use in C. elegans. This assay can rapidly and unambiguously identify protein-protein interactions between pairs of fluorescently tagged proteins. In the LARIAT protein-protein interaction assay, cryptochrome 2 (CRY2) and cryptochrome-interacting bHLH 1 (CIB1) bind each other in a blue-light dependent manner, forming small but densely packed protein clusters within the cell. Fusion of an anti-GFP nanobody to the CRY2 protein traps any GFP-tagged protein in the clusters. Co-clustering of a mCherry/mScarlet fluorescent protein can be monitored for inclusion in the CRY2/GFP clusters, indicating an interaction between the two fluorescently tagged proteins. We tested the system in C. elegans with an array of positive and negative reference protein pairs, using endogenously tagged GFP and mCherry/mScarlet alleles. CRY2/CIB1 proteins were expressed from extrachromosomal arrays since genome integration is unnecessary for the assay. Light-induced clusters form rapidly within seconds in multiple tissue types. Assay performance was extremely robust with no false positives detected in the negative reference pairs, and no lethality associated with the clustering, even with proteins essential for development and cell polarity. We have generated a toolkit containing plasmids with different promoters, an anti-mCherry nanobody variant, and a plasmid with an empty promoter module to enable one-step cloning of any particular promoter using SapTrap assembly. The light induced co-clustering assay is a powerful and rapid technique that will be valuable for the C. elegans community, especially due to the fact that the assay uses a universal plasmid for every protein pair, and existing fluorescently tagged strains can be used without need for additional cloning or genetic modification of the genome.

Geisler, Florian et al. (2021) International Worm Meeting "Perturbed intermediate filament regulation causes aggregate toxicity"

Leube, Rudolf, Remmelzwaal, Sanne, Richardson, Christine, Geisler, Florian, Boxem, Mike

[International Worm Meeting, 2021]

Intermediate filaments are major components of the metazoan cytoskeleton. A long-standing debate concerns the question whether intermediate filament network organization only reflects or also determines cell and tissue function and dysfunction. This is particularly relevant for aggregate-forming diseases involving intermediate filaments. Using C. elegans as a genetic model organism, we have recently described mutants of signaling and stress response pathways with perturbed intermediate filament network organization. In a mutagenesis screen, we now identify the intermediate filament polypeptide IFB-2 as a highly efficient suppressor of these phenotypes restoring not only intestinal morphology but also rescuing compromised development, growth, reproduction and stress resilience. Ultrastructural analyses show that downregulation of IFB-2 leads to depletion of the aggregated intermediate filaments. The findings provide compelling evidence for the toxic function of deranged intermediate filaments and reveal novel insights into the cross talk between signaling and structural functions of the intermediate filament cytoskeleton.