Biology subjects

Remmelzwaal, S.

Publications and source records attributed to Remmelzwaal, S..

3 recordsLinked to original sources

bioRxiv2022

Intermediate filament network perturbation in the C. elegans intestine causes systemic toxicity

Intermediate filaments (IFs) are major components of the metazoan cytoskeleton. A long-standing debate concerns the question whether IF network organization only reflects or also determines cell and tissue function. Using C. elegans, we have recently described mutants of the MAPK SMA-5, which perturb the organization of the intestinal IF cytoskeleton resulting in luminal widening and cytoplasmic invaginations. Besides these structural phenotypes, systemic dysfunctions were also observed. We now identify the IF polypeptide IFB-2 as a highly efficient suppressor of both the structural and functional deficiencies by removing the aberrant IF network. Mechanistically, IF network morphogenesis is linked to the phosphorylated IFB-2 aminoterminus. The rescuing capability is IF isotype-specific and not restricted to SMA-5 mutants but extends to other regulators of IF network morphogenesis, i.e. the cytoskeletal linker IFO-1 and the IF-associated protein BBLN1. The findings provide strong evidence for a gain-of-toxic function of the deranged IF networks with implications for diseases that are characterized by altered IF network organization.

cell biology↗

bioRxiv2021

CeLINC, a fluorescence-based protein-protein interaction assay in C. elegans

Interactions among proteins are fundamental for life and determining whether two particular proteins physically interact can be essential for fully understanding a proteins function. We present C. elegans light-induced co-clustering (CeLINC), an optical binary protein-protein interaction assay to determine whether two proteins interact in vivo. Based on CRY2/CIB1 light-dependent oligomerization, CeLINC can rapidly and unambiguously identify protein-protein interactions between pairs of fluorescently tagged proteins. A fluorescently tagged bait protein is captured using a nanobody directed against the fluorescent protein (GFP or mCherry) and brought into artificial clusters within the cell. Co-localization of a fluorescently tagged prey protein in the cluster indicates a protein interaction. We tested the system with an array of positive and negative reference protein pairs. Assay performance was extremely robust with no false positives detected in the negative reference pairs. We then used the system to test for interactions among apical and basolateral polarity regulators. We confirmed interactions seen between PAR-6, PKC-3, and PAR-3, but observed no physical interactions among the basolateral Scribble module proteins LET-413, DLG-1, and LGL-1. We have generated a plasmid toolkit that allows use of custom promoters or CRY2 variants to promote flexibility of the system. The CeLINC assay is a powerful and rapid technique that can be widely applied in C. elegans due to the universal plasmids that can be used with existing fluorescently tagged strains without need for additional cloning or genetic modification of the genome. SummaryWe have developed a protein-protein interaction assay for C. elegans to investigate whether pairs of proteins interact in vivo. C. elegans light-induced co-clustering (CeLINC) is based on trapping a fluorescently-tagged bait protein into artificial clusters, and observing whether candidate interacting prey proteins co-cluster with the bait protein. CeLINC can be widely applied as a single set of universal plasmids can be used with existing strains expressing fluorescently-tagged proteins.

developmental biology↗

bioRxiv2020

BUBL-1 is essential for intermediate filamentorganization and apical membrane morphology

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. SummaryWe identify BBLN-1 as an evolutionary conserved regulator of lumen morphology in the C. elegans intestine. Loss of bbln-1 causes intermediate filament network reorganization that induces severe apical morphology defects. We also identify bublin (BBLN) as the mammalian ortholog, which can compensate for the loss of BBLN-1 in C. elegans.

developmental biology↗