Publications by authors named "João J Ramalho"

5 Publications

  • Page 1 of 1

BBLN-1 is essential for intermediate filament organization and apical membrane morphology.

Curr Biol 2021 06 14;31(11):2334-2346.e9. Epub 2021 Apr 14.

Developmental Biology, Institute of Biodynamics and Biocomplexity, Department of Biology, Faculty of Science, Utrecht University, Padualaan 8, 3584 CH Utrecht, the Netherlands. Electronic address:

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, BBLN recapitulates the localization pattern of BBLN-1 and can compensate for the loss of BBLN-1 in early larvae. In mouse intestinal organoids, BBLN 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.
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http://dx.doi.org/10.1016/j.cub.2021.03.069DOI Listing
June 2021

C-terminal phosphorylation modulates ERM-1 localization and dynamics to control cortical actin organization and support lumen formation during development.

Development 2020 07 22;147(14). Epub 2020 Jul 22.

Division of Developmental Biology, Institute of Biodynamics and Biocomplexity, Department of Biology, Faculty of Science, Utrecht University, Padualaan 8, 3584 CH, Utrecht, The Netherlands

ERM proteins are conserved regulators of cortical membrane specialization that function as membrane-actin linkers and molecular hubs. The activity of ERM proteins requires a conformational switch from an inactive cytoplasmic form into an active membrane- and actin-bound form, which is thought to be mediated by sequential PIP binding and phosphorylation of a conserved C-terminal threonine residue. Here, we use the single ERM ortholog, ERM-1, to study the contribution of these regulatory events to ERM activity and tissue formation Using CRISPR/Cas9-generated mutant alleles, we demonstrate that a PIP-binding site is crucially required for ERM-1 function. By contrast, dynamic regulation of C-terminal T544 phosphorylation is not essential but modulates ERM-1 apical localization and dynamics in a tissue-specific manner, to control cortical actin organization and support lumen formation in epithelial tubes. Our work highlights the dynamic nature of ERM protein regulation during tissue morphogenesis and the importance of C-terminal phosphorylation in fine-tuning ERM activity in a tissue-specific context.
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http://dx.doi.org/10.1242/dev.188011DOI Listing
July 2020

JMJD-5/KDM8 regulates H3K36me2 and is required for late steps of homologous recombination and genome integrity.

PLoS Genet 2017 02 16;13(2):e1006632. Epub 2017 Feb 16.

Biotech Research and Innovation Centre (BRIC), University of Copenhagen, Copenhagen, Denmark.

The eukaryotic genome is organized in a three-dimensional structure called chromatin, constituted by DNA and associated proteins, the majority of which are histones. Post-translational modifications of histone proteins greatly influence chromatin structure and regulate many DNA-based biological processes. Methylation of lysine 36 of histone 3 (H3K36) is a post-translational modification functionally relevant during early steps of DNA damage repair. Here, we show that the JMJD-5 regulates H3K36 di-methylation and it is required at late stages of double strand break repair mediated by homologous recombination. Loss of jmjd-5 results in hypersensitivity to ionizing radiation and in meiotic defects, and it is associated with aberrant retention of RAD-51 at sites of double strand breaks. Analyses of jmjd-5 genetic interactions with genes required for resolving recombination intermediates (rtel-1) or promoting the resolution of RAD-51 double stranded DNA filaments (rfs-1 and helq-1) suggest that jmjd-5 prevents the formation of stalled postsynaptic recombination intermediates and favors RAD-51 removal. As these phenotypes are all recapitulated by a catalytically inactive jmjd-5 mutant, we propose a novel role for H3K36me2 regulation during late steps of homologous recombination critical to preserve genome integrity.
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http://dx.doi.org/10.1371/journal.pgen.1006632DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5336306PMC
February 2017

A tissue-specific protein purification approach in Caenorhabditis elegans identifies novel interaction partners of DLG-1/Discs large.

BMC Biol 2016 08 9;14:66. Epub 2016 Aug 9.

Developmental Biology, Department of Biology, Faculty of Science, Utrecht University, Padualaan 8, 3584 CH, Utrecht, The Netherlands.

Background: Affinity purification followed by mass spectrometry (AP/MS) is a widely used approach to identify protein interactions and complexes. In multicellular organisms, the accurate identification of protein complexes by AP/MS is complicated by the potential heterogeneity of complexes in different tissues. Here, we present an in vivo biotinylation-based approach for the tissue-specific purification of protein complexes from Caenorhabditis elegans. Tissue-specific biotinylation is achieved by the expression in select tissues of the bacterial biotin ligase BirA, which biotinylates proteins tagged with the Avi peptide.

Results: We generated N- and C-terminal tags combining GFP with the Avi peptide sequence, as well as four BirA driver lines expressing BirA ubiquitously and specifically in the seam and hyp7 epidermal cells, intestine, or neurons. We validated the ability of our approach to identify bona fide protein interactions by identifying the known LGL-1 interaction partners PAR-6 and PKC-3. Purification of the Discs large protein DLG-1 identified several candidate interaction partners, including the AAA-type ATPase ATAD-3 and the uncharacterized protein MAPH-1.1. We have identified the domains that mediate the DLG-1/ATAD-3 interaction, and show that this interaction contributes to C. elegans development. MAPH-1.1 co-purified specifically with DLG-1 purified from neurons, and shared limited homology with the microtubule-associated protein MAP1A, a known neuronal interaction partner of mammalian DLG4/PSD95. A CRISPR/Cas9-engineered GFP::MAPH-1.1 fusion was broadly expressed and co-localized with microtubules.

Conclusions: The method we present here is able to purify protein complexes from specific tissues. We uncovered a series of DLG-1 interactors, and conclude that ATAD-3 is a biologically relevant interaction partner of DLG-1. Finally, we conclude that MAPH-1.1 is a microtubule-associated protein of the MAP1 family and a candidate neuron-specific interaction partner of DLG-1.
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http://dx.doi.org/10.1186/s12915-016-0286-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4977824PMC
August 2016

A combined binary interaction and phenotypic map of C. elegans cell polarity proteins.

Nat Cell Biol 2016 Mar 18;18(3):337-46. Epub 2016 Jan 18.

Division of Developmental Biology, Department of Biology, Faculty of Science, Utrecht University, 3584 CH, Utrecht, The Netherlands.

The establishment of cell polarity is an essential process for the development of multicellular organisms and the functioning of cells and tissues. Here, we combine large-scale protein interaction mapping with systematic phenotypic profiling to study the network of physical interactions that underlies polarity establishment and maintenance in the nematode Caenorhabditis elegans. Using a fragment-based yeast two-hybrid strategy, we identified 439 interactions between 296 proteins, as well as the protein regions that mediate these interactions. Phenotypic profiling of the network resulted in the identification of 100 physically interacting protein pairs for which RNAi-mediated depletion caused a defect in the same polarity-related process. We demonstrate the predictive capabilities of the network by showing that the physical interaction between the RhoGAP PAC-1 and PAR-6 is required for radial polarization of the C. elegans embryo. Our network represents a valuable resource of candidate interactions that can be used to further our insight into cell polarization.
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http://dx.doi.org/10.1038/ncb3300DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4767559PMC
March 2016
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