Publications by authors named "Mike Boxem"

44 Publications

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

Genetics 2021 Dec;219(4)

Division of Developmental Biology, Department of Biology, Faculty of Science, Institute of Biodynamics and Biocomplexity, Utrecht University, 3584 CH Utrecht, the Netherlands.

Interactions among proteins are fundamental for life and determining whether two particular proteins physically interact can be essential for fully understanding a protein's function. We present Caenorhabditis elegans light-induced coclustering (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. Colocalization 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.
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http://dx.doi.org/10.1093/genetics/iyab163DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8664570PMC
December 2021

Caenorhabditis elegans LET-413 Scribble is essential in the epidermis for growth, viability, and directional outgrowth of epithelial seam cells.

PLoS Genet 2021 10 21;17(10):e1009856. Epub 2021 Oct 21.

Division of Developmental Biology, Institute of Biodynamics and Biocomplexity, Department of Biology, Faculty of Science, Utrecht University, Utrecht, The Netherlands.

The conserved adapter protein Scribble (Scrib) plays essential roles in a variety of cellular processes, including polarity establishment, proliferation, and directed cell migration. While the mechanisms through which Scrib promotes epithelial polarity are beginning to be unraveled, its roles in other cellular processes including cell migration remain enigmatic. In C. elegans, the Scrib ortholog LET-413 is essential for apical-basal polarization and junction formation in embryonic epithelia. However, whether LET-413 is required for postembryonic development or plays a role in migratory events is not known. Here, we use inducible protein degradation to investigate the functioning of LET-413 in larval epithelia. We find that LET-413 is essential in the epidermal epithelium for growth, viability, and junction maintenance. In addition, we identify a novel role for LET-413 in the polarized outgrowth of the epidermal seam cells. These stem cell-like epithelial cells extend anterior and posterior directed apical protrusions in each larval stage to reconnect to their neighbors. We show that the role of LET-413 in seam cell outgrowth is likely mediated largely by the junctional component DLG-1 discs large, which we demonstrate is also essential for directed outgrowth of the seam cells. Our data uncover multiple essential functions for LET-413 in larval development and show that the polarized outgrowth of the epithelial seam cells is controlled by LET-413 Scribble and DLG-1 Discs large.
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http://dx.doi.org/10.1371/journal.pgen.1009856DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8570498PMC
October 2021

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

Epidermal PAR-6 and PKC-3 are essential for larval development of and organize non-centrosomal microtubules.

Elife 2020 12 10;9. Epub 2020 Dec 10.

Division of Developmental Biology, Institute of Biodynamics and Biocomplexity, Department of Biology, Faculty of Science, Utrecht University, Utrecht, Netherlands.

The cortical polarity regulators PAR-6, PKC-3, and PAR-3 are essential for the polarization of a broad variety of cell types in multicellular animals. In , the roles of the PAR proteins in embryonic development have been extensively studied, yet little is known about their functions during larval development. Using inducible protein degradation, we show that PAR-6 and PKC-3, but not PAR-3, are essential for postembryonic development. PAR-6 and PKC-3 are required in the epidermal epithelium for animal growth, molting, and the proper pattern of seam-cell divisions. Finally, we uncovered a novel role for PAR-6 in organizing non-centrosomal microtubule arrays in the epidermis. PAR-6 was required for the localization of the microtubule organizer NOCA-1/Ninein, and defects in a mutant are highly similar to those caused by epidermal PAR-6 depletion. As NOCA-1 physically interacts with PAR-6, we propose that PAR-6 promotes non-centrosomal microtubule organization through localization of NOCA-1/Ninein.
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http://dx.doi.org/10.7554/eLife.62067DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7755398PMC
December 2020

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

Split Intein-Mediated Protein Ligation for detecting protein-protein interactions and their inhibition.

Nat Commun 2020 05 15;11(1):2440. Epub 2020 May 15.

Donnelly Centre, University of Toronto, Toronto, ON, Canada.

Here, to overcome many limitations accompanying current available methods to detect protein-protein interactions (PPIs), we develop a live cell method called Split Intein-Mediated Protein Ligation (SIMPL). In this approach, bait and prey proteins are respectively fused to an intein N-terminal fragment (IN) and C-terminal fragment (IC) derived from a re-engineered split intein GP41-1. The bait/prey binding reconstitutes the intein, which splices the bait and prey peptides into a single intact protein that can be detected by regular protein detection methods such as Western blot analysis and ELISA, serving as readouts of PPIs. The method is robust and can be applied not only in mammalian cell lines but in animal models such as C. elegans. SIMPL demonstrates high sensitivity and specificity, and enables exploration of PPIs in different cellular compartments and tracking of kinetic interactions. Additionally, we establish a SIMPL ELISA platform that enables high-throughput screening of PPIs and their inhibitors.
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http://dx.doi.org/10.1038/s41467-020-16299-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7229206PMC
May 2020

Host interactors of effector proteins of the lettuce downy mildew Bremia lactucae obtained by yeast two-hybrid screening.

PLoS One 2020 12;15(5):e0226540. Epub 2020 May 12.

Plant-Microbe Interactions, Department of Biology, Utrecht University, Utrecht, The Netherlands.

Plant pathogenic bacteria, fungi and oomycetes secrete effector proteins to manipulate host cell processes to establish a successful infection. Over the last decade the genomes and transcriptomes of many agriculturally important plant pathogens have been sequenced and vast candidate effector repertoires were identified using bioinformatic analyses. Elucidating the contribution of individual effectors to pathogenicity is the next major hurdle. To advance our understanding of the molecular mechanisms underlying lettuce susceptibility to the downy mildew Bremia lactucae, we mapped physical interactions between B. lactucae effectors and lettuce candidate target proteins. Using a lettuce cDNA library-based yeast-two-hybrid system, 61 protein-protein interactions were identified, involving 21 B. lactucae effectors and 46 unique lettuce proteins. The top ten interactors based on the number of independent colonies identified in the Y2H and two interactors that belong to gene families involved in plant immunity, were further characterized. We determined the subcellular localization of the fluorescently tagged lettuce proteins and their interacting effectors. Importantly, relocalization of effectors or their interactors to the nucleus was observed for four protein-pairs upon their co-expression, supporting their interaction in planta.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0226540PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7217486PMC
September 2020

New insights into apical-basal polarization in epithelia.

Curr Opin Cell Biol 2020 02 7;62:1-8. Epub 2019 Sep 7.

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

The establishment of an apical-basal axis of polarity is essential for the organization and functioning of epithelial cells. Polarization of epithelial cells is orchestrated by a network of conserved polarity regulators that establish opposing cortical domains through mutually antagonistic interactions and positive feedback loops. While our understanding is still far from complete, the molecular details behind these interactions continue to be worked out. Here, we highlight recent findings on the mechanisms that control the activity and localization of apical-basal polarity regulators, including oligomerization and higher-order complex formation, auto-inhibitory interactions, and electrostatic interactions with the plasma membrane.
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http://dx.doi.org/10.1016/j.ceb.2019.07.017DOI Listing
February 2020

Functional Dissection of bZip-Protein CEBP-1 Reveals Novel Structural Motifs Required for Axon Regeneration and Nuclear Import.

Front Cell Neurosci 2019 31;13:348. Epub 2019 Jul 31.

Convergence Program of Material Science for Medicine and Pharmaceutics, Department of Life Science, Multidisciplinary Genome Institute, Hallym University, Chuncheon, South Korea.

The basic leucine-zipper (bZIP) domain transcription factors CCAAT/enhancer-binding proteins (C/EBP) have a variety of roles in cell proliferation, differentiation, and stress response. In the nervous system, several isoforms of C/EBP function in learning and memory, neuronal plasticity, neuroinflammation, and axon regeneration. We previously reported that the C/EBP homolog, CEBP-1, is essential for axon regeneration. CEBP-1 consists of 319 amino acids, with its bZIP domain at the C-terminus and a long N-terminal fragment with no known protein motifs. Here, using forward genetic screening with targeted genome editing, we have identified a unique domain in the N-terminus that is critical for its function. Additionally, we characterized three nuclear localization signals (NLS) in CEBP-1 that act together to mediate CEBP-1's nuclear import. Moreover, the Importin-α, IMA-3, can bind to CEBP-1 via one of the NLS. is ubiquitously expressed in all somatic cells, and null mutants are larval lethal. Using Cre-lox dependent neuron-specific deletion strategy, we show that is not critical for axon development, but is required for axon regeneration in adults. Together, these data advance our understanding of CEBP-1's function, and suggest new regulators that remain to be identified to expand the CEBP-1 protein interactome.
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http://dx.doi.org/10.3389/fncel.2019.00348DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6685058PMC
July 2019

Cell Polarity: Getting the PARty Started.

Curr Biol 2019 07;29(13):R637-R639

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

Polarity establishment is a key developmental process, but what determines its timing is poorly understood. New research in Caenorhabditis elegans demonstrates that the PAR polarity system extensively reconfigures before becoming competent to polarize. By inhibiting membrane localization of anterior PAR proteins, AIR-1 (aurora A) and PLK-1 (polo kinase) prevent premature polarization.
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http://dx.doi.org/10.1016/j.cub.2019.05.032DOI Listing
July 2019

Protein interactome mapping in .

Curr Opin Syst Biol 2019 Feb;13:1-9

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

The systematic identification of all protein-protein interactions that take place in an organism (the 'interactome') is an important goal in modern biology. The nematode was one of the first multicellular models for which a proteome-wide interactome mapping project was initiated. Most interactome mapping efforts have utilized the yeast two-hybrid system, yielding an extensive binary interactome, while recent developments in mass spectrometry-based approaches hold great potential for further improving our understanding of protein interactome networks in a multicellular context. For example, methods like co-fractionation, proximity labeling, and tissue-specific protein purification not only identify protein-protein interactions, but have the potential to provide crucial insight into when and where interactions take place. Here we review current standards and recent improvements in protein interaction mapping in
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http://dx.doi.org/10.1016/j.coisb.2018.08.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7493430PMC
February 2019

Genetic and cellular sensitivity of to the chemotherapeutic agent cisplatin.

Dis Model Mech 2018 06 21;11(6). Epub 2018 Jun 21.

Modeling human diseases in C. elegans. Genes, Diseases and Therapies Program, Bellvitge Biomedical Research Institute - IDIBELL, L'Hospitalet de Llobregat, 08908 Barcelona, Spain

Cisplatin and derivatives are commonly used as chemotherapeutic agents. Although the cytotoxic action of cisplatin on cancer cells is very efficient, clinical oncologists need to deal with two major difficulties, namely the onset of resistance to the drug and the cytotoxic effect in patients. Here, we used to investigate factors influencing the response to cisplatin in multicellular organisms. In this hermaphroditic model organism, we observed that sperm failure is a major cause of cisplatin-induced infertility. RNA sequencing data indicate that cisplatin triggers a systemic stress response, in which DAF-16/FOXO and SKN-1/NRF2, two conserved transcription factors, are key regulators. We determined that inhibition of the DNA damage-induced apoptotic pathway does not confer cisplatin protection to the animal. However, mutants for the pro-apoptotic BH3-only gene are sensitive to cisplatin, suggesting a protective role of the intrinsic apoptotic pathway. Finally, we demonstrated that our system can also be used to identify mutations providing resistance to cisplatin and therefore potential biomarkers of innate cisplatin-refractory patients. We show that mutants for the redox regulator , ortholog of the mammalian thioredoxin reductase 1 , display cisplatin resistance. By CRISPR/Cas9, we determined that such resistance relies on the presence of the single selenocysteine residue in TRXR-1.This article has an associated First Person interview with the first author of the paper.
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http://dx.doi.org/10.1242/dmm.033506DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6031354PMC
June 2018

Mapping the Polarity Interactome.

J Mol Biol 2018 09 29;430(19):3521-3544. Epub 2017 Dec 29.

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

Interactions between proteins are an essential part of biology, and the desire to identify these interactions has led to the development of numerous technologies to systematically map protein-protein interactions at a large scale. As in most cellular processes, protein interactions are central to the control of cell polarity, and a full understanding of polarity will require comprehensive knowledge of the protein interactions involved. At its core, cell polarity is established through carefully regulated mutually inhibitory interactions between several groups of cortical proteins. While several interactions have been identified, the dynamics and molecular mechanisms that control these interactions are not well understood. Cell polarity also needs to be integrated with cellular processes including junction formation, cytoskeletal organization, organelle positioning, protein trafficking, and functional specialization of membrane domains. Moreover, polarized cells need to respond to external cues that coordinate polarity at the tissue level. Identifying the protein-protein interactions responsible for integrating polarity with all of these processes remains a major challenge, in part because the mechanisms of polarity control vary in different contexts and with developmental times. Because of their unbiased nature, systematic large-scale protein-protein interaction mapping approaches can be particularly helpful to identify such mechanisms. Here, we discuss methods commonly used to generate proteome-wide interactome maps, with an emphasis on advances in our understanding of cell polarity that have been achieved through application of such methods.
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http://dx.doi.org/10.1016/j.jmb.2017.12.017DOI Listing
September 2018

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

Multisite Phosphorylation of NuMA-Related LIN-5 Controls Mitotic Spindle Positioning in C. elegans.

PLoS Genet 2016 Oct 6;12(10):e1006291. Epub 2016 Oct 6.

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

During cell division, the mitotic spindle segregates replicated chromosomes to opposite poles of the cell, while the position of the spindle determines the plane of cleavage. Spindle positioning and chromosome segregation depend on pulling forces on microtubules extending from the centrosomes to the cell cortex. Critical in pulling force generation is the cortical anchoring of cytoplasmic dynein by a conserved ternary complex of Gα, GPR-1/2, and LIN-5 proteins in C. elegans (Gα-LGN-NuMA in mammals). Previously, we showed that the polarity kinase PKC-3 phosphorylates LIN-5 to control spindle positioning in early C. elegans embryos. Here, we investigate whether additional LIN-5 phosphorylations regulate cortical pulling forces, making use of targeted alteration of in vivo phosphorylated residues by CRISPR/Cas9-mediated genetic engineering. Four distinct in vivo phosphorylated LIN-5 residues were found to have critical functions in spindle positioning. Two of these residues form part of a 30 amino acid binding site for GPR-1, which we identified by reverse two-hybrid screening. We provide evidence for a dual-kinase mechanism, involving GSK3 phosphorylation of S659 followed by phosphorylation of S662 by casein kinase 1. These LIN-5 phosphorylations promote LIN-5-GPR-1/2 interaction and contribute to cortical pulling forces. The other two critical residues, T168 and T181, form part of a cyclin-dependent kinase consensus site and are phosphorylated by CDK1-cyclin B in vitro. We applied a novel strategy to characterize early embryonic defects in lethal T168,T181 knockin substitution mutants, and provide evidence for sequential LIN-5 N-terminal phosphorylation and dephosphorylation in dynein recruitment. Our data support that phosphorylation of multiple LIN-5 domains by different kinases contributes to a mechanism for spatiotemporal control of spindle positioning and chromosome segregation.
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http://dx.doi.org/10.1371/journal.pgen.1006291DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5053539PMC
October 2016

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

Axon injury triggers EFA-6 mediated destabilization of axonal microtubules via TACC and doublecortin like kinase.

Elife 2015 Sep 4;4. Epub 2015 Sep 4.

Neurobiology Section, Division of Biological Sciences, University of California, San Diego, La Jolla, United States.

Axon injury triggers a series of changes in the axonal cytoskeleton that are prerequisites for effective axon regeneration. In Caenorhabditis elegans the signaling protein Exchange Factor for ARF-6 (EFA-6) is a potent intrinsic inhibitor of axon regrowth. Here we show that axon injury triggers rapid EFA-6-dependent inhibition of axonal microtubule (MT) dynamics, concomitant with relocalization of EFA-6. EFA-6 relocalization and axon regrowth inhibition require a conserved 18-aa motif in its otherwise intrinsically disordered N-terminal domain. The EFA-6 N-terminus binds the MT-associated proteins TAC-1/Transforming-Acidic-Coiled-Coil, and ZYG-8/Doublecortin-Like-Kinase, both of which are required for regenerative growth cone formation, and which act downstream of EFA-6. After injury TAC-1 and EFA-6 transiently relocalize to sites marked by the MT minus end binding protein PTRN-1/Patronin. We propose that EFA-6 acts as a bifunctional injury-responsive regulator of axonal MT dynamics, acting at the cell cortex in the steady state and at MT minus ends after injury.
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http://dx.doi.org/10.7554/eLife.08695DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4596636PMC
September 2015

The C. elegans Crumbs family contains a CRB3 homolog and is not essential for viability.

Biol Open 2015 Feb 6;4(3):276-84. Epub 2015 Feb 6.

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

Crumbs proteins are important regulators of epithelial polarity. In C. elegans, no essential role for the two described Crumbs homologs has been uncovered. Here, we identify and characterize an additional Crumbs family member in C. elegans, which we termed CRB-3 based on its similarity in size and sequence to mammalian CRB3. We visualized CRB-3 subcellular localization by expressing a translational GFP fusion. CRB-3::GFP was expressed in several polarized tissues in the embryo and larval stages, and showed apical localization in the intestine and pharynx. To identify the function of the Crumbs family in C. elegans development, we generated a triple Crumbs deletion mutant by sequentially removing the entire coding sequence for each crumbs homolog using a CRISPR/Cas9-based approach. Remarkably, animals lacking all three Crumbs homologs are viable and show normal epithelial polarity. Thus, the three C. elegans Crumbs family members do not appear to play an essential role in epithelial polarity establishment.
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http://dx.doi.org/10.1242/bio.201410744DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4359734PMC
February 2015

Rb and FZR1/Cdh1 determine CDK4/6-cyclin D requirement in C. elegans and human cancer cells.

Nat Commun 2015 Jan 6;6:5906. Epub 2015 Jan 6.

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

Cyclin-dependent kinases 4 and 6 (CDK4/6) in complex with D-type cyclins promote cell cycle entry. Most human cancers contain overactive CDK4/6-cyclin D, and CDK4/6-specific inhibitors are promising anti-cancer therapeutics. Here, we investigate the critical functions of CDK4/6-cyclin D kinases, starting from an unbiased screen in the nematode Caenorhabditis elegans. We found that simultaneous mutation of lin-35, a retinoblastoma (Rb)-related gene, and fzr-1, an orthologue to the APC/C co-activator Cdh1, completely eliminates the essential requirement of CDK4/6-cyclin D (CDK-4/CYD-1) in C. elegans. CDK-4/CYD-1 phosphorylates specific residues in the LIN-35 Rb spacer domain and FZR-1 amino terminus, resembling inactivating phosphorylations of the human proteins. In human breast cancer cells, simultaneous knockdown of Rb and FZR1 synergistically bypasses cell division arrest induced by the CDK4/6-specific inhibitor PD-0332991. Our data identify FZR1 as a candidate CDK4/6-cyclin D substrate and point to an APC/C(FZR1) activity as an important determinant in response to CDK4/6-inhibitors.
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http://dx.doi.org/10.1038/ncomms6906DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4354291PMC
January 2015

Controlled sumoylation of the mevalonate pathway enzyme HMGS-1 regulates metabolism during aging.

Proc Natl Acad Sci U S A 2014 Sep 3;111(37):E3880-9. Epub 2014 Sep 3.

Division of Biology and Biological Engineering and Howard Hughes Medical Institute, California Institute of Technology, Pasadena, CA 91125;

Many metabolic pathways are critically regulated during development and aging but little is known about the molecular mechanisms underlying this regulation. One key metabolic cascade in eukaryotes is the mevalonate pathway. It catalyzes the synthesis of sterol and nonsterol isoprenoids, such as cholesterol and ubiquinone, as well as other metabolites. In humans, an age-dependent decrease in ubiquinone levels and changes in cholesterol homeostasis suggest that mevalonate pathway activity changes with age. However, our knowledge of the mechanistic basis of these changes remains rudimentary. We have identified a regulatory circuit controlling the sumoylation state of Caenorhabditis elegans HMG-CoA synthase (HMGS-1). This protein is the ortholog of human HMGCS1 enzyme, which mediates the first committed step of the mevalonate pathway. In vivo, HMGS-1 undergoes an age-dependent sumoylation that is balanced by the activity of ULP-4 small ubiquitin-like modifier protease. ULP-4 exhibits an age-regulated expression pattern and a dynamic cytoplasm-to-mitochondria translocation. Thus, spatiotemporal ULP-4 activity controls the HMGS-1 sumoylation state in a mechanism that orchestrates mevalonate pathway activity with the age of the organism. To expand the HMGS-1 regulatory network, we combined proteomic analyses with knockout studies and found that the HMGS-1 level is also governed by the ubiquitin-proteasome pathway. We propose that these conserved molecular circuits have evolved to govern the level of mevalonate pathway flux during aging, a flux whose dysregulation is associated with numerous age-dependent cardiovascular and cancer pathologies.
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http://dx.doi.org/10.1073/pnas.1414748111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4169931PMC
September 2014

PID-1 is a novel factor that operates during 21U-RNA biogenesis in Caenorhabditis elegans.

Genes Dev 2014 Apr;28(7):683-8

Institute of Molecular Biology, D-55128 Mainz, Germany;

The Piwi-piRNA pathway represents a small RNA-based mechanism responsible for the recognition and silencing of invading DNA. Biogenesis of piRNAs (21U-RNAs) is poorly understood. In Caenorhabditis elegans, the piRNA-binding Argonaute protein PRG-1 is the only known player acting downstream from precursor transcription. From a screen aimed at the isolation of piRNA-induced silencing-defective (Pid) mutations, we identified, among known Piwi pathway components, PID-1 as a novel player. PID-1 is a mostly cytoplasmic, germline-specific factor essential for 21U-RNA biogenesis, affecting an early step in the processing or transport of 21U precursor transcripts. We also show that maternal 21U-RNAs are essential to initiate silencing.
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http://dx.doi.org/10.1101/gad.238220.114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4015495PMC
April 2014

Engineering the Caenorhabditis elegans genome with CRISPR/Cas9.

Methods 2014 Aug 28;68(3):381-8. Epub 2014 Mar 28.

Department of Biology, Utrecht University, 3584 CH Utrecht, The Netherlands. Electronic address:

The development in early 2013 of CRISPR/Cas9-based genome engineering promises to dramatically advance our ability to alter the genomes of model systems at will. A single, easily produced targeting RNA guides the Cas9 endonuclease to a specific DNA sequence where it creates a double strand break. Imprecise repair of the break can yield mutations, while homologous recombination with a repair template can be used to effect specific changes to the genome. The tremendous potential of this system led several groups to independently adapt it for use in Caenorhabditiselegans, where it was successfully used to generate mutations and to create tailored genome changes through homologous recombination. Here, we review the different approaches taken to adapt CRISPR/Cas9 for C. elegans, and provide practical guidelines for CRISPR/Cas9-based genome engineering.
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http://dx.doi.org/10.1016/j.ymeth.2014.03.024DOI Listing
August 2014

The EBAX-type Cullin-RING E3 ligase and Hsp90 guard the protein quality of the SAX-3/Robo receptor in developing neurons.

Neuron 2013 Sep;79(5):903-16

Neurobiology Section, Division of Biological Sciences, University of California, San Diego, La Jolla, CA 92093, USA.

Although protein quality control (PQC) is generally perceived as important for the development of the nervous system, the specific mechanisms of neuronal PQC have remained poorly understood. Here, we report that C. elegans Elongin BC-binding axon regulator (EBAX-1), a conserved BC-box protein, regulates axon guidance through PQC of the SAX-3/Robo receptor. EBAX-1 buffers guidance errors against temperature variations. As a substrate-recognition subunit in the Elongin BC-containing Cullin-RING ubiquitin ligase (CRL), EBAX-1 also binds to DAF-21, a cytosolic Hsp90 chaperone. The EBAX-type CRL and DAF-21 collaboratively regulate SAX-3-mediated axon pathfinding. Biochemical and imaging assays indicate that EBAX-1 specifically recognizes misfolded SAX-3 and promotes its degradation in vitro and in vivo. Importantly, vertebrate EBAX also shows substrate preference toward aberrant Robo3 implicated in horizontal gaze palsy with progressive scoliosis (HGPPS). Together, our findings demonstrate a triage PQC mechanism mediated by the EBAX-type CRL and DAF-21/Hsp90 that maintains the accuracy of neuronal wiring.
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http://dx.doi.org/10.1016/j.neuron.2013.06.035DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3779136PMC
September 2013

CRISPR/Cas9-targeted mutagenesis in Caenorhabditis elegans.

Genetics 2013 Nov 26;195(3):1187-91. Epub 2013 Aug 26.

Department of Biology, Utrecht University, 3584 CH, Utrecht, The Netherlands.

The generation of genetic mutants in Caenorhabditis elegans has long relied on the selection of mutations in large-scale screens. Directed mutagenesis of specific loci in the genome would greatly speed up analysis of gene function. Here, we adapt the CRISPR/Cas9 system to generate mutations at specific sites in the C. elegans genome.
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http://dx.doi.org/10.1534/genetics.113.156299DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3813849PMC
November 2013

Identification of human protein interaction domains using an ORFeome-based yeast two-hybrid fragment library.

J Proteome Res 2013 Jul 17;12(7):3181-92. Epub 2013 Jun 17.

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

Physical interactions between proteins are essential for biological processes. Hence, there have been major efforts to elucidate the complete networks of protein-protein interactions, or "interactomes", of various organisms. Detailed descriptions of protein interaction networks should include information on the discrete domains that mediate these interactions, yet most large-scale efforts model interactions between whole proteins only. We previously developed a yeast two-hybrid-based strategy to systematically map interaction domains and generated a domain-based interactome network for 750 proteins involved in C. elegans early embryonic development. Here, we expand the concept of Y2H-based interaction domain mapping to the genome-wide level. We generated a human fragment library by randomly fragmenting the full-length open reading frames (ORFs) present in the human ORFeome collection. Screens using several proteins required for cell division or polarity establishment as baits demonstrate the ability to accurately identify interaction domains for human proteins using this approach, while the experimental quality of the Y2H data was independently verified in coaffinity purification assays. The library generation strategy can easily be adapted to generate libraries from full-length ORF collections of other organisms.
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http://dx.doi.org/10.1021/pr400047pDOI Listing
July 2013

F-actin asymmetry and the endoplasmic reticulum-associated TCC-1 protein contribute to stereotypic spindle movements in the Caenorhabditis elegans embryo.

Mol Biol Cell 2013 Jul 22;24(14):2201-15. Epub 2013 May 22.

Developmental Biology, Bijvoet Center for Biomolecular Research.

The microtubule spindle apparatus dictates the plane of cell cleavage in animal cells. During development, dividing cells control the position of the spindle to determine the size, location, and fate of daughter cells. Spindle positioning depends on pulling forces that act between the cell periphery and astral microtubules. This involves dynein recruitment to the cell cortex by a heterotrimeric G-protein α subunit in complex with a TPR-GoLoco motif protein (GPR-1/2, Pins, LGN) and coiled-coil protein (LIN-5, Mud, NuMA). In this study, we searched for additional factors that contribute to spindle positioning in the one-cell Caenorhabditis elegans embryo. We show that cortical actin is not needed for Gα-GPR-LIN-5 localization and pulling force generation. Instead, actin accumulation in the anterior actually reduces pulling forces, possibly by increasing cortical rigidity. Examining membrane-associated proteins that copurified with GOA-1 Gα, we found that the transmembrane and coiled-coil domain protein 1 (TCC-1) contributes to proper spindle movements. TCC-1 localizes to the endoplasmic reticulum membrane and interacts with UNC-116 kinesin-1 heavy chain in yeast two-hybrid assays. RNA interference of tcc-1 and unc-116 causes similar defects in meiotic spindle positioning, supporting the concept of TCC-1 acting with kinesin-1 in vivo. These results emphasize the contribution of membrane-associated and cortical proteins other than Gα-GPR-LIN-5 in balancing the pulling forces that position the spindle during asymmetric cell division.
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http://dx.doi.org/10.1091/mbc.E13-02-0076DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3708726PMC
July 2013

Caenorhabditis elegans cyclin D/CDK4 and cyclin E/CDK2 induce distinct cell cycle re-entry programs in differentiated muscle cells.

PLoS Genet 2011 Nov 10;7(11):e1002362. Epub 2011 Nov 10.

Developmental Biology, Utrecht University, Utrecht, The Netherlands.

Cell proliferation and differentiation are regulated in a highly coordinated and inverse manner during development and tissue homeostasis. Terminal differentiation usually coincides with cell cycle exit and is thought to engage stable transcriptional repression of cell cycle genes. Here, we examine the robustness of the post-mitotic state, using Caenorhabditis elegans muscle cells as a model. We found that expression of a G1 Cyclin and CDK initiates cell cycle re-entry in muscle cells without interfering with the differentiated state. Cyclin D/CDK4 (CYD-1/CDK-4) expression was sufficient to induce DNA synthesis in muscle cells, in contrast to Cyclin E/CDK2 (CYE-1/CDK-2), which triggered mitotic events. Tissue-specific gene-expression profiling and single molecule FISH experiments revealed that Cyclin D and E kinases activate an extensive and overlapping set of cell cycle genes in muscle, yet failed to induce some key activators of G1/S progression. Surprisingly, CYD-1/CDK-4 also induced an additional set of genes primarily associated with growth and metabolism, which were not activated by CYE-1/CDK-2. Moreover, CYD-1/CDK-4 expression also down-regulated a large number of genes enriched for catabolic functions. These results highlight distinct functions for the two G1 Cyclin/CDK complexes and reveal a previously unknown activity of Cyclin D/CDK-4 in regulating metabolic gene expression. Furthermore, our data demonstrate that many cell cycle genes can still be transcriptionally induced in post-mitotic muscle cells, while maintenance of the post-mitotic state might depend on stable repression of a limited number of critical cell cycle regulators.
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http://dx.doi.org/10.1371/journal.pgen.1002362DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3213155PMC
November 2011

aPKC phosphorylates NuMA-related LIN-5 to position the mitotic spindle during asymmetric division.

Nat Cell Biol 2011 Aug 21;13(9):1132-8. Epub 2011 Aug 21.

Developmental Biology, Utrecht University, 3584 CH Utrecht, The Netherlands.

The position of the mitotic spindle controls the plane of cell cleavage and determines whether polarized cells divide symmetrically or asymmetrically. In animals, an evolutionarily conserved pathway of LIN-5 (homologues: Mud and NuMA), GPR-1/2 (homologues: Pins, LGN, AGS-3) and Gα mediates spindle positioning, and acts downstream of the conserved PAR-3-PAR-6-aPKC polarity complex. However, molecular interactions between polarity proteins and LIN-5-GPR-Gα remain to be identified. Here we describe a quantitative mass spectrometry approach for in vivo identification of protein kinase substrates. Applying this strategy to Caenorhabditis elegans embryos, we found that depletion of the polarity kinase PKC-3 results in markedly decreased levels of phosphorylation of a cluster of four LIN-5 serine residues. These residues are directly phosphorylated by PKC-3 in vitro. Phospho-LIN-5 co-localizes with PKC-3 at the anterior cell cortex and temporally coincides with a switch from anterior- to posterior-directed spindle movements in the one-cell embryo. LIN-5 mutations that prevent phosphorylation increase the extent of anterior-directed spindle movements, whereas phosphomimetic mutations decrease spindle migration. Our results indicate that anterior-located PKC-3 inhibits cortical microtubule pulling forces through direct phosphorylation of LIN-5. This molecular interaction between polarity and spindle-positioning proteins may be used broadly in cell cleavage plane determination.
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http://dx.doi.org/10.1038/ncb2315DOI Listing
August 2011

Independently evolved virulence effectors converge onto hubs in a plant immune system network.

Science 2011 Jul;333(6042):596-601

Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.

Plants generate effective responses to infection by recognizing both conserved and variable pathogen-encoded molecules. Pathogens deploy virulence effector proteins into host cells, where they interact physically with host proteins to modulate defense. We generated an interaction network of plant-pathogen effectors from two pathogens spanning the eukaryote-eubacteria divergence, three classes of Arabidopsis immune system proteins, and ~8000 other Arabidopsis proteins. We noted convergence of effectors onto highly interconnected host proteins and indirect, rather than direct, connections between effectors and plant immune receptors. We demonstrated plant immune system functions for 15 of 17 tested host proteins that interact with effectors from both pathogens. Thus, pathogens from different kingdoms deploy independently evolved virulence proteins that interact with a limited set of highly connected cellular hubs to facilitate their diverse life-cycle strategies.
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http://dx.doi.org/10.1126/science.1203659DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3170753PMC
July 2011
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