The c-Jun N-terminal kinase (JNK) regulates various important physiological processes. Although the JNK pathway has been under intense investigation for over 20 yr, its complexity is still perplexing, with multiple protein partners underlying the diversity of its activity. We show that JNK is associated with the basal bodies in both primary and motile cilia.
View Article and Find Full Text PDFFront Cell Dev Biol
January 2023
Neural tube closure is a fundamental process during vertebrate embryogenesis, which leads to the formation of the central nervous system. Defective neural tube closure leads to neural tube defects which are some of the most common human birth defects. While the intrinsic morphogenetic events shaping the neuroepithelium have been studied extensively, how tissues mechanically coupled with the neural plate influence neural tube closure remains poorly understood.
View Article and Find Full Text PDFThe Ciliary Adhesion (CA) complex forms in close association with the basal bodies of cilia during the early stages of ciliogenesis and is responsible for mediating complex interactions with the actin networks of multiciliated cells (MCCs). However, its precise localization with respect to basal body accessory structures and the interactions that lead to its establishment in MCCs are not well understood. Here, we studied the distribution of the CA proteins using super-resolution imaging and possible interactions with the microtubule network.
View Article and Find Full Text PDFCadherins and integrins are intrinsically linked through the actin cytoskeleton and are largely responsible for the mechanical integrity and organization of tissues. We show that cadherin clustering stimulates and spatially guides integrin activation. Adherens junction (AJ)-associated integrin activation depends on locally generated tension and does not require extracellular matrix ligands.
View Article and Find Full Text PDFNeural tube closure (NTC) is a fundamental process during vertebrate development and is indispensable for the formation of the central nervous system. Here, using Xenopus laevis embryos, live imaging, single-cell tracking, optogenetics and loss-of-function experiments, we examine the roles of convergent extension and apical constriction, and define the role of the surface ectoderm during NTC. We show that NTC is a two-stage process with distinct spatiotemporal contributions of convergent extension and apical constriction at each stage.
View Article and Find Full Text PDFBackground: Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that is overexpressed or activated in several advanced-stage solid cancers. It is known to play both kinase-dependent and -independent roles in promoting tumor progression and metastasis. Numerous inhibitors, targeting either the enzymatic or scaffolding activities of FAK have been generated, with varying degree of success.
View Article and Find Full Text PDFCorrect selection of the cell division axis is important for cell differentiation, tissue and organ morphogenesis, and homeostasis. Both integrins, which mediate interactions with extracellular matrix (ECM) components such as fibronectin, and cadherins, which mediate interactions between cells, are implicated in the determination of spindle orientation. We found that both cadherin- and integrin-based adhesion resulted in cell divisions parallel to the attachment plane and elicited identical spindle responses to spatial adhesive cues.
View Article and Find Full Text PDFCalpains are a family of calcium-dependent intracellular cysteine proteases that regulate important physiological processes by substrate cleavage. Despite the fact that the role of calpains in cell migration and other processes has been extensively studied in vitro, the same does not apply to cell migration and morphogenetic events during embryogenesis, in vivo. Herein, we describe the use of three different methods to selectively block calpain activity in vivo in order to investigate the impact on Xenopus gastrulation and neurulation, namely, a calpain inhibitor, a dominant negative, and a morpholino antisense oligonucleotide (MO).
View Article and Find Full Text PDFCalpains are a family of calcium-dependent intracellular cysteine proteases that regulate important physiological processes by substrate cleavage. Despite the fact that Calpains have been identified in the Xenopus genome, their expression patterns and role have not been characterized. Therefore, herein, we describe two methods to determine temporal and spatial expression of Calpain 2 during Xenopus development, namely, RT-PCR and whole-mount in situ hybridization (WISH).
View Article and Find Full Text PDFWe previously identified focal adhesion kinase (FAK) as an important regulator of ciliogenesis in multiciliated cells. FAK and other focal adhesion (FA) proteins associate with the basal bodies and their striated rootlets and form complexes named ciliary adhesions (CAs). CAs display similarities with FAs but are established in an integrin independent fashion and are responsible for anchoring basal bodies to the actin cytoskeleton during ciliogenesis as well as in mature multiciliated cells.
View Article and Find Full Text PDFControl of spindle orientation is a fundamental process for embryonic development, morphogenesis and tissue homeostasis, while defects are associated with tumorigenesis and other diseases. Force sensing is one of the mechanisms through which division orientation is determined. Here we show that integrin β1 plays a critical role in this process, becoming activated at the lateral regions of the cell cortex in a ligand-independent manner.
View Article and Find Full Text PDFNeurulation is a critical period in all vertebrates and results in the formation of the neural tube, which gives rise to the CNS. Apical constriction is one of the fundamental morphogenetic movements that drives neural tube closure. Using live imaging, we show that apical constriction during the neurulation is a stepwise process driven by cell-autonomous and asynchronous contraction pulses followed by stabilization steps.
View Article and Find Full Text PDFSpindle orientation is critical for proper morphogenesis of organs and tissues as well as for the maintenance of tissue morphology. Although significant progress has been made in understanding the mechanisms linking the cell cortex to the spindle and the well-documented role that extracellular forces play in spindle orientation, how such forces are transduced to the cortex remains poorly understood. Here we report that focal adhesion kinase (FAK) is necessary for correct spindle orientation and as a result, indispensable for proper epithelial morphogenesis in the vertebrate embryo.
View Article and Find Full Text PDFHeterogeneous nuclear ribonucleoproteins (hnRNPs) comprise a large group of modular RNA-binding proteins classified according to their conserved domains. This modular nature, coupled with a large choice of alternative splice variants generates functional diversity. Here, we investigate the biological differences between 40LoVe, its splice variant Samba and its pseudoallele hnRNP AB in neural development.
View Article and Find Full Text PDFCilia have been associated with diverse developmental and physiological processes, and defects in cilia underlie a number of genetic conditions. Several lines of evidence support a critical role of the actin cytoskeleton in ciliogenesis and ciliary function. Here, we show that well-characterized focal adhesion (FA) proteins, including FAK, Paxillin, and Vinculin, associate with the basal bodies of multiciliated cells and form complexes (CAs) that interact with the actin cytoskeleton.
View Article and Find Full Text PDFCalpains are a family of calcium-dependent intracellular cysteine proteases that regulate several physiological processes by limited cleavage of different substrates. The role of Calpain2 in embryogenesis is not clear with conflicting evidence from a number of mouse knockouts. Here we report the temporal and spatial expression of Calpain2 in Xenopus laevis embryos and address its role in Xenopus development.
View Article and Find Full Text PDFFAK is a non-receptor tyrosine kinase involved in a wide variety of biological processes and crucial for embryonic development. In this manuscript, we report the generation of a new FAK dominant negative (FF), composed of the C terminus (FRNK) and the FERM domain of the protein. FF, unlike FRNK and FERM, mimics the localization of active FAK in the embryo, demonstrating that both domains are necessary to target FAK to its complexes in vivo.
View Article and Find Full Text PDFNucleotide binding protein 1 (Nubp1) is a highly conserved phosphate loop (P-loop) ATPase involved in diverse processes including iron-sulfur protein assembly, centrosome duplication and lung development. Here, we report the cloning, expression and functional characterization of Xenopus laevis Nubp1. We show that xNubp1 is expressed maternally, displays elevated expression in neural tissues and is required for convergent extension movements and neural tube closure.
View Article and Find Full Text PDFBackground: The Focal Adhesion Kinase is a well studied tyrosine kinase involved in a wide number of cellular processes including cell adhesion and migration. It has also been shown to play important roles during embryonic development and targeted disruption of the FAK gene in mice results in embryonic lethality by day 8.5.
View Article and Find Full Text PDFThe ability to target proteins with nanostructures and/or nanodevices in vivo is important for understanding and controlling their biological function. Quantum dots (QDs) serve as an ideal model nanostructure due to their superior optical properties that permit visual confirmation of in vivo targeting and localization and due to their potential as a bio-imaging tool. Here, we describe the site-specific covalent conjugation of quantum dots to target proteins in vivo using an intein-based method.
View Article and Find Full Text PDFThe photostability and narrow emission spectra of nanometer-scale semiconductor crystallites (QDs) make them desirable candidates for whole-mount fluorescent in situ hybridization to detect mRNA transcripts in morphologically preserved intact embryos. We describe a method for direct QD labeling of modified oligonucleotide probes through streptavidin-biotin and antibody-mediated interactions (anti-FITC and anti-digoxigenin). To overcome permeability issues and allow QD conjugate penetration, embryos were treated with proteinase K.
View Article and Find Full Text PDFBackground: Proteins labelled with Quantum Dots (QDs) can be imaged over long periods of time with ultrahigh spatial and temporal resolution, yielding important information on the spatiotemporal dynamics of proteins within live cells or in vivo. However one of the major problems regarding the use of QDs for biological imaging is the difficulty of targeting QDs onto proteins. We have recently developed a DnaE split intein-based method to conjugate Quantum Dots (QDs) to the C-terminus of target proteins in vivo.
View Article and Find Full Text PDFJ Nanobiotechnology
December 2009
We describe an intein based method to site-specifically conjugate Quantum Dots (QDs) to target proteins in vivo. This approach allows the covalent conjugation of any nanostructure and/or nanodevice to any protein and thus the targeting of such material to any intracellular compartment or signalling complex within the cells of the developing embryo. We genetically fused a pleckstrin-homology (PH) domain with the N-terminus half of a split intein (IN).
View Article and Find Full Text PDFMesoderm migration is a well studied morphogenetic movement that takes place during Xenopus gastrulation. The study of mesoderm migration and other morphogenetic movements has been primarily based on in vitro assays due to the inability to image deep tissue movements in the opaque embryo. We are the first to report the use of Near Infra Red Quantum Dots (NIR QD's) to image mesoderm migration in vivo with single cell resolution and provide quantitative in vivo data regarding migration rates.
View Article and Find Full Text PDF