Chanjae Lee

Shroom family proteins regulate γ-tubulin distribution and microtubule architecture during epithelial cell shape change

Cell shape changes require the coordination of actin and microtubule cytoskeletons. The molecular mechanisms by which such coordination is achieved remain obscure, particularly in the context of epithelial cells within developing vertebrate embryos. We have identified a novel role for the actin-binding protein Shroom3 as a regulator of the microtubule cytoskeleton during epithelial morphogenesis. We show that Shroom3 is sufficient and also necessary to induce a redistribution of the microtubule regulatorγ -tubulin. Moreover, this change in γ-tubulin distribution underlies the assembly of aligned arrays of microtubules that drive apicobasal cell elongation. Finally, experiments with the related protein, Shroom1, demonstrate that γ-tubulin regulation is a conserved feature of this protein family. Together, the data demonstrate that Shroom family proteins govern epithelial cell behaviors by coordinating the assembly of both microtubule and actin cytoskeletons.

The shroom family proteins play broad roles in the morphogenesis of thickened epithelial sheets.

Thickened epithelial sheets are found in a wide variety of organ systems and the mechanisms governing their morphogenesis remain poorly defined. We show here, through expression patterns and functional studies, that Shroom family proteins are broadly involved in generating thickened epithelial sheets. Through in situ hybridization, we report the temporal and spatial expression patterns of the four Shroom family members during early Xenopus development, from oocytes to tadpole stage embryos. Further, we show that Shroom1 and 2 mRNAs are maternally expressed, while Shroom3 and Shroom4 are zygotic transcripts. In addition, maternal Shroom1 and 2 mRNAs localize in the animal hemisphere of the Xenopus egg and early blastula. During later development, all four Shroom family proteins are broadly expressed in developing epithelial organs, and the epithelial cells that express Shrooms are elongated. Moreover, we show that ectopic expression of Shroom2, like Shroom3, is able to increase cell height and that loss of Shroom2 function results in a failure of cell elongation in the neural epithelium. Together, these data suggest that Shroom family proteins play an important role in the morphogenesis of several different epithelial tissues during development. Developmental Dynamics 238:1480–1491, 2009.

Shroom2 (APXL) regulates melanosome biogenesis and localization in the retinal pigment epithelium

Shroom family proteins have been implicated in the control of the actin cytoskeleton, but so far only a single family member has been studied in the context of developing embryos. Here, we show that the Shroom-family protein, Shroom2 (previously known as APXL) is both necessary and sufficient to govern the localization of pigment granules at the apical surface of epithelial cells. In Xenopus embryos that lack Shroom2 function, we observed defects in pigmentation of the eye that stem from failure of melanosomes to mature and to associate with the apical cell surface. Ectopic expression of Shroom2 in naïve epithelial cells facilitates apical pigment accumulation, and this activity specifically requires the Rab27a GTPase. Most interestingly, we find that Shroom2, like Shroom3 (previously called Shroom), is sufficient to induce a dramatic apical accumulation of the microtubule-nucleating protein γ-tubulin at the apical surfaces of naïve epithelial cells. Together, our data identify Shroom2 as a central regulator of RPE pigmentation, and suggest that, despite their diverse biological roles, Shroom family proteins share a common activity. Finally, because the locus encoding human SHROOM2 lies within the critical region for two distinct forms of ocular albinism, it is possible that SHROOM2 mutations may be a contributing factor in these human visual system disorders.

The ciliopathy-associated CPLANE proteins direct basal body recruitment of intraflagellar transport machinery

Cilia use microtubule-based intraflagellar transport (IFT) to organize intercellular signaling. Ciliopathies are a spectrum of human diseases resulting from defects in cilia structure or function. The mechanisms regulating the assembly of ciliary multiprotein complexes and the transport of these complexes to the base of cilia remain largely unknown. Combining proteomics, in vivo imaging and genetic analysis of proteins linked to planar cell polarity (Inturned, Fuzzy and Wdpcp), we identified and characterized a new genetic module, which we term CPLANE (ciliogenesis and planar polarity effector), and an extensive associated protein network. CPLANE proteins physically and functionally interact with the poorly understood ciliopathy-associated protein Jbts17 at basal bodies, where they act to recruit a specific subset of IFT-A proteins. In the absence of CPLANE, defective IFT-A particles enter the axoneme and IFT-B trafficking is severely perturbed. Accordingly, mutation of CPLANE genes elicits specific ciliopathy phenotypes in mouse models and is associated with ciliopathies in human patients.

Integration of over 9,000 mass spectrometry experiments builds a global map of human protein complexes

Macromolecular protein complexes carry out many of the essential functions of cells, and many genetic diseases arise from disrupting the functions of such complexes. Currently, there is great interest in defining the complete set of human protein complexes, but recent published maps lack comprehensive coverage. Here, through the synthesis of over 9,000 published mass spectrometry experiments, we present hu.MAP, the most comprehensive and accurate human protein complex map to date, containing > 4,600 total complexes, > 7,700 proteins, and > 56,000 unique interactions, including thousands of confident protein interactions not identified by the original publications. hu.MAP accurately recapitulates known complexes withheld from the learning procedure, which was optimized with the aid of a new quantitative metric (k‐cliques) for comparing sets of sets. The vast majority of complexes in our map are significantly enriched with literature annotations, and the map overall shows improved coverage of many disease‐associated proteins, as we describe in detail for ciliopathies. Using hu.MAP, we predicted and experimentally validated candidate ciliopathy disease genes in vivo in a model vertebrate, discovering CCDC138, WDR90, and KIAA1328 to be new cilia basal body/centriolar satellite proteins, and identifying ANKRD55 as a novel member of the intraflagellar transport machinery. By offering significant improvements to the accuracy and coverage of human protein complexes, hu.MAP (http://proteincomplexes.org) serves as a valuable resource for better understanding the core cellular functions of human proteins and helping to determine mechanistic foundations of human disease.

High-Magnification In Vivo Imaging of Xenopus Embryos for Cell and Developmental Biology

Embryos of the frog Xenopus laevis are an ideal model system for in vivo imaging of dynamic biological processes, from the inner workings of individual cells to the reshaping of tissues during embryogenesis. Their externally developing embryos are more amenable to in vivo analysis than internally developing mammalian embryos, and the large size of the embryos make them particularly suitable for time-lapse analysis of tissue-level morphogenetic events. In addition, individual cells in Xenopus embryos are larger than those in other vertebrate models, making them ideal for imaging cell behavior and subcellular processes (e.g., following the dynamics of fluorescent fusion proteins in living or fixed cells and tissues). Xenopus embryos are amenable to simple manipulations of gene function, including knockdown and misexpression, and the large number of embryos available allows even an inexperienced researcher to perform hundreds of such manipulations per day. Transgenesis is quite effective as well. Finally, because the fate map of Xenopus embryos is stereotypical, simple targeted microinjections can reliably deliver reagents into specific tissues and cell types for gene manipulation or for imaging. Although yolk opacity can hinder deep imaging in intact embryos, almost any cell in the early embryo can be placed into organotypic culture, such that the cells of interest are directly apposed to the cover glass. Furthermore, live imaging techniques can be complemented with immunostaining and in situ hybridization approaches in fixed tissues. This protocol describes methods for labeling and high-magnification time-lapse imaging of cell biological and developmental processes in Xenopus embryos by confocal microscopy.

Embryogenesis and laboratory maintenance of the foam-nesting túngara frogs, genus Engystomops (= Physalaemus).

The vast majority of embryological research on amphibians focuses on just a single genus of frogs, Xenopus. To attain a more comprehensive understanding of amphibian development, experimentation on non‐model frogs will be essential. Here, we report on the early development, rearing, and embryological analysis of túngara frogs (genus Engystomops, also called Physalaemus). The frogs Engystomops pustulosus, Engystomops coloradorum, and Engystomops randi construct floating foam‐nests with small eggs. We define a table of 23 stages for the developmental period in the foam‐nest. Embryos were immunostained against Lim1, neural, and somite‐specific proteins and the expression pattern of RetinoBlastoma Binding Protein 6 (RBBP6) was analyzed by in situ hybridization. Due to their brief life‐cycle, frogs belonging to the genus Engystomops are attractive for comparative and genetic studies of development. Developmental Dynamics 238:1444–1454, 2009.

TTC25 deficiency results in defects of the outer dynein arm docking machinery and primary ciliary dyskinesia with left-right body asymmetry randomization

Multiprotein complexes referred to as outer dynein arms (ODAs) develop the main mechanical force to generate the ciliary and flagellar beat. ODA defects are the most common cause of primary ciliary dyskinesia (PCD), a congenital disorder of ciliary beating, characterized by recurrent infections of the upper and lower airways, as well as by progressive lung failure and randomization of left-right body asymmetry. Using a whole-exome sequencing approach, we identified recessive loss-of-function mutations within TTC25 in three individuals from two unrelated families affected by PCD. Mice generated by CRISPR/Cas9 technology and carrying a deletion of exons 2 and 3 in Ttc25 presented with laterality defects. Consistently, we observed immotile nodal cilia and missing leftward flow via particle image velocimetry. Furthermore, transmission electron microscopy (TEM) analysis in TTC25-deficient mice revealed an absence of ODAs. Consistent with our findings in mice, we were able to show loss of the ciliary ODAs in humans via TEM and immunofluorescence (IF) analyses. Additionally, IF analyses revealed an absence of the ODA docking complex (ODA-DC), along with its known components CCDC114, CCDC151, and ARMC4. Co-immunoprecipitation revealed interaction between the ODA-DC component CCDC114 and TTC25. Thus, here we report TTC25 as a new member of the ODA-DC machinery in humans and mice.

Whole-Mount Fluorescence Immunocytochemistry on Xenopus Embryos

INTRODUCTIONImmunocytochemistry (ICC) is widely exploited in studying mammalian systems, but is underutilized among Xenopus developmental biologists. This stems, in part, from the relatively small number of Xenopus antibodies available for use in research. Common misconceptions about ICC in Xenopus embryos also prevail, discouraging researchers from trying the procedure. However, ICC with Xenopus is simple and effective. This article describes methods for whole-mount ICC in Xenopus embryos. Also included are simple procedures to quench autofluorescence of Xenopus and to remove surface pigment from embryos which may interfere with fluorescence imaging. The methods described here are useful for detecting tissue-specific probes (e.g., 12/101 to detect somites). They are also effective for imaging the cytoskeleton (e.g., α-tubulin to detect microtubules) or localizing specific proteins at the subcellular level (e.g., ZO-1 to detect tight junctions). In addition, combining ICC with in situ hybridization is simple and highly effective.

An ENU mutagenesis screen in zebrafish for visual system mutants identifies a novel splice-acceptor site mutation in patched2 that results in Colobomas.

PURPOSE To identify recessive mutations affecting development and/or maintenance of the zebrafish visual system. METHODS A three-generation ENU (N-Nitroso-N-ethylurea)-based forward genetic screen was performed. F3 embryos were screened visually from 1 to 5 days postfertilization (dpf) for ocular abnormalities, and 5 dpf embryos were fixed and processed for cryosectioning, after which eye sections were screened for defects in cellular organization within the retina, lens, and cornea. A combination of PCR and DNA sequencing, in situ hybridization, and pharmacological treatments were used to clone and characterize a coloboma mutant. RESULTS A total of 126 F2 families were screened, and, from these, 18 recessive mutations were identified that affected eye development. Phenotypes included lens malformations and cataracts, photoreceptor defects, oculocutaneous albinism, microphthalmia, and colobomas. Analysis of one such coloboma mutant, uta(1), identified a splice-acceptor mutation in the patched2 gene that resulted in an in-frame deletion of 19 amino acids that are predicted to contribute to the first extracellular loop of Patched2. ptch2(uta1) mutants possessed elevated Hedgehog (Hh) pathway activity, and blocking the Hh pathway with cyclopamine prevented colobomas in ptch2(uta1) mutant embryos. CONCLUSIONS We have identified 18 recessive mutations affecting development of the zebrafish visual system and we have characterized a novel splice-acceptor site mutation in patched2 that results in enhanced Hh pathway activity and colobomas.