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Lgr4 and Lgr5 Drive the Formation of Long Actin-Rich Cytoneme-Like

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Lgr4 and Lgr5 Drive the Formation of Long Actin-Rich Cytoneme-Like ß 2015. Published by The Company of Biologists Ltd | Journal of Cell Science (2015) 128, 1230–1240 doi:10.1242/jcs.166322 RESEARCH ARTICLE Lgr4 and Lgr5 drive the formation of long actin-rich cytoneme-like membrane protrusions Joshua C. Snyder1, Lauren K. Rochelle1,Se´bastien Marion1, H. Kim Lyerly2, Larry S. Barak1 and Marc G. Caron1,* ABSTRACT with Lgr5-expressing hair follicle stem cells (Snippert et al., 2010). The clinical relevance of these discoveries has been Embryonic development and adult tissue homeostasis require underscored by the fact that Lgr5-expressing cells possess greater precise information exchange between cells and their tumorigenic potential than their differentiated progeny (Barker microenvironment to coordinate cell behavior. A specialized class et al., 2010; Barker et al., 2009), and the demonstration that Lgr5- of ultra-long actin-rich filopodia, termed cytonemes, provides one expressing cells in intestinal adenomas are cancer stem cells mechanism for this spatiotemporal regulation of extracellular cues. (Schepers et al., 2012). We provide here a mechanism whereby the stem-cell marker Lgr5, The complement of membrane receptors on stem cells and its family member Lgr4, promote the formation of cytonemes. might confer them with intrinsic regulatory capacity by tightly Lgr4- and Lgr5-induced cytonemes exceed lengths of 80 mm, are controlling their response to extracellular cues. Lgr4–6 signal by generated through stabilization of nascent filopodia from an a non-canonical G-protein-independent mechanism by binding underlying lamellipodial-like network and functionally provide a to R-spondins (Carmon et al., 2012; de Lau et al., 2011) or pipeline for the transit of signaling effectors. As proof-of-principle, Norrin (Deng et al., 2013) and potentiating Wnt–bcatenin signal we demonstrate that Lgr5-induced cytonemes act as conduits for transduction. However, it is likely that other signaling pathways cell signaling by demonstrating that the actin motor and filopodial and biological processes are engaged by Lgr4–6. In fact, GPCR cargo carrier protein myosin X (Myo10) and the G-protein-coupled signaling is multifaceted in nature and is tightly regulated by receptor (GPCR) signaling effector b-arrestin-2 (Arrb2) transit into endocytosis of activated receptors (Rajagopal et al., 2010). We cytonemes. This work delineates a biological function for Lgr4 and have reported on the cellular trafficking properties of Lgr5 using Lgr5 and provides the rationale to fully investigate Lgr4 and Lgr5 EGFP-tagged forms of the receptor and found that the C-terminal function and cytonemes in mammalian stem cell and cancer stem tail regulates its unique constitutive internalization and retrograde cell behavior. trafficking to the trans-Golgi network in the apparent absence of ligand (Snyder et al., 2013b). Therefore, we hypothesized that KEY WORDS: Lgr5, GPCR, Cytoneme, Stem cell inhibiting this constitutive internalization would provide new insight into the cellular function of Lgr5. Surprisingly, we INTRODUCTION discovered that the plasma membrane expression of Lgr5 and The G-protein-coupled receptor (GPCR) Lgr5 genetically Lgr4 provide a mechanism for driving the formation of ultra-long identifies a population of intestinal crypt stem cells (Barker actin-rich membrane protrusions that reach lengths approaching et al., 2007). Since this observation, the ability to distinguish stem 80 mm. These filopodia are phenotypically and molecularly cells in other tissues on the basis of Lgr5 expression has proved to similar to a specialized class of filopodia previously described be a successful strategy in the stomach (Barker et al., 2010), skin by Kornberg in the Drosophila wing imaginal disc as ‘cell (Jaks et al., 2008), liver (Huch et al., 2013b), pancreas (Huch threads’ or cytonemes (Ramı´rez-Weber and Kornberg, 1999). et al., 2013a), kidney (Barker et al., 2012), mammary gland (de Filopodia are typically of modest length (often ,10 mm) Visser et al., 2012; Plaks et al., 2013) and developing owing to the biophysical forces required to deform the plasma hematopoietic system (Liu et al., 2014). Lgr5 is the founding membrane (Mogilner and Rubinstein, 2005). By contrast, ultra- member of a subclass of the leucine-rich G-protein-coupled long actin-rich filopodia were first observed in sea urchin receptors (Lgrs) that include Lgr4 and Lgr6 (Hsu et al., 2000; Hsu embryogenesis by Wolpert in 1961 (Gustafson, 1963) and et al., 1998). In addition to Lgr5, Lgr4 and Lgr6 are also used as further investigated by McClay in 1995 (Miller et al., 1995). markers of the stem and progenitor cell lineages within tissues. Since their initial description, these structures have been named Lgr4 expression identifies an expanded pool of progenitor cells in cytonemes to distinguish them from shorter filopodia. Cytonemes the intestinal crypt, which also includes those cells expressing have many distinctive characteristics including their fragility, Lgr5 (de Lau et al., 2011). Lgr6 expression identifies a more width and length; all features shared by Lgr4 and Lgr5- primordial stem cell with restricted cellular expression compared induced protrusions. Cytonemes can compartmentalize signaling pathways (Roy et al., 2011) and direct the transfer of morphogens 1Department of Cell Biology, Duke University Medical Center, Durham, NC 27710, between cells (Roy et al., 2014). In Drosophila, cytonemes USA. 2Department of Surgery, Duke University Medical Center, Durham, NC orient towards a morphogen-producing cell or emanate from a 27710, USA. morphogen-producing cell towards a target cell. Recent studies *Author for correspondence ([email protected]) in Drosophila indicate that cytonemes provide a platform for transmitting morphogens in the stem cell niche of the Drosophila Received 18 November 2014; Accepted 27 January 2015 germarium (Rojas-Rı´os et al., 2012). Therefore, cytonemes can Journal of Cell Science 1230 RESEARCH ARTICLE Journal of Cell Science (2015) 128, 1230–1240 doi:10.1242/jcs.166322 be employed to exquisitely regulate the scope and precision of RESULTS signaling during tissue development and maintenance. Lgr5 and Lgr4 expression in mammalian cells induces the Despite these elegant studies, only a few reports have robust formation of membrane protrusions investigated similar structures in mammalian cells and, until Normally, Lgr5 is constitutively internalized, resulting in recently, even effectors responsible for their formation in intracellular localization of EGFP-tagged receptor (Fig. 1A–C) Drosophila were missing entirely (Roy et al., 2014). Therefore, (Snyder et al., 2013b). However, when the internalization of Lgr5 questions regarding the mechanisms that drive cytoneme was blocked either by overexpression of dominant-negative formation and their utility in signaling remain largely dynamin-1 (K44A), by C-terminal tail truncation at position unexplored in mammalian systems (Affolter and Basler, 2011; 834 (834del) or by exchanging the C-terminal tail for that of Kornberg and Roy, 2014). Our study answers these questions. We the human vasopressin V2 receptor (V2R; Lgr5–V2Rtail) demonstrate that Lgr5 and Lgr4 provide a receptor-based (Fig. 1A,B,D–F), we discovered the robust formation of mechanism for triggering the formation of cytonemes and membrane protrusions at the interface between the cell surface further illustrate that these cytonemes can be scaffolds for and the substrate by confocal microscopy (Fig. 1H–J). Unlike signaling effectors in a mammalian cell system. These findings wild-type Lgr5, wild-type Lgr4 was expressed more robustly at suggest that stem cells might possess the hardware for regulating the plasma membrane and cells transfected with wild-type Lgr4 signaling at a distance. displayed extensive protrusions (Fig. 1A,B,G,K). Lgr4 and Lgr5 Fig. 1. Cell-surface expression of Lgr5 or Lgr4 induces the robust formation of membrane protrusions. (A) An on-cell enzyme-linked immunosorbent assay (ELISA) was performed to measure the membrane (live cell stained) and total (fixed, permeabilized and stained) expression of each receptor in HEK cells. The images shown are representative of more than three experiments. (B) Relative membrane expression was determined by calculating the ratio of membraneto total expression values for each receptor and then normalizing to Lgr5FL across multiple experiments. The data show the mean6s.e.m.; *P,0.05 (one-way ANOVA with Bonferroni post-hoc testing). Confocal optical sections of live cells transiently expressing (C) Lgr5FL, (D) Lgr5FL + dynamin-1 K44A (K44A), (E) 834del, (F) Lgr5–V2Rtail (Lgr5/V2Rtail) or (G) Lgr4 to determine their expression at the plasma membrane. HEK cells were transiently transfected with EGFP-tagged (H) Lgr5 +K44A, (I) 834del, (J) Lgr5–V2Rtail or (K) Lgr4, imaged live on a confocal microscope and rendered in three dimensions. Scale bars: 10 mm. FL Journal of Cell Science 1231 RESEARCH ARTICLE Journal of Cell Science (2015) 128, 1230–1240 doi:10.1242/jcs.166322 membrane protrusions are very fragile, and can be severed by Morphological and biochemical characterization of Lgr4- and over-fixation or even by mild physical perturbations. These Lgr5-induced membrane protrusions structures were often found to be directed towards the basolateral The EGFP tag on the receptor enabled a remarkable visualization surface and branched upon their contact with the substratum of the membrane protrusions. These structures go unnoticed if the (supplementary material Movie 1). These data demonstrate
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