Tankyrase regulates epithelial lumen formation via suppression of Rab11 GEFs

Rab11 GTPase proteins are required for cytokinesis, ciliogenesis, and lumenogenesis. Rab11a is critical for apical delivery of podocalyxin (PODXL) during lumen formation in epithelial cells. SH3BP5 and SH3BP5L are guanine nucleotide exchange factors (GEFs) for Rab11. We show that SH3BP5 and SH3BP5L are required for activation of Rab11a and cyst lumen formation. Using proximity-dependent biotin identification (BioID) interaction proteomics, we have identified SH3BP5 and its paralogue SH3BP5L as new substrates of the poly-ADP-ribose polymerase Tankyrase and the E3 ligase RNF146. We provide data demonstrating that epithelial polarity via cyst lumen formation is governed by Tankyrase, which inhibits Rab11a activation through the suppression of SH3BP5 and SH3BP5L. RNF146 reduces Tankyrase protein abundance and restores Rab11a activation and lumen formation. Thus, Rab11a activation is controlled by a signaling pathway composed of the sequential inhibition of SH3BP5 paralogues by Tankyrase, which is itself suppressed by RNF146.     

FIP5 phosphorylation during mitosis regulates apical trafficking and lumenogenesis

Apical lumen formation is a key step during epithelial morphogenesis. The establishment of the apical lumen is a complex process that involves coordinated changes in plasma membrane composition, endocytic transport, and cytoskeleton organization. These changes are accomplished, at least in part, by the targeting and fusion of Rab11/FIP5‐containing apical endosomes with the apical membrane initiation site (AMIS). Although AMIS formation and polarized transport of Rab11/FIP5‐containing endosomes are crucial for the formation of a single apical lumen, the spatiotemporal regulation of this process remains poorly understood. Here, we demonstrate that the formation of the midbody during cytokinesis is a symmetry‐breaking event that establishes the location of the AMIS. The interaction of FIP5 with SNX18, which is required for the formation of apical endocytic carriers, is inhibited by GSK‐3 phosphorylation at FIP5‐T276. Importantly, we show that FIP5‐T276 phosphorylation occurs specifically during metaphase and anaphase, to ensure the fidelity and timing of FIP5‐endosome targeting to the AMIS during apical lumen formation.     

Actin remodeling by Nck regulates endothelial lumen formation

Multiple angiogenic cues modulate phosphotyrosine signaling to promote vasculogenesis and angiogenesis. Despite its functional and clinical importance, how vascular cells integrate phosphotyrosine-dependent signaling to elicit cytoskeletal changes required for endothelial morphogenesis remains poorly understood. The family of Nck adaptors couples phosphotyrosine signals with actin dynamics and therefore is well positioned to orchestrate cellular processes required in vascular formation and remodeling. Culture of endothelial cells in three-dimensional collagen matrices in the presence of VEGF stimulation was combined with molecular genetics, optical imaging, and biochemistry to show that Nck-dependent actin remodeling promotes endothelial cell elongation and proper organization of VE-cadherin intercellular junctions. Major morphogenetic defects caused by abrogation of Nck signaling included loss of endothelial apical-basal polarity and impaired lumenization. Time-lapse imaging using a Förster resonance energy transfer biosensor, immunostaining with phospho-specific antibodies, and GST pull-down assays showed that Nck determines spatiotemporal patterns of Cdc42/aPKC activation during endothelial morphogenesis. Our results demonstrate that Nck acts as an important hub integrating angiogenic cues with cytoskeletal changes that enable endothelial apical-basal polarization and lumen formation. These findings point to Nck as an emergent target for effective antiangiogenic therapy.     

Membrane remodeling by the PX-BAR protein SNX18 promotes autophagosome formation

The membrane remodeling events required for autophagosome biogenesis are still poorly understood. Because PX domain proteins mediate membrane remodeling and trafficking, we conducted an imaging-based siRNA screen for autophagosome formation targeting human PX proteins. The PX-BAR protein SNX18 was identified as a positive regulator of autophagosome formation, and its Drosophila melanogaster homologue SH3PX1 was found to be required for efficient autophagosome formation in the larval fat body. We show that SNX18 is required for recruitment of Atg16L1-positive recycling endosomes to a perinuclear area and for delivery of Atg16L1- and LC3-positive membranes to autophagosome precursors. We identify a direct interaction of SNX18 with LC3 and show that the pro-autophagic activity of SNX18 depends on its membrane binding and tubulation capacity. We also show that the function of SNX18 in membrane tubulation and autophagy is negatively regulated by phosphorylation of S233. We conclude that SNX18 promotes autophagosome formation by virtue of its ability to remodel membranes and provide membrane to forming autophagosomes.     

Detachment-induced autophagy during anoikis and lumen formation in epithelial acini

The individual units (called acini) comprising glandular epithelium possess a hollow lumen that is filled in early epithelial cancers. While investigating the generation of this hollow lumen using an in vitro three-dimensional (3D) epithelial cell culture model, we observed extensive autophagy in dying cells during lumen formation, and thus, proposed that excessive self-eating may promote cell death in the lumen. However, we now reassess this hypothesis. Because cells in the lumen die due to extracellular matrix (ECM) deprivation (anoikis), we tested if autophagy is regulated by ECM detachment. We discovered that detachment strongly induces autophagy in epithelial cells, even when apoptosis is inhibited by Bcl-2 expression. RNAi-mediated depletion of autophagy-related (ATG) genes inhibits detachment-induced autophagy, resulting in increased apoptosis and reduced clonogenic recovery following anoikis. Similarly, during 3D morphogenesis, ATG-depletion enhances luminal apoptosis and fails to elicit long-term luminal survival and filling, even when combined with apoptotic inhibition. Thus, autophagy promotes epithelial cell survival during anoikis. These results broach the possibility that autophagy contributes to the survival of tumor cells lacking appropriate matrix contact, either during early carcinoma formation or in the later stages of dissemination and metastasis.     

SNX18 regulates ATG9A trafficking from recycling endosomes by recruiting Dynamin‐2

Trafficking of mammalian ATG9A between the Golgi apparatus, endosomes and peripheral ATG9A compartments is important for autophagosome biogenesis. Here, we show that the membrane remodelling protein SNX18, previously identified as a positive regulator of autophagy, regulates ATG9A trafficking from recycling endosomes. ATG9A is recruited to SNX18‐induced tubules generated from recycling endosomes and accumulates in juxtanuclear recycling endosomes in cells lacking SNX18. Binding of SNX18 to Dynamin‐2 is important for ATG9A trafficking from recycling endosomes and for formation of ATG16L1‐ and WIPI2‐positive autophagosome precursor membranes. We propose a model where upon autophagy induction, SNX18 recruits Dynamin‐2 to induce budding of ATG9A and ATG16L1 containing membranes from recycling endosomes that traffic to sites of autophagosome formation.     

Cell-polarity dynamics controls the mechanism of lumen formation in epithelial morphogenesis

Many organs consist of tubes of epithelial cells enclosing a central lumen. How the space of this lumen is generated is a key question in morphogenesis. Two predominant mechanisms of de novo lumen formation have been observed: hollowing and cavitation. In hollowing, the lumen is formed by exocytosis and membrane separation, whereas, in cavitation, the lumen is generated by apoptosis of cells in the middle of the structure [1, 2]. Using MDCK cells in three-dimensional cultures, we found an inverse correlation between polarization efficiency and apoptosis. When cells were grown in collagen, where cells polarized slowly, apoptosis was needed for lumen formation. However, in the presence of Matrigel, which allowed rapid polarization, lumens formed without apoptosis. If polarization in Matrigel was perturbed by blocking formation of the apical surface by RNAi of Cdc42, lumens formed by apoptosis. In a complementary approach, we plated cells at high density so that aggregates formed with little polarity. These aggregates required apoptosis to form lumens, whereas cells plated at low density formed cysts with rapidly polarizing cells and did not need apoptosis to form lumens. The mechanism of lumen formation in the 3D-MDCK model can shift between hollowing and cavitation, depending on cell polarization.     

Two distinct integrin-mediated mechanisms contribute to apical lumen formation in epithelial cells

Background

Formation of apical compartments underlies the morphogenesis of most epithelial organs during development. The extracellular matrix (ECM), particularly the basement membrane (BM), plays an important role in orienting the apico-basal polarity and thereby the positioning of apical lumens. Integrins have been recognized as essential mediators of matrix-derived polarity signals. The importance of β1-integrins in epithelial polarization is well established but the significance of the accompanying α-subunits have not been analyzed in detail.

Principal Findings

Here we demonstrate that two distinct integrin-dependent pathways regulate formation of apical lumens to ensure robust apical membrane biogenesis under different microenvironmental conditions; 1) α2β1- and α6β4-integrins were required to establish a basal cue that depends on Rac1-activity and guides apico-basal cell polarization. 2) α3β1-integrins were implicated in positioning of mitotic spindles in cysts, a process that is essential for Cdc42-driven epithelial hollowing.

Significance

Identification of the separate processes driven by particular integrin receptors clarifies the functional hierarchies between the different integrins co-expressed in epithelial cells and provides valuable insight into the complexity of cell-ECM interactions thereby guiding future studies addressing the molecular basis of epithelial morphogenesis during development and disease.     

Pak1 regulates the orientation of apical polarization and lumen formation by distinct pathways

The development of the basic architecture of branching tubules enclosing a central lumen that characterizes most epithelial organs crucially depends on the apico-basolateral polarization of epithelial cells. Signals from the extracellular matrix control the orientation of the apical surface, so that it faces the lumen interior, opposite to cell-matrix adhesion sites. This orientation of the apical surface is thought to be intrinsically linked to the formation of single lumens. We previously demonstrated in three-dimensional cyst cultures of Madin-Darby canine kidney (MDCK) cells that signaling by β1 integrins regulates the orientation of the apical surface, via a mechanism that depends on the activity of the small GTPase Rac1. Here, we investigated whether the Rac1 effector Pak1 is a downstream effector in this pathway. Expression of constitutive active Pak1 phenocopies the effect of β1 integrin inhibition in that it misorients the apical surface and induces a multilumen phenotype. The misorientation of apical surfaces depends on the interaction of active Pak1 with PIX proteins and is linked to defects in basement membrane assembly. In contrast, the multilumen phenotype was independent of PIX and the basement membrane. Therefore, Pak1 likely regulates apical polarization and lumen formation by two distinct pathways.     

Coordination of epithelial branching and salivary gland lumen formation by Wnt and FGF signals

Branching morphogenesis is a molecularly conserved mechanism that is adopted by several organs, such as the lung, kidney, mammary gland and salivary gland, to maximize the surface area of a tissue within a small volume. Branching occurs through repetitive clefting and elongation of spherical epithelial structures, called endbuds, which invade the surrounding mesenchyme. In the salivary gland, lumen formation takes place alongside branching morphogenesis, but in a controlled manner, so that branching is active at the distal ends of epithelial branches while lumen formation initiates at the proximal ends, and spreads distally. We present here data showing that interaction between FGF signaling and the canonical (β-catenin dependent) and non-canonical branches of Wnt signaling coordinates these two processes. Using the Axin2^lacZ reporter mice, we find Wnt/β-catenin signaling activity first in the mesenchyme and later, at the time of lumen formation, in the ductal epithelium. Gain and loss of function experiments reveal that this pathway exerts an inhibitory effect on salivary gland branching morphogenesis. We have found that endbuds remain devoid of Wnt/β-catenin signaling activity, a hallmark of ductal structures, through FGF-mediated inhibition of this pathway. Our data also show that FGF signaling has a major role in the control of lumen formation by preventing premature hollowing of epithelial endbuds and slowing down the canalization of presumptive ducts. Concomitantly, FGF signaling strongly represses the ductal marker Cp2l1, most likely via repression of Wnt5b and non-canonical Wnt signaling. Inhibition of canonical and non-canonical Wnt signaling in endbuds by FGF signaling occurs at least in part through sFRP1, a secreted inhibitor of Wnt signaling and downstream target of FGF signaling. Altogether, these findings point to a key function of FGF signaling in the maintenance of an undifferentiated state in endbud cells by inhibition of a ductal fate.     

Three-dimensional modeling of mechanical forces in the extracellular matrix during epithelial lumen formation

Mechanical interactions between cells and extracellular matrix (ECM) mediate epithelial cyst formation. This work relies on the combination of numerical modeling with live cell imaging, to piece together a novel nonintrusive method for determining three-dimensional (3D) mechanical forces caused by shape changes of a multicellular aggregate at the early stages of epithelial cyst formation. We analyzed the evolution of Madin-Darby canine kidney cells in 3D cultures using time-lapse microscopy, with type I collagen gel forming the ECM. The evolving 3D interface between the ECM and the cell aggregate was obtained from microscopy images, and the stress on the surface of a proliferating aggregate and in the surrounding ECM was calculated using the finite element method. The viscoelastic properties of the ECM (a needed input for the finite element method solver) were obtained through oscillatory shear flow experiments on a rheometer. For validation purpose, the forces exerted by an aggregate on a force-sensor array were measured and compared against the computational results.     

PATJ regulates tight junction formation and polarity in mammalian epithelial cells

Recent studies have revealed an important role for tight junction protein complexes in epithelial cell polarity. One of these complexes contains the apical transmembrane protein, Crumbs, and two PSD95/discs large/zonula occludens domain proteins, protein associated with Lin seven 1 (PALS1)/Stardust and PALS1-associated tight junction protein (PATJ). Although Crumbs and PALS1/Stardust are known to be important for cell polarization, recent studies have suggested that Drosophila PATJ is not essential and its function is unclear. Here, we find that PATJ is targeted to the apical region and tight junctions once cell polarization is initiated. We show using RNAi techniques that reduction in PATJ expression leads to delayed tight junction formation as well as defects in cell polarization. These effects are reversed by reintroduction of PATJ into these RNAi cells. This study provides new functional information on PATJ as a polarity protein and increases our understanding of the Crumbs-PALS1-PATJ complex function in epithelial polarity.     

Bone morphogenetic protein-4 abrogates lumen formation by mammary epithelial cells and promotes invasive growth

Bone morphogenetic proteins (BMPs) are multifunctional cytokines that regulate key developmental processes, but are also overexpressed in many carcinomas. To assess whether BMPs would influence the three-dimensional architecture of epithelial structures, we took advantage of an in vitro model in which mammary epithelial cells form alveolar-like spherical cysts in collagen gels. We found that BMP-4 has a dramatic, biphasic effect on the organization of epithelial cysts. When added in the concentration range of 1-10 ng/ml, the cytokine abrogates lumen formation and induces the outgrowth of multiple invasive cord-like structures. At higher concentrations (20-100 ng/ml), BMP-4 additionally disrupts cell-cell adhesion, resulting in cyst disintegration and scattering of individual cells into the surrounding collagen matrix. The finding that BMP-4 subverts the ability of mammary epithelial cells to form polarized lumen-containing structures and endows them with invasive properties supports the involvement of this cytokine in the progression of breast cancer.     

Interaction of phospholipase C-gamma1 with villin regulates epithelial cell migration

Tyrosine-phosphorylated villin regulates actin dynamics, cell morphology, and cell migration. Previously, we identified four tyrosine phosphorylation sites in the amino-terminal domain of villin. In this study we report six new sites in the carboxyl-terminal region of the villin core. With this study we document all phosphorylatable tyrosine residues in villin and map them to functions of villin. In this study, we identify for the first time the functional relevance of the carboxyl-terminal domains of the villin core. Expression of the carboxyl-terminal phosphorylation site mutant, as well as the villin truncation mutant S1-S3, inhibited cell migration in HeLa and Madin-Darby canine kidney Tet-Off cells, confirming the role of the carboxyl-terminal phosphorylation sites in villin-induced cell migration. The carboxyl-terminal phosphorylation sites were found to be critical for the interaction of villin with its ligand phospholipase C-gamma1 and for its localization to the developing lamellipodia in a motile cell. The results presented here elucidate the molecular basis for tyrosine-phosphorylated villin-induced changes in cell motility.     

The DHR1 domain of DOCK180 binds to SNX5 and regulates cation-independent mannose 6-phosphate receptor transport

DOCK180 is the archetype of the DOCK180-family guanine nucleotide exchange factor for small GTPases Rac1 and Cdc42. DOCK180-family proteins share two conserved domains, called DOCK homology region (DHR)-1 and -2. Although the function of DHR2 is to activate Rac1, DHR1 is required for binding to phosphoinositides. To better understand the function of DHR1, we searched for its binding partners by direct nanoflow liquid chromatography/tandem mass spectrometry, and we identified sorting nexins (SNX) 1, 2, 5, and 6, which make up a multimeric protein complex mediating endosome-to-trans-Golgi-network (TGN) retrograde transport of the cation-independent mannose 6-phosphate receptor (CI-MPR). Among these SNX proteins, SNX5 was coimmunoprecipitated with DOCK180 most efficiently. In agreement with this observation, DOCK180 colocalized with SNX5 at endosomes. The RNA interference-mediated knockdowns of SNX5 and DOCK180, but not Rac1, resulted in the redistribution of CI-MPR from TGN to endosomes. Furthermore, expression of the DOCK180 DHR1 domain was sufficient to restore the perturbed CI-MPR distribution in DOCK180 knockdown cells. These data suggest that DOCK180 regulates CI-MPR trafficking via SNX5 and that this function is independent of its guanine nucleotide exchange factor activity toward Rac1.     

Bim regulation of lumen formation in cultured mammary epithelial acini is targeted by oncogenes

Epithelial cells organize into cyst-like structures that contain a spherical monolayer of cells that enclose a central lumen. Using a three-dimensional basement membrane culture model in which mammary epithelial cells form hollow, acinus-like structures, we previously demonstrated that lumen formation is achieved, in part, through apoptosis of centrally localized cells. We demonstrate that the proapoptotic protein Bim may selectively trigger apoptosis of the centrally localized acinar cells, leading to temporally controlled lumen formation. Bim is not detectable during early stages of three-dimensional mammary acinar morphogenesis and is then highly upregulated in all cells of acini, coincident with detection of apoptosis in the centrally localized acinar cells. Inhibition of Bim expression by RNA interference transiently blocks luminal apoptosis and delays lumen formation. Oncogenes that induce acinar luminal filling, such as ErbB2 and v-Src, suppress expression of Bim through a pathway dependent on Erk-mitogen-activated protein kinase; however, HPV 16 E7, an oncogene that stimulates cell proliferation but not luminal filling, is unable to reduce Bim expression. Thus, Bim is a critical regulator of luminal apoptosis during mammary acinar morphogenesis in vitro and may be an important target of oncogenes that disrupt glandular epithelial architecture.     

SNX10 is required for osteoclast formation and resorption activity

RANKL-stimulation of osteoclast precursors results in up-regulation of genes involved in the process of differentiation and activation. In this report we describe the expression and functional characterization of Sorting Nexin 10 (snx10). Snx10 belongs to the sorting nexin (SNX) family, a diverse group of proteins with a common feature: the PX domain, which is involved in membrane trafficking and cargo sorting in endosomes. Snx10 is strongly up-regulated during RANKL-induced osteoclast differentiation in vitro and expressed in osteoclasts in vivo. qPCR analysis confirmed a significant increase in the expression of snx10 in in vitro-derived osteoclasts, as well as in femur and calvaria. Immunohistochemical analysis of mouse embryo sections showed expression in long bone, calvariae, and developing teeth. The expression was limited to cells that also expressed TRAP, demonstrating osteoclastic localization. Confocal immunofluorescence and subcellular fractionation analysis revealed Snx10 localization in the nucleus and in the endoplasmic reticulum (ER). To study a possible role for snx10 in osteoclast differentiation and function we silenced snx10 expression and found that snx10 silencing inhibited RANKL-induced osteoclast formation and osteoclast resorption on hydroxyapatite. Silencing also inhibited TRAP secretion. Taken together, these results confirm that snx10 is expressed in osteoclasts and is required for osteoclast differentiation and activity in vitro. Since inhibition of vesicular trafficking is essential for osteoclast formation and activity and SNX10 is involved in intracellular vesicular trafficking, these studies may identify a new candidate gene involved in the development of human bone diseases including osteoporosis.     

SNX9 regulates dynamin assembly and is required for efficient clathrin-mediated endocytosis

Dynamin, a central player in clathrin-mediated endocytosis, interacts with several functionally diverse SH3 domain-containing proteins. However, the role of these interactions with regard to dynamin function is poorly defined. We have investigated a recently identified protein partner of dynamin, SNX9, sorting nexin 9. SNX9 binds directly to both dynamin-1 and dynamin-2. Moreover by stimulating dynamin assembly, SNX9 stimulates dynamin's basal GTPase activity and potentiates assembly-stimulated GTPase activity on liposomes. In fixed cells, we observe that SNX9 partially localizes to clathrin-coated pits. Using total internal reflection fluorescence microscopy in living cells, we detect a transient burst of EGFP-SNX9 recruitment to clathrin-coated pits that occurs during the late stages of vesicle formation and coincides spatially and temporally with a burst of dynamin-mRFP fluorescence. Transferrin internalization is inhibited in HeLa cells after siRNA-mediated knockdown of SNX9. Thus, our results establish that SNX9 is required for efficient clathrin-mediated endocytosis and suggest that it functions to regulate dynamin activity.