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Sandra Citi - One of the best experts on this subject based on the ideXlab platform.
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the tight junction protein Cingulin regulates the vascular response to burn injury in a mouse model
Microvascular Research, 2020Co-Authors: Kristina Zhuravleva, Sandra Citi, Laurent Guillemot, Peter Petzelbauer, Ole Goertz, Gerald Wolkart, Marcus Lehnhardt, Kurt Schmidt, Klaudia SchossleitnerAbstract:Abstract Edema formation due to the collapse of physiological barriers and the associated delayed healing process is still a central problem in the treatment of burn injuries. In healthy individuals, tight junctions form a barrier to fluid and small molecules. Cingulin is a cytoplasmic component of tight junctions and is involved in the regulation of the paracellular barrier. Endothelial specific Cingulin knock-out mice provide new insight into the influence of tight junction proteins on edema formation and angiogenesis during wound healing. Knock-out mice lacking the head domain of Cingulin in endothelial cells (CgnΔEC) were created by breeding Cgnfl/fl mice with Tie1-cre mice. Using a no-touch hot air jet a burn trauma was induced on the ear of the mouse. Over a period of 12 days microcirculatory parameters such as edema formation, angiogenesis and leukocyte-endothelial interactions were visualized using intravital fluorescence microscopy. At baseline, CgnΔEC mice surprisingly showed significantly less tracer extravasation compared to Cgnfl/fl littermates, whereas, after burn injury, edema was consistently higher in CgnΔEC mice. Non-perfused area after wounding was increased, but there was no difference in vessel diameters, contraction or dilation of arteries in CgnΔEC mice. Moreover, Cingulin knock-out did not cause a difference in leukocyte adhesion after burn injury. In summary, Cingulin limits non-perfused area after burn injury and maintains the paracellular barrier of blood vessels. Since edema formation with serious systemic effects is a central problem of burn wounds, understanding the importance of tight junction proteins might help to find new treatment strategies for burn wounds.
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role of Cingulin in agonist induced vascular endothelial permeability
Journal of Biological Chemistry, 2016Co-Authors: Yufeng Tian, Sandra Citi, Grzegorz Gawlak, Xinyong Tian, Alok S Shah, Nicolene Sarich, Anna A BirukovaAbstract:Agonist-induced activation of Rho GTPase signaling leads to endothelial cell (EC) permeability and may culminate in pulmonary edema, a devastating complication of acute lung injury. Cingulin is an adaptor protein first discovered in epithelium and is involved in the organization of the tight junctions. This study investigated the role of Cingulin in control of agonist-induced lung EC permeability via interaction with RhoA-specific activator GEF-H1. The siRNA-induced Cingulin knockdown augmented thrombin-induced EC permeability monitored by measurements of transendothelial electrical resistance and endothelial cell permeability for macromolecules. Increased thrombin-induced permeability in ECs with depleted Cingulin was associated with increased activation of GEF-H1 and RhoA detected in pulldown activation assays. Increased GEF-H1 association with Cingulin was essential for down-regulation of thrombin-induced RhoA barrier disruptive signaling. Using Cingulin-truncated mutants, we determined that GEF-H1 interaction with the rod + tail domain of Cingulin was required for inactivation of GEF-H1 and endothelial cell barrier preservation. The results demonstrate the role for association of GEF-H1 with Cingulin as the mechanism of RhoA pathway inactivation and rescue of EC barrier after agonist challenge.
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Cingulin and actin mediate midbody dependent apical lumen formation during polarization of epithelial cells
Nature Communications, 2016Co-Authors: Anthony Mangan, Sandra Citi, Daniel V Sietsema, Jeffrey K Moore, Rytis PrekerisAbstract:Coordinated polarization of epithelial cells is a key step during morphogenesis that leads to the formation of an apical lumen. Rab11 and its interacting protein FIP5 are necessary for the targeting of apical endosomes to the midbody and apical membrane initiation site (AMIS) during lumenogenesis. However, the machinery that mediates AMIS establishment and FIP5-endosome targeting remains unknown. Here we identify a FIP5-interacting protein, Cingulin, which localizes to the AMIS and functions as a tether mediating FIP5-endosome targeting. We analysed the machinery mediating AMIS recruitment to the midbody and determined that both branched actin and microtubules are required for establishing the site of the nascent lumen. We demonstrate that the Rac1-WAVE/Scar complex mediates Cingulin recruitment to the AMIS by inducing branched actin formation, and that Cingulin directly binds to microtubule C-terminal tails through electrostatic interactions. We propose a new mechanism for apical endosome targeting and AMIS formation around the midbody during epithelial lumenogenesis.
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evidence that Cingulin regulates endothelial barrier function in vitro and in vivo
Arteriosclerosis Thrombosis and Vascular Biology, 2016Co-Authors: Klaudia Schossleitner, Sabine Rauscher, Marion Groger, Heinz Peter Friedl, Richard Finsterwalder, Andreas Habertheuer, Maria Sibilia, Christine Brostjan, Dagmar Fodinger, Sandra CitiAbstract:Objective—Cingulin is a cytoplasmic component of tight junctions. Although modulation of Cingulin levels in cultured epithelial model systems has no significant effect on barrier function, evidence from Cingulin knockout mice suggests that Cingulin may be involved in the regulation of the behavior of epithelial or endothelial cells. Here, we investigate the role of Cingulin in the barrier function of endothelial cells. Approach and Results—We show that Cingulin is expressed in human endothelial cells of the skin, brain, and lung in vivo and in vitro. Endothelial Cingulin colocalizes and coimmunoprecipitates with the tight junction proteins zonula occludens-1 and guanine nucleotide exchange factor-H1. Cingulin overexpression in human umbilical vein endothelial cell induces tight junction formation, increases transendothelial electric resistance, and strengthens barrier function for low and high molecular weight tracers. In contrast, cultured endothelial cells lacking Cingulin are more permeable for low mol...
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Specificity of anti-PLEKHA7 monoclonal antibody for human PLEKHA7.
2015Co-Authors: Jean-christophe Tille, Jimit Shah, Olivia Seyde, Thomas A. Mckee, Sandra CitiAbstract:(A) Immunoblot analysis of lysates from cultured human epithelial cells (SKCO), following treatment with siRNA for PLEKHA7 (siRNA3), control siRNA (untreated and mock-transfected cells for control) using monoclonal antibody 37-8F1. (B) Immunofluorescence analysis of si-PLEKHA7 treated cultures, showing decreased PLEKHA7 junctional (red, Cy3) labeling in PLEKHA7-depleted cells (asterisks, siRNA2) with respect to normal cells. Anti-Cingulin antibody (green, Alexa488) was used as control to normalize junctional labeling. The same results were used with cells depleted of PLEKHA7 by three different siRNAs (see Materials and Methods).
Laurent Guillemot - One of the best experts on this subject based on the ideXlab platform.
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the tight junction protein Cingulin regulates the vascular response to burn injury in a mouse model
Microvascular Research, 2020Co-Authors: Kristina Zhuravleva, Sandra Citi, Laurent Guillemot, Peter Petzelbauer, Ole Goertz, Gerald Wolkart, Marcus Lehnhardt, Kurt Schmidt, Klaudia SchossleitnerAbstract:Abstract Edema formation due to the collapse of physiological barriers and the associated delayed healing process is still a central problem in the treatment of burn injuries. In healthy individuals, tight junctions form a barrier to fluid and small molecules. Cingulin is a cytoplasmic component of tight junctions and is involved in the regulation of the paracellular barrier. Endothelial specific Cingulin knock-out mice provide new insight into the influence of tight junction proteins on edema formation and angiogenesis during wound healing. Knock-out mice lacking the head domain of Cingulin in endothelial cells (CgnΔEC) were created by breeding Cgnfl/fl mice with Tie1-cre mice. Using a no-touch hot air jet a burn trauma was induced on the ear of the mouse. Over a period of 12 days microcirculatory parameters such as edema formation, angiogenesis and leukocyte-endothelial interactions were visualized using intravital fluorescence microscopy. At baseline, CgnΔEC mice surprisingly showed significantly less tracer extravasation compared to Cgnfl/fl littermates, whereas, after burn injury, edema was consistently higher in CgnΔEC mice. Non-perfused area after wounding was increased, but there was no difference in vessel diameters, contraction or dilation of arteries in CgnΔEC mice. Moreover, Cingulin knock-out did not cause a difference in leukocyte adhesion after burn injury. In summary, Cingulin limits non-perfused area after burn injury and maintains the paracellular barrier of blood vessels. Since edema formation with serious systemic effects is a central problem of burn wounds, understanding the importance of tight junction proteins might help to find new treatment strategies for burn wounds.
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mgcracgap interacts with Cingulin and paraCingulin to regulate rac1 activation and development of the tight junction barrier during epithelial junction assembly
Molecular Biology of the Cell, 2014Co-Authors: Laurent Guillemot, Lionel Jond, Domenica Spadaro, Diego Guerrera, Rocio Tapia, Sandra CitiAbstract:The regulation of Rho-family GTPases is crucial to direct the formation of cell-cell junctions and tissue barriers. Cingulin (CGN) and paraCingulin (CGNL1) control RhoA activa- tion in epithelial cells by interacting with RhoA guanidine exchange factors. CGNL1 depletion also inhibits Rac1 activation during junction assembly. Here we show that, unexpectedly, Madin-Darby canine kidney epithelial cells depleted of both CGN and CGNL1 (double-KD cells) display normal Rac1 activation and tight junction (TJ) formation, despite decreased junctional recruitment of the Rac1 activator Tiam1. The expression of the Rac1 inhibitor MgcRacGAP is decreased in double-KD cells, and the barrier development and Rac1 activa- tion phenotypes are rescued by exogenous expression of MgcRacGAP. MgcRacGAP colocal- izes with CGN and CGNL1 at TJs and forms a complex and interacts directly in vitro with CGN and CGNL1. Depletion of either CGN or CGNL1 in epithelial cells results in decreased junc- tional localization of MgcRacGAP but not of ECT2, a centralspindlin-interacting Rho GEF. These results provide new insight into coordination of Rho-family GTPase activities at junctions, since apical accumulation of CGN and CGNL1 at TJs during junction maturation provides a mechanism to spatially restrict down-regulation of Rac1 activation through the recruitment of MgcRacGAP.
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the junctional proteins Cingulin and paraCingulin modulate the expression of tight junction protein genes through gata 4
PLOS ONE, 2013Co-Authors: Laurent Guillemot, Domenica Spadaro, Sandra CitiAbstract:The cytoplamic junctional proteins Cingulin and paraCingulin have been implicated in the regulation of gene expression in different cultured cell models. In renal epithelial MDCK cells, depletion of either protein results in a Rho-dependent increase in the expression of claudin-2. Here we examined MDCK cell clones depleted of both Cingulin and paraCingulin (double-KD cells), and we found that unexpectedly the expression of claudin-2, and also the expression of ZO-3 and claudin-3, were decreased, while RhoA activity was still higher than in control cells. The decreased expression of claudin-2 and other TJ proteins in double–KD cells correlated with reduced levels of the transcription factor GATA-4, and was rescued by overexpression of GATA-4, but not by inhibiting RhoA activity. These results indicate that in MDCK cells GATA-4 is required for the expression of claudin-2 and other TJ proteins, and that maintenance of GATA-4 expression requires either Cingulin or paraCingulin. These results and previous studies suggest a model whereby Cingulin and paraCingulin redundantly control the expression of specific TJ proteins through distinct GATA-4- and RhoA-dependent mechanisms, and that in the absence of sufficient levels of GATA-4 the RhoA-mediated upregulation of claudin-2 is inhibited.
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Hypothetical schematic model for the regulation of claudin-2 expression in MDCK cells.
2013Co-Authors: Laurent Guillemot, Domenica Spadaro, Sandra CitiAbstract:GATA-4 is required for optimal transcription of claudin-2 (and other TJ proteins, not shown for simplicity) (double arrow in scheme). Cingulin and paraCingulin redundantly control the mRNA and protein expression of GATA-4, and independently contribute to the down-regulation of active RhoA in confluent cells. Active RhoA increases claudin-2 expression, either through GATA-4, and/or other, unknown mechanisms (question marks), but is ineffective when GATA-4 is down-regulated.
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Down-regulation of claudin-2, ZO-3, and claudin-3 in Cingulin/paraCingulin double knockdown MDCK cells.
2013Co-Authors: Laurent Guillemot, Domenica Spadaro, Sandra CitiAbstract:(A) Histogram showing relative mRNA levels, determined by qRT-PCR, for the indicated transcripts, in wild-type MDCK cells (WT), in a MDCK cell clone expressing a control shRNA (Control), in a Cingulin single-KD cell clone (CGN(-)), in a paraCingulin single-KD cell clone (CGNL1(-)), and in a double-KD cell clone (CGN(-)/CGNL1(-)). The relative mRNA levels were calculated as the ratio of the mRNA in experimental samples versus WT cells (taken as 100%). *p
Karl Matter - One of the best experts on this subject based on the ideXlab platform.
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spatially restricted activation of rhoa signalling at epithelial junctions by p114rhogef drives junction formation and morphogenesis
Nature Cell Biology, 2011Co-Authors: Stephen J. Terry, Ahmed Elbediwy, Ceniz Zihni, Maria S. Balda, Elisa Vitiello, Isabelle Leefa Chong V San, Karl MatterAbstract:Signalling by the GTPase RhoA, a key regulator of epithelial cell behaviour, can stimulate opposing processes: RhoA can promote junction formation and apical constriction, and reduce adhesion and cell spreading. Molecular mechanisms are thus required that ensure spatially restricted and process-specific RhoA activation. For many fundamental processes, including assembly of the epithelial junctional complex, such mechanisms are still unknown. Here we show that p114RhoGEF is a junction-associated protein that drives RhoA signalling at the junctional complex and regulates tight-junction assembly and epithelial morphogenesis. p114RhoGEF is required for RhoA activation at cell-cell junctions, and its depletion stimulates non-junctional Rho signalling and induction of myosin phosphorylation along the basal domain. Depletion of GEF-H1, a RhoA activator inhibited by junctional recruitment, does not reduce junction-associated RhoA activation. p114RhoGEF associates with a complex containing myosin II, Rock II and the junctional adaptor Cingulin, indicating that p114RhoGEF is a component of a junction-associated Rho signalling module that drives spatially restricted activation of RhoA to regulate junction formation and epithelial morphogenesis.
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Regulation of tight junction assembly and epithelial morphogenesis by the heat shock protein Apg-2-4
2011Co-Authors: Saima Aijaz, Maria S. Balda, Elena Sanchez-heras, Karl MatterAbstract:Copyright information:Taken from "Regulation of tight junction assembly and epithelial morphogenesis by the heat shock protein Apg-2"http://www.biomedcentral.com/1471-2121/8/49BMC Cell Biology 2007;8():49-49.Published online 20 Nov 2007PMCID:PMC2211299.antibodies against the transmembrane proteins occludin (A) and claudin-4 (B), the junctional plaque components Cingulin (C) and GEF-H1 (D), and the adherens junction proteins α-catenin (E) and E-cadherin (F). Shown are images derived from samples fixed after 1 hour and 27 hours of junction formation. Note, retardation of junctional recruitment was observed for occludin, Cingulin, α-catenin and E-cadherin. GEF-H1 became only concentrated at tight junctions after longer time points of junction formation. Claudin-4 was present at the plasma membrane at all time points but only appeared to be concentrated at junctions at later time points. GEF-H1 and claudin-4 distributions were not affected by depletion of Apg-2
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Regulation of tight junction assembly and epithelial morphogenesis by the heat shock protein Apg-2-3
2011Co-Authors: Saima Aijaz, Maria S. Balda, Elena Sanchez-heras, Karl MatterAbstract:Copyright information:Taken from "Regulation of tight junction assembly and epithelial morphogenesis by the heat shock protein Apg-2"http://www.biomedcentral.com/1471-2121/8/49BMC Cell Biology 2007;8():49-49.Published online 20 Nov 2007PMCID:PMC2211299.on was allowed to proceed for the indicated amount of time (A) or for one hour (B). The cells were then stained for either ZO-1 (A) or f-actin, occludin, claudin-4, Cingulin, GEF-H1, α-catenin or E-cadherin (B). The fractions of cells expressing the labelled markers along the entire lateral cell membrane were then determined by counting. At least five different fields derived from two independent experiments were counted for each marker and time point. Note, as a large fraction of claudin-4 is continuously at the plasma membrane even before the addition of calcium, its early presence at the plasma membrane does not indicate tight junction formation
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binding of gef h1 to the tight junction associated adaptor Cingulin results in inhibition of rho signaling and g1 s phase transition
Developmental Cell, 2005Co-Authors: Saima Aijaz, Sandra Citi, Maria S. Balda, Fabio Datri, Karl MatterAbstract:The activity of Rho GTPases is carefully timed to control epithelial proliferation and differentiation. RhoA is downregulated when epithelial cells reach confluence, resulting in inhibition of signaling pathways that stimulate proliferation. Here we show that GEF-H1/Lfc, a guanine nucleotide exchange factor for RhoA, directly interacts with Cingulin, a junctional adaptor. Cingulin binding inhibits RhoA activation and signaling, suggesting that the increase in Cingulin expression in confluent cells causes downregulation of RhoA by inhibiting GEF-H1/Lfc. In agreement, RNA interference of GEF-H1 or transfection of GEF-H1 binding Cingulin mutants inhibit G1/S phase transition of MDCK cells, and depletion of Cingulin by regulated RNA interference results in irregular monolayers and RhoA activation. These results indicate that forming epithelial tight junctions contribute to the downregulation of RhoA in epithelia by inactivating GEF-H1 in a Cingulin-dependent manner, providing a molecular mechanism whereby tight junction formation is linked to inhibition of RhoA signaling.
Andrea Lippoldt - One of the best experts on this subject based on the ideXlab platform.
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tight junctions of the blood brain barrier development composition and regulation
Vascular Pharmacology, 2002Co-Authors: Hartwig Wolburg, Andrea LippoldtAbstract:1. The blood-brain barrier is essential for the maintenance and regulation of the neural microenvironment. The main characteristic features of blood-brain barrier endothelial cells are an extremely low rate of transcytotic vesicles and a restrictive paracellular diffusion barrier. 2. Endothelial blood-brain barrier tight junctions differ from epithelial tight junctions, not only by distinct morphological and molecular properties, but also by the fact that endothelial tight junctions are more sensitive to microenvironmental than epithelial factors. 3. Many ubiquitous molecular tight junction components have been identified and characterized including claudins, occludin, ZO-1, ZO-2, ZO-3, Cingulin and 7H6. Signaling pathways involved in tight junction regulation include G-proteins, serine-, threonine- and tyrosine-kinases, extra and intracellular calcium levels, cAMP levels, proteases and cytokines. Common to most of these pathways is the modulation of cytoskeletal elements and the connection of tight junction transmembrane molecules to the cytoskeleton. Additionally, crosstalk between components of the tight junction- and the cadherin-catenin system of the adherens junction suggests a close functional interdependence of the two cell-cell contact systems. 4. Important new molecular aspects of tight junction regulation were recently elucidated. This review provides an integration of these new results.
Fabio Datri - One of the best experts on this subject based on the ideXlab platform.
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binding of gef h1 to the tight junction associated adaptor Cingulin results in inhibition of rho signaling and g1 s phase transition
Developmental Cell, 2005Co-Authors: Saima Aijaz, Sandra Citi, Maria S. Balda, Fabio Datri, Karl MatterAbstract:The activity of Rho GTPases is carefully timed to control epithelial proliferation and differentiation. RhoA is downregulated when epithelial cells reach confluence, resulting in inhibition of signaling pathways that stimulate proliferation. Here we show that GEF-H1/Lfc, a guanine nucleotide exchange factor for RhoA, directly interacts with Cingulin, a junctional adaptor. Cingulin binding inhibits RhoA activation and signaling, suggesting that the increase in Cingulin expression in confluent cells causes downregulation of RhoA by inhibiting GEF-H1/Lfc. In agreement, RNA interference of GEF-H1 or transfection of GEF-H1 binding Cingulin mutants inhibit G1/S phase transition of MDCK cells, and depletion of Cingulin by regulated RNA interference results in irregular monolayers and RhoA activation. These results indicate that forming epithelial tight junctions contribute to the downregulation of RhoA in epithelia by inactivating GEF-H1 in a Cingulin-dependent manner, providing a molecular mechanism whereby tight junction formation is linked to inhibition of RhoA signaling.
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evidence for a functional interaction between Cingulin and zo 1 in cultured cells
Journal of Biological Chemistry, 2002Co-Authors: Fabio Datri, Sandra Citi, Fabio NadaluttiAbstract:Cingulin, a protein component of the submembrane plaque of tight junctions (TJ), contains globular and coiled-coil domains and interacts in vitro with several TJ and cytoskeletal proteins, including the PDZ protein ZO-1. Overexpression of Xenopus Cingulin in transfected Xenopus A6 cells resulted in the disruption of endogenous ZO-1 localization, suggesting that Cingulin functionally interacts with ZO-1. GlutathioneS-transferase pull-down experiments showed that a conserved ZO-1 interaction motif (ZIM) at the NH2 terminus of Cingulin is required for Cingulin-ZO-1 interaction in vitro. An NH2-terminal region of Cingulin, containing the ZIM, was sufficient, when fused to coiled-coil sequences, to target transfected Cingulin to junctions. However, deletion of the ZIM did not abolish junctional localization of transfected Cingulin in A6 cells, suggesting that Cingulin can be recruited to TJ through multiple protein interactions. Interestingly, the ZIM was required for Cingulin recruitment into ZO-1-containing adherens junctions of Rat-1 fibroblasts, indicating that Cingulin junctional recruitment does not require the molecular context of TJ. Cingulin coiled-coil sequences enhanced the junctional accumulation of expressed Cingulin head region in A6 cells, but purified recombinant Cingulin did not form filaments under physiological conditions in vitro, suggesting that the Cingulin coiled-coil domain acts primarily by promoting dimerization.
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human and xenopus Cingulin share a modular organization of the coiled coil rod domain predictions for intra and intermolecular assembly
Journal of Structural Biology, 2000Co-Authors: Sandra Citi, Fabio Datri, David A D ParryAbstract:The complete nucleotide and derived amino acid sequences of Homo sapiens Cingulin cDNA (5143 bp) were determined by sequencing two distinct EST clones that showed significant sequence homology to Xenopus laevis Cingulin. Protein sequence analysis indicates that the molecule contains two chains and has a tripartite structure with N-terminal (head) domains, a coiled-coil rod domain (length, 120 nm), and short C-terminal (tail) domains. Human and Xenopus Cingulin heads are only 33% identical, yet a human Cingulin N-terminal fragment still interacts with canine ZO-1 and ZO-2 in vitro. The rod domain contains two A and two B subdomains, though it lacks the third B subdomain present in Xenopus Cingulin. The heptad substructures of Xenopus and human Cingulins were further characterized by computer analysis and indicated that the two-stranded coiled-coil structure contained chains that were parallel and in axial register. Fast Fourier transform analysis and a scoring technique designed to recognize potential interactions between different supramolecular arrangements suggests that Cingulin dimers may further assemble through antiparallel interactions between the last ∼100 amino acids of the coiled-coil region. Cingulin mRNA (∼5.2 kb) was detected by Northern blotting in epithelial tissues. A human Cingulin EST was mapped to chromosome 1q21 using the UniGene database.
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Cingulin contains globular and coiled coil domains and interacts with zo 1 zo 2 zo 3 and myosin
Journal of Cell Biology, 1999Co-Authors: Michelangelo Cordenonsi, Sandra Citi, Eva Hammar, John Kendrickjones, Fabio Datri, David A D Parry, David ShoreAbstract:We characterized the sequence and protein interactions of Cingulin, an Mr 140–160-kD phosphoprotein localized on the cytoplasmic surface of epithelial tight junctions (TJ). The derived amino acid sequence of a full-length Xenopus laevis Cingulin cDNA shows globular head (residues 1–439) and tail (1,326–1,368) domains and a central α-helical rod domain (440–1,325). Sequence analysis, electron microscopy, and pull-down assays indicate that the Cingulin rod is responsible for the formation of coiled-coil parallel dimers, which can further aggregate through intermolecular interactions. Pull-down assays from epithelial, insect cell, and reticulocyte lysates show that an NH2-terminal fragment of Cingulin (1–378) interacts in vitro with ZO-1 (Kd ∼5 nM), ZO-2, ZO-3, myosin, and AF-6, but not with symplekin, and a COOH-terminal fragment (377–1,368) interacts with myosin and ZO-3. ZO-1 and ZO-2 immunoprecipitates contain Cingulin, suggesting in vivo interactions. Full-length Cingulin, but not NH2-terminal and COOH-terminal fragments, colocalizes with endogenous Cingulin in transfected MDCK cells, indicating that sequences within both head and rod domains are required for TJ localization. We propose that Cingulin is a functionally important component of TJ, linking the submembrane plaque domain of TJ to the actomyosin cytoskeleton.