RESUMEN
The ability to remodel and move cellular membranes, and the cargoes regulated by these membranes, allows for specialised functions to occur in distinct regions of the cell in a process known as cellular polarisation. The ability to collectively co-ordinate such polarisation between cells allows for the genesis of multicellularity, such as the formation of organs. During tumourigenesis, the rules for such tissue polarisation become dysregulated, allowing for collective polarity rearrangements that can drive metastasis. In this review, we focus on how membrane trafficking underpins collective cell invasion and metastasis in cancer. We examine this through the lens of the ADP-ribosylation factor (ARF) subfamily of small GTPases, focusing on how the ARF regulatory network - ARF activators, inactivators, effectors, and modifications - controls ARF GTPase function.
Asunto(s)
Factores de Ribosilacion-ADP , Carcinogénesis , Humanos , Membrana Celular , Transformación Celular NeoplásicaRESUMEN
Dysregulation of the PI3K/AKT pathway is a common occurrence in high-grade serous ovarian carcinoma (HGSOC), with the loss of the tumour suppressor PTEN in HGSOC being associated with poor prognosis. The cellular mechanisms of how PTEN loss contributes to HGSOC are largely unknown. We here utilise time-lapse imaging of HGSOC spheroids coupled to a machine learning approach to classify the phenotype of PTEN loss. PTEN deficiency induces PI(3,4,5)P3 -rich and -dependent membrane protrusions into the extracellular matrix (ECM), resulting in a collective invasion phenotype. We identify the small GTPase ARF6 as a crucial vulnerability of HGSOC cells upon PTEN loss. Through a functional proteomic CRISPR screen of ARF6 interactors, we identify the ARF GTPase-activating protein (GAP) AGAP1 and the ECM receptor ß1-integrin (ITGB1) as key ARF6 interactors in HGSOC regulating PTEN loss-associated invasion. ARF6 functions to promote invasion by controlling the recycling of internalised, active ß1-integrin to maintain invasive activity into the ECM. The expression of the CYTH2-ARF6-AGAP1 complex in HGSOC patients is inversely associated with outcome, allowing the identification of patient groups with improved versus poor outcome. ARF6 may represent a therapeutic vulnerability in PTEN-depleted HGSOC.
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Proteínas de Unión al GTP Monoméricas , Neoplasias Ováricas , Humanos , Femenino , Integrinas/metabolismo , Proteómica , Fosfatidilinositol 3-Quinasas/metabolismo , Neoplasias Ováricas/genética , Neoplasias Ováricas/patología , Proteínas de Unión al GTP Monoméricas/metabolismo , Fosfohidrolasa PTEN/genética , Fosfohidrolasa PTEN/metabolismoRESUMEN
ARF GTPases are central regulators of membrane trafficking that control local membrane identity and remodeling facilitating vesicle formation. Unraveling their function is complicated by the overlapping association of ARFs with guanine nucleotide exchange factors (GEFs), GTPase-activating proteins (GAPs), and numerous interactors. Through a functional genomic screen of three-dimensional (3D) prostate cancer cell behavior, we explore the contribution of ARF GTPases, GEFs, GAPs, and interactors to collective invasion. This revealed that ARF3 GTPase regulates the modality of invasion, acting as a switch between leader cell-led chains of invasion or collective sheet movement. Functionally, the ability of ARF3 to control invasion modality is dependent on association and subsequent control of turnover of N-cadherin. In vivo, ARF3 levels acted as a rheostat for metastasis from intraprostatic tumor transplants and ARF3/N-cadherin expression can be used to identify prostate cancer patients with metastatic, poor-outcome disease. Our analysis defines a unique function for the ARF3 GTPase in controlling how cells collectively organize during invasion and metastasis.
Asunto(s)
Factores de Ribosilacion-ADP , Proteínas Activadoras de GTPasa , Proteínas de Unión al GTP Monoméricas , Neoplasias de la Próstata , Humanos , Masculino , Factores de Ribosilacion-ADP/genética , Cadherinas/genética , Endocitosis , Proteínas Activadoras de GTPasa/genética , Neoplasias de la Próstata/genéticaRESUMEN
The glycocalyx component and sialomucin podocalyxin (PODXL) is required for normal tissue development by promoting apical membranes to form between cells, triggering lumen formation. Elevated PODXL expression is also associated with metastasis and poor clinical outcome in multiple tumor types. How PODXL presents this duality in effect remains unknown. We identify an unexpected function of PODXL as a decoy receptor for galectin-3 (GAL3), whereby the PODXL-GAL3 interaction releases GAL3 repression of integrin-based invasion. Differential cortical targeting of PODXL, regulated by ubiquitination, is the molecular mechanism controlling alternate fates. Both PODXL high and low surface levels occur in parallel subpopulations within cancer cells. Orthotopic intraprostatic xenograft of PODXL-manipulated cells or those with different surface levels of PODXL define that this axis controls metastasis in vivo. Clinically, interplay between PODXL-GAL3 stratifies prostate cancer patients with poor outcome. Our studies define the molecular mechanisms and context in which PODXL promotes invasion and metastasis.
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Glicocálix , Sialoglicoproteínas , Masculino , Humanos , Glicocálix/metabolismo , Sialoglicoproteínas/metabolismo , Xenoinjertos , Trasplante HeterólogoRESUMEN
High-grade serous (HGS) ovarian cancer is the most lethal gynaecological disease in the world and metastases is a major cause. The omentum is the preferential metastatic site in HGS ovarian cancer patients and in vitro models that recapitulate the original environment of this organ at cellular and molecular level are being developed to study basic mechanisms that underpin this disease. The tumour extracellular matrix (ECM) plays active roles in HGS ovarian cancer pathology and response to therapy. However, most of the current in vitro models use matrices of animal origin and that do not recapitulate the complexity of the tumour ECM in patients. Here, we have developed omentum gel (OmGel), a matrix made from tumour-associated omental tissue of HGS ovarian cancer patients that has unprecedented similarity to the ECM of HGS omental tumours and is simple to prepare. When used in 2D and 3D in vitro assays to assess cancer cell functions relevant to metastatic ovarian cancer, OmGel performs as well as or better than the widely use Matrigel and does not induce additional phenotypic changes to ovarian cancer cells. Surprisingly, OmGel promotes pronounced morphological changes in cancer associated fibroblasts (CAFs). These changes were associated with the upregulation of proteins that define subsets of CAFs in tumour patient samples, highlighting the importance of using clinically and physiologically relevant matrices for in vitro studies. Hence, OmGel provides a step forward to study the biology of HGS omental metastasis. Metastasis in the omentum are also typical of other cancer types, particularly gastric cancer, implying the relevance of OmGel to study the biology of other highly lethal cancers.
RESUMEN
Single cell profiling by genetic, proteomic and imaging methods has expanded the ability to identify programmes regulating distinct cell states. The 3-dimensional (3D) culture of cells or tissue fragments provides a system to study how such states contribute to multicellular morphogenesis. Whether cells plated into 3D cultures give rise to a singular phenotype or whether multiple biologically distinct phenotypes arise in parallel is largely unknown due to a lack of tools to detect such heterogeneity. Here we develop Traject3d (Trajectory identification in 3D), a method for identifying heterogeneous states in 3D culture and how these give rise to distinct phenotypes over time, from label-free multi-day time-lapse imaging. We use this to characterise the temporal landscape of morphological states of cancer cell lines, varying in metastatic potential and drug resistance, and use this information to identify drug combinations that inhibit such heterogeneity. Traject3d is therefore an important companion to other single-cell technologies by facilitating real-time identification via live imaging of how distinct states can lead to alternate phenotypes that occur in parallel in 3D culture.
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Neoplasias , Proteómica , Diagnóstico por Imagen , Humanos , Neoplasias/diagnóstico por imagen , FenotipoRESUMEN
Patient-derived organoids and cellular spheroids recapitulate tissue physiology with remarkable fidelity. We investigated how engagement with a reconstituted basement membrane in three dimensions (3D) supports the polarized, stress resilient tissue phenotype of mammary epithelial spheroids. Cells interacting with reconstituted basement membrane in 3D had reduced levels of total and actin-associated filamin and decreased cortical actin tension that increased plasma membrane protrusions to promote negative plasma membrane curvature and plasma membrane protein associations linked to protein secretion. By contrast, cells engaging a reconstituted basement membrane in 2D had high cortical actin tension that forced filamin unfolding and endoplasmic reticulum (ER) associations. Enhanced filamin-ER interactions increased levels of PKR-like ER kinase effectors and ER-plasma membrane contact sites that compromised calcium homeostasis and diminished cell viability. Consequently, cells with decreased cortical actin tension had reduced ER stress and survived better. Consistently, cortical actin tension in cellular spheroids regulated polarized basement membrane membrane deposition and sensitivity to exogenous stress. The findings implicate cortical actin tension-mediated filamin unfolding in ER function and underscore the importance of tissue mechanics in organoid homeostasis.
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Actinas , Retículo Endoplásmico , Actinas/metabolismo , Retículo Endoplásmico/metabolismo , Estrés del Retículo Endoplásmico , Células Epiteliales/metabolismo , Filaminas/metabolismo , FenotipoRESUMEN
The three-dimensional culture of epithelial cells allows the characterization of processes required for collective epithelial polarization, such as formation of an epithelial lumen. Madin-Darby Canine Kidney (MDCK) cells have been instrumental in pioneering 3-Dimensional culture analysis methods. Here we describe methods for MDCK cell three-dimensional culture, generation of stable engineered cell lines, immunolabeling, and imaging approaches that allow for analysis of apical-basal polarity during lumen formation in this model.
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Polaridad Celular , Células Epiteliales , Animales , Técnicas de Cultivo de Célula , Línea Celular , Perros , Células de Riñón Canino Madin Darby , MorfogénesisRESUMEN
February is LGBT+ history month, and to celebrate, Journal of Cell Science Editorial Advisory Board member David Bryant organised a conversation with a selection of scientists to explore their experiences of being LGBT+ in academia.
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Liderazgo , Minorías Sexuales y de Género , Movilidad Laboral , Comunicación , HumanosRESUMEN
RAS-like (RAL) GTPases function in Wnt signalling-dependent intestinal stem cell proliferation and regeneration. Whether RAL proteins work as canonical RAS effectors in the intestine and the mechanisms of how they contribute to tumourigenesis remain unclear. Here, we show that RAL GTPases are necessary and sufficient to activate EGFR/MAPK signalling in the intestine, via induction of EGFR internalisation. Knocking down Drosophila RalA from intestinal stem and progenitor cells leads to increased levels of plasma membrane-associated EGFR and decreased MAPK pathway activation. Importantly, in addition to influencing stem cell proliferation during damage-induced intestinal regeneration, this role of RAL GTPases impacts on EGFR-dependent tumourigenic growth in the intestine and in human mammary epithelium. However, the effect of oncogenic RAS in the intestine is independent from RAL function. Altogether, our results reveal previously unrecognised cellular and molecular contexts where RAL GTPases become essential mediators of adult tissue homeostasis and malignant transformation.
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Proliferación Celular , Transformación Celular Neoplásica/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimología , Receptores ErbB/metabolismo , Mucosa Intestinal/metabolismo , Proteínas de Unión al GTP Monoméricas/metabolismo , Receptores de Péptidos de Invertebrados/metabolismo , Células Madre/metabolismo , Proteínas de Unión al GTP ral/metabolismo , Animales , Animales Modificados Genéticamente , Neoplasias de la Mama/enzimología , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Línea Celular Tumoral , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/patología , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Endocitosis , Receptores ErbB/genética , Femenino , Humanos , Hiperplasia , Mucosa Intestinal/patología , Neoplasias Pulmonares/enzimología , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Glándulas Mamarias Humanas/enzimología , Glándulas Mamarias Humanas/patología , Ratones Endogámicos C57BL , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas de Unión al GTP Monoméricas/genética , Receptores de Péptidos de Invertebrados/genética , Transducción de Señal , Células Madre/patología , Proteínas de Unión al GTP ral/genéticaRESUMEN
The signalling pathways underpinning cell growth and invasion use overlapping components, yet how mutually exclusive cellular responses occur is unclear. Here, we report development of 3-Dimensional culture analyses to separately quantify growth and invasion. We identify that alternate variants of IQSEC1, an ARF GTPase Exchange Factor, act as switches to promote invasion over growth by controlling phosphoinositide metabolism. All IQSEC1 variants activate ARF5- and ARF6-dependent PIP5-kinase to promote PI(3,4,5)P3-AKT signalling and growth. In contrast, select pro-invasive IQSEC1 variants promote PI(3,4,5)P3 production to form invasion-driving protrusions. Inhibition of IQSEC1 attenuates invasion in vitro and metastasis in vivo. Induction of pro-invasive IQSEC1 variants and elevated IQSEC1 expression occurs in a number of tumour types and is associated with higher-grade metastatic cancer, activation of PI(3,4,5)P3 signalling, and predicts long-term poor outcome across multiple cancers. IQSEC1-regulated phosphoinositide metabolism therefore is a switch to induce invasion over growth in response to the same external signal. Targeting IQSEC1 as the central regulator of this switch may represent a therapeutic vulnerability to stop metastasis.
Asunto(s)
Factores de Intercambio de Guanina Nucleótido/metabolismo , Metástasis de la Neoplasia , Fosfatidilinositoles/metabolismo , Transducción de Señal , Factor 6 de Ribosilación del ADP , Factores de Ribosilacion-ADP/metabolismo , Animales , Carcinogénesis/genética , Carcinogénesis/metabolismo , Línea Celular Tumoral , Factores de Intercambio de Guanina Nucleótido/genética , Xenoinjertos , Humanos , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad/metabolismo , Masculino , Ratones , Ratones Desnudos , Metástasis de la Neoplasia/genética , Neoplasias/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismoRESUMEN
Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer with poor prognosis and high rates of relapse. The lack of actionable targets for TNBC has contributed to the high mortality rates of this disease, and new candidate molecules for potential manipulation are urgently required. Here, we show that macrophage-stimulating protein (MSP) and its tyrosine kinase receptor, Recepteur d'origine nantais (RON), are potent drivers of cancer cell growth and tumor progression in a mouse model of TNBC driven by the loss of Trp53 and Brca1. After comparison of two genetically engineered mouse models of TNBC, we found that mammary tumors from K14-Cre;Brca1F/F ;Trp53F/F (KB1P) mice exhibit high endogenous levels of MSP and RON expression. We show that MSP stimulates serine/threonine kinase 1 and extracellular regulated MAPK activation as well as cancer cell growth in cell lines derived from the two mouse models, while genetic and pharmacological inhibition of RON prevents these effects. Similarly, KB1P tumor progression in mice was robustly attenuated by treatment with a RON inhibitor with accompanied reduction in the proliferation marker, Ki-67. Analysis of human gene expression data confirmed that the genes encoding MSP and RON are robustly expressed in human TNBC as well as other subsets of breast cancer. Our findings uncover a mouse model where MSP expression and RON expression are naturally increased, and they provide evidence that this receptor and its ligand are viable candidate molecules for targeted treatment of breast cancer.
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Factor de Crecimiento de Hepatocito/metabolismo , Modelos Biológicos , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Transducción de Señal , Neoplasias de la Mama Triple Negativas/patología , Animales , Línea Celular Tumoral , Proliferación Celular , Regulación Neoplásica de la Expresión Génica , Humanos , Sistema de Señalización de MAP Quinasas , Ratones , Proteínas Proto-Oncogénicas c-akt/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/metabolismoRESUMEN
Intratumoral hypoxia causes the formation of dysfunctional blood vessels, which contribute to tumor metastasis and reduce the efficacy of therapeutic treatments. Blood vessels are embedded in the tumor stroma of which cancer-associated fibroblasts (CAFs) constitute a prominent cellular component. We found that hypoxic human mammary CAFs promoted angiogenesis in CAF-endothelial cell cocultures in vitro. Mass spectrometry-based proteomic analysis of the CAF secretome unraveled that hypoxic CAFs contributed to blood vessel abnormalities by altering their secretion of various pro- and anti-angiogenic factors. Hypoxia induced pronounced remodeling of the CAF proteome, including proteins that have not been previously related to this process. Among those, the uncharacterized protein NCBP2-AS2 that we renamed HIAR (hypoxia-induced angiogenesis regulator) was the protein most increased in abundance in hypoxic CAFs. Silencing of HIAR abrogated the pro-angiogenic and pro-migratory function of hypoxic CAFs by decreasing secretion of the pro-angiogenic factor VEGFA and consequently reducing VEGF/VEGFR downstream signaling in the endothelial cells. Our study has identified a regulator of angiogenesis and provides a map of hypoxia-induced molecular alterations in mammary CAFs.
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Fibroblastos Asociados al Cáncer/metabolismo , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Neovascularización Patológica/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Células Cultivadas , Técnicas de Cocultivo , Regulación Neoplásica de la Expresión Génica , Células HEK293 , Humanos , Hipoxia , Neovascularización Patológica/genética , Proteoma/metabolismo , Proteómica/métodos , Transducción de Señal/genética , Factor A de Crecimiento Endotelial Vascular/genéticaRESUMEN
Apical-basal polarization is essential for epithelial tissue formation, segregating cortical domains to perform distinct physiological functions. Cortical lipid asymmetry has emerged as a determinant of cell polarization. We report a network of phosphatidylinositol phosphate (PIP)-modifying enzymes, some of which are transcriptionally induced upon embedding epithelial cells in extracellular matrix, and that are essential for apical-basal polarization. Unexpectedly, we find that PI(3,4)P2 localization and function is distinct from the basolateral determinant PI(3,4,5)P3. PI(3,4)P2 localizes to the apical surface, and Rab11a-positive apical recycling endosomes. PI(3,4)P2 is produced by the 5-phosphatase SHIP1 and Class-II PI3-Kinases to recruit the endocytic regulatory protein SNX9 to basolateral domains that are being remodeled into apical surfaces. Perturbing PI(3,4)P2 levels results in defective polarization through subcortical retention of apically destined vesicles at apical membrane initiation sites. We conclude that PI(3,4)P2 is a determinant of apical membrane identity.
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Fosfatidilinositoles/metabolismo , Animales , Perros , Endosomas/metabolismo , Membranas Intracelulares/metabolismo , Células de Riñón Canino Madin Darby , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatasas/metabolismoRESUMEN
Based on a molecular classification of prostate cancer using gene expression pathway signatures, we derived a set of 48 genes in critical pathways that significantly predicts clinical outcome in all tested patient cohorts. We tested these genes in a functional genomics screen in a panel of three prostate cancer cell lines (LNCaP, PC3, DU145), using RNA interference. The screen revealed several genes whose knockdown caused strong growth inhibition in all cell lines. Additionally, we tested the gene set in the presence of docetaxel to see whether any gene exhibited additive or synergistic effects with the drug. We observed a strong synergistic effect between DLGAP5 knockdown and docetaxel in the androgen-sensitive line LNCaP, but not in the two other androgen-independent lines. We then tested whether this effect was connected to androgen pathways and found that knockdown of the androgen receptor by si-RNA attenuated the synergy significantly. Similarly, androgen desensitized LNCaP-AI cells had a higher IC50 to docetaxel and did not exhibit the synergistic interaction. Short-term exposure to enzalutamide did not significantly alter the behaviour of parental LNCaP cells. An immunofluorescence analysis in LNCaP cells suggests that under the double insult of DLGAP5 knockdown and docetaxel, cells predominantly arrest in metaphase. In contrast, the knockdown of the androgen receptor by siRNA appears to assist cells to progress through metaphase in to anaphase, even in the presence of docetaxel. Our data suggest that DLGAP5 has a unique function in stabilizing spindle formation and surviving microtubule assault from docetaxel, in an androgen-regulated cell cycle system.
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Docetaxel/farmacología , Genómica/métodos , Proteínas de Neoplasias/genética , Neoplasias de la Próstata/genética , Receptores Androgénicos/metabolismo , Benzamidas , Proteínas Cdc20/genética , Proteínas de Ciclo Celular/genética , Proliferación Celular/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Humanos , Masculino , Metafase , Proteínas Asociadas a Microtúbulos/genética , Nitrilos , Células PC-3 , Feniltiohidantoína/análogos & derivados , Feniltiohidantoína/farmacología , Receptores Androgénicos/genética , TransfecciónRESUMEN
Actin-based protrusions are reinforced through positive feedback, but it is unclear what restricts their size, or limits positive signals when they retract or split. We identify an evolutionarily conserved regulator of actin-based protrusion: CYRI (CYFIP-related Rac interactor) also known as Fam49 (family of unknown function 49). CYRI binds activated Rac1 via a domain of unknown function (DUF1394) shared with CYFIP, defining DUF1394 as a Rac1-binding module. CYRI-depleted cells have broad lamellipodia enriched in Scar/WAVE, but reduced protrusion-retraction dynamics. Pseudopods induced by optogenetic Rac1 activation in CYRI-depleted cells are larger and longer lived. Conversely, CYRI overexpression suppresses recruitment of active Scar/WAVE to the cell edge, resulting in short-lived, unproductive protrusions. CYRI thus focuses protrusion signals and regulates pseudopod complexity by inhibiting Scar/WAVE-induced actin polymerization. It thus behaves like a 'local inhibitor' as predicted in widely accepted mathematical models, but not previously identified in cells. CYRI therefore regulates chemotaxis, cell migration and epithelial polarization by controlling the polarity and plasticity of protrusions.
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Movimiento Celular , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Seudópodos/metabolismo , Proteína de Unión al GTP rac1/metabolismo , Actinas/genética , Actinas/metabolismo , Animales , Células COS , Línea Celular Tumoral , Quimiotaxis/genética , Chlorocebus aethiops , Perros , Células HEK293 , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Células de Riñón Canino Madin Darby , Polimerizacion , Unión Proteica , Seudópodos/genética , Transducción de Señal/genética , Proteína de Unión al GTP rac1/genéticaRESUMEN
Fibroblast growth factor receptors (FGFRs) are a family of receptor tyrosine kinases that control a diverse range of biological processes during development and in adult tissues. We recently reported that somatic FGFR2 mutations are associated with shorter survival in endometrial cancer. However, little is known about how these FGFR2 mutations contribute to endometrial cancer metastasis. Here, we report that expression of the activating mutations FGFR2N550K and FGFR2Y376C in an endometrial cancer cell model induce Golgi fragmentation, and loss of polarity and directional migration. In mutant FGFR2-expressing cells, this was associated with an inability to polarise intracellular pools of FGFR2 towards the front of migrating cells. Such polarization defects were exacerbated in three-dimensional culture, where FGFR2 mutant cells were unable to form well-organised acini, instead undergoing exogenous ligand-independent invasion. Our findings uncover collective cell polarity and invasion as common targets of disease-associated FGFR2 mutations that lead to poor outcome in endometrial cancer patients.
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Movimiento Celular/fisiología , Polaridad Celular/fisiología , Neoplasias Endometriales/metabolismo , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/metabolismo , Biotinilación , Línea Celular Tumoral , Movimiento Celular/genética , Polaridad Celular/genética , Proliferación Celular/genética , Proliferación Celular/fisiología , Quimiotaxis/genética , Quimiotaxis/fisiología , Neoplasias Endometriales/genética , Femenino , Técnica del Anticuerpo Fluorescente , Células HEK293 , Humanos , Immunoblotting , Lentivirus/genética , Mutación/genética , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/genéticaRESUMEN
In many types of tubules, continuity of the lumen is paramount to tubular function, yet how tubules generate lumen continuity in vivo is not known. We recently found that the F-actin-binding protein afadin is required for lumen continuity in developing renal tubules, though its mechanism of action remains unknown. Here, we demonstrate that afadin is required for lumen continuity by orienting the mitotic spindle during cell division. Using an in vitro 3D cyst model, we find that afadin localizes to the cell cortex adjacent to the spindle poles and orients the mitotic spindle. In tubules, cell division may be oriented relative to two axes: longitudinal and apical-basal. Unexpectedly, in vivo examination of early-stage developing nephron tubules reveals that cell division is not oriented in the longitudinal (or planar-polarized) axis. However, cell division is oriented perpendicular to the apical-basal axis. Absence of afadin in vivo leads to misorientation of apical-basal cell division in nephron tubules. Together, these results support a model whereby afadin determines lumen placement by directing apical-basal spindle orientation, resulting in a continuous lumen and normal tubule morphogenesis.