Inmaculada Bañón-Rodríguez

Synaptotagmin-like proteins control the formation of a single apical membrane domain in epithelial cells

The formation of epithelial tissues requires both the generation of apical–basal polarity and the coordination of this polarity between neighbouring cells to form a central lumen. During de novo lumen formation, vectorial membrane transport contributes to the formation of a singular apical membrane, resulting in the contribution of each cell to only a single lumen. Here, from a functional screen for genes required for three-dimensional epithelial architecture, we identify key roles for synaptotagmin-like proteins 2-a and 4-a (Slp2-a/4-a) in the generation of a single apical surface per cell. Slp2-a localizes to the luminal membrane in a PtdIns(4,5)P2-dependent manner, where it targets Rab27-loaded vesicles to initiate a single lumen. Vesicle tethering and fusion is controlled by Slp4-a, in conjunction with Rab27/Rab3/Rab8 and the SNARE syntaxin-3. Together, Slp2-a/4-a coordinate the spatiotemporal organization of vectorial apical transport to ensure that only a single apical surface, and thus the formation of a single lumen, occurs per cell.

Role of WASP in cell polarity and podosome dynamics of myeloid cells

The integrin-dependent migration of myeloid cells requires tight coordination between actin-based cell membrane protrusion and integrin-mediated adhesion to form a stable leading edge. Under this mode of migration, polarised myeloid cells including dendritic cells, macrophages and osteoclasts develop podosomes that sustain the extending leading edge. Podosome integrity and dynamics vary in response to changes in the physical and biochemical properties of the cell environment. In the current article we discuss the role of various factors in initiation and stability of podosomes and the roles of the Wiskott Aldrich Syndrome Protein (WASP) in this process. We discuss recent data indicating that in a cellular context WASP is crucial not only for localised actin polymerisation at the leading edge and in podosome cores but also for coordination of integrin clustering and activation during podosome formation and disassembly.

The cortactin-binding domain of WIP is essential for podosome formation and extracellular matrix degradation by murine dendritic cells

In immature dendritic cells (DCs) podosomes form and turn over behind the leading edge of migrating cells. The Arp2/3 complex activator Wiskott-Aldrich Syndrome Protein (WASP) localises to the actin core of forming podosomes together with WASP-Interacting Protein (WIP). A second weaker Arp2/3 activator, cortactin, is also found at podosomes where it has been proposed to participate in matrix metalloproteinase (MMP) secretion. We have previously shown that WIP(-/-) DCs are unable to make podosomes. WIP binds to cortactin and in this report we address whether WIP regulates cortactin-mediated MMP activity. Using DCs derived from splenic murine precursors, we found that wild-type cells were able to localise MMPs at podosomes where matrix degradation takes place. In contrast, WIP(-/-) DCs remain able to synthesise MMPs but do not degrade the extracellular matrix. Infection of WIP KO DCs with lentivirus expressing WIP restored both podosome formation and their ability to degrade the extracellular matrix, implicating WIP-induced podosomes as foci of functional MMP location. When WIP KO DCs were infected with a mutant form of WIP lacking the cortactin-binding domain (WIPΔ110-170) DCs were only able to elaborate disorganised podosomes that were unable to support MMP-mediated matrix degradation. Taken together, these results suggest a role for WIP not only in WASP-mediated actin polymerisation and podosome formation, but also in cortactin-mediated extracellular matrix degradation by MMPs.

EGFR controls IQGAP basolateral membrane localization and mitotic spindle orientation during epithelial morphogenesis

Establishing the correct orientation of the mitotic spindle is an essential step in epithelial cell division in order to ensure that epithelial tubules form correctly during organ development and regeneration. While recent findings have identified some of the molecular mechanisms that underlie spindle orientation, many aspects of this process remain poorly understood. Here, we have used the 3D‐MDCK model system to demonstrate a key role for a newly identified protein complex formed by IQGAP1 and the epithelial growth factor receptor (EGFR) in controlling the orientation of the mitotic spindle. IQGAP1 is a scaffolding protein that regulates many cellular pathways, from cell‐cell adhesion to microtubule organization, and its localization in the basolateral membrane ensures correct spindle orientation. Through its IQ motifs, IQGAP1 binds to EGFR, which is responsible for maintaining IQGAP1 in the basolateral membrane domain. Silencing IQGAP1, or disrupting the basolateral localization of either IQGAP1 or EGFR, results in a non‐polarized distribution of NuMA, mitotic spindle misorientation and defects in single lumen formation.

Semaphorin-Plexin Signaling Controls Mitotic Spindle Orientation during Epithelial Morphogenesis and Repair

Morphogenesis, homeostasis, and regeneration of epithelial tissues rely on the accurate orientation of cell divisions, which is specified by the mitotic spindle axis. To remain in the epithelial plane, symmetrically dividing epithelial cells align their mitotic spindle axis with the plane. Here, we show that this alignment depends on epithelial cell-cell communication via semaphorin-plexin signaling. During kidney morphogenesis and repair, renal tubular epithelial cells lacking the transmembrane receptor Plexin-B2 or its semaphorin ligands fail to correctly orient the mitotic spindle, leading to severe defects in epithelial architecture and function. Analyses of a series of transgenic and knockout mice indicate that Plexin-B2 controls the cell division axis by signaling through its GTPase-activating protein (GAP) domain and Cdc42. Our data uncover semaphorin-plexin signaling as a central regulatory mechanism of mitotic spindle orientation necessary for the alignment of epithelial cell divisions with the epithelial plane.

WIP regulates persistence of cell migration and ruffle formation in both mesenchymal and amoeboid modes of motility.

The spatial distribution of signals downstream from receptor tyrosine kinases (RTKs) or G-protein coupled receptors (GPCR) regulates fundamental cellular processes that control cell migration and growth. Both pathways rely significantly on actin cytoskeleton reorganization mediated by nucleation-promoting factors such as the WASP-(Wiskott-Aldrich Syndrome Protein) family. WIP (WASP Interacting Protein) is essential for the formation of a class of polarised actin microdomain, namely dorsal ruffles, downstream of the RTK for PDGF (platelet-derived growth factor) but the underlying mechanism is poorly understood. Using lentivirally-reconstituted WIP-deficient murine fibroblasts we define the requirement for WIP interaction with N-WASP (neural WASP) and Nck for efficient dorsal ruffle formation and of WIP-Nck binding for fibroblast chemotaxis towards PDGF-AA. The formation of both circular dorsal ruffles in PDGF-AA-stimulated primary fibroblasts and lamellipodia in CXCL13-treated B lymphocytes are also compromised by WIP-deficiency. We provide data to show that a WIP-Nck signalling complex interacts with RTK to promote polarised actin remodelling in fibroblasts and provide the first evidence for WIP involvement in the control of migratory persistence in both mesenchymal (fibroblast) and amoeboid (B lymphocytes) motility.

WIP Is a Negative Regulator of Neuronal Maturation and Synaptic Activity

Wiskott-Aldrich syndrome protein (WASP) -interacting protein (WIP) is an actin-binding protein involved in the regulation of actin polymerization in cells, such as fibroblasts and lymphocytes. Despite its recognized function in non-neuronal cells, the role of WIP in the central nervous system has not been examined previously. We used WIP-deficient mice to examine WIP function both in vivo and in vitro. We report here that WIP(-)(/-) hippocampal neurons exhibit enlargement of somas as well as overgrowth of neuritic and dendritic branches that are more evident in early developmental stages. Dendritic arborization and synaptogenesis, which includes generation of postsynaptic dendritic spines, are actin-dependent processes that occur in parallel at later stages. WIP deficiency also increases the amplitude and frequency of miniature excitatory postsynaptic currents, suggesting that WIP(-)(/-) neurons have more mature synapses than wild-type neurons. These findings reveal WIP as a previously unreported regulator of neuronal maturation and synaptic activity.

WIP modulates dendritic spine actin cytoskeleton by transcriptional control of lipid metabolic enzymes

We identify Wiskott-Aldrich syndrome protein (WASP)-interacting protein (WIP) as a novel component of neuronal synapses whose absence increases dendritic spine size and filamentous actin levels in an N-WASP/Arp2/3-independent, RhoA/ROCK/profilinIIa-dependent manner. These effects depend on the reduction of membrane sphingomyelin (SM) due to transcriptional upregulation of neutral sphingomyelinase (NSM) through active RhoA; this enhances RhoA binding to the membrane, raft partitioning and activation in steady state but prevents RhoA changes in response to stimulus. Inhibition of NSM or SM addition reverses RhoA, filamentous actin and functional anomalies in synapses lacking WIP. Our findings characterize WIP as a link between membrane lipid composition and actin cytoskeleton at dendritic spines. They also contribute to explain cognitive deficits shared by individuals bearing mutations in the region assigned to the gene encoding for WIP.

Integrin linked kinase (ILK) regulates podosome maturation and stability in dendritic cells

Podosomes are integrin-based adhesions fundamental for stabilisation of the leading lamellae in migrating dendritic cells (DCs) and for extracellular matrix (ECM) degradation. We have previously shown that soluble factors and chemokines such as SDF 1-a trigger podosome initiation whereas integrin ligands promote podosome maturation and stability in DCs. The exact intracellular signalling pathways that regulate the sequential organisation of podosomal components in response to extracellular cues remain largely undetermined. The Wiskott Aldrich Syndrome Protein (WASP) mediates actin polymerisation and the initial recruitment of integrins and associated proteins in a circular configuration surrounding the core of filamentous actin (F-actin) during podosome initiation. We have now identified integrin linked kinase (ILK) surrounding the podosomal actin core. We report that DC polarisation in response to chemokines and the assembly of actin cores during podosome initiation require PI3K-dependent clustering of the Wiskott Aldrich Syndrome Protein (WASP) in puncta independently of ILK. ILK is essential for the clustering of integrins and associated proteins leading to podosome maturation and stability that are required for degradation of the subjacent extracellular matrix and the invasive motility of DCs across connective tissue barriers. We conclude that WASP regulates DCs polarisation for migration and initiation of actin polymerisation downstream of PI3K in nascent podosomes. Subsequently, ILK mediates the accumulation of integrin-associated proteins during podosome maturation and stability for efficient degradation of the subjacent ECM during the invasive migration of DCs.

WIP, What an Interesting Protein for leukocyte activation, migration and inflammation

Resumen La inflamacion es un proceso fisiologico dirigido a prevenir o reparar los danos producidos por agresiones externas, infecciones o intoxicacion. Este proceso depende de la coordinacion entre la activacion y migracion de los leucocitos hacia la region danada y por tanto esta asociado con cambios en la morfologia celular y con la reorganizacion del citoesqueleto de actina. El citoesqueleto de actina tambien contribuye a otros cambios morfologicos esenciales necesarios para la vida de la celula. WIP (WASP Interacting Protein) es una proteina que se une a la actina y participa en procesos inflamatorios. Dicha union estabiliza los microfilamentos de actina y regula su organizacion especial y temporal. La ausencia de WIP induce la formacion de redes de actina deficientes en linfocitos T y B, lo que conduce a la modificacion de la capacidad linfocitaria de activacion y migracion y desemboca en alteraciones autoinmunes con infiltracion leucocitaria en organos vitales y muerte del animal.