Sven Bogdan

Drosophila Cip4 and WASp Define a Branch of the Cdc42-Par6-aPKC Pathway Regulating E-Cadherin Endocytosis

BACKGROUND Integral to the function and morphology of the epithelium is the lattice of cell-cell junctions known as adherens junctions (AJs). AJ stability and plasticity relies on E-Cadherin exocytosis and endocytosis. A mechanism regulating E-Cadherin (E-Cad) exocytosis to the AJs has implicated proteins of the exocyst complex, but mechanisms regulating E-Cad endocytosis from the AJs remain less well understood. RESULTS Here we show that Cdc42, Par6, or aPKC loss of function is accompanied by the accumulation of apical E-Cad intracellular punctate structures and the disruption of AJs in Drosophila epithelial cells. These punctate structures derive from large and malformed endocytic vesicles that emanate from the AJs; a phenotype that is also observed upon blocking vesicle scission in dynamin mutant cells. We demonstrate that the Drosophila Cdc42-interacting protein 4 (Cip4) is a Cdc42 effector that interacts with Dynamin and the Arp2/3 activator WASp in Drosophila. Accordingly, Cip4, WASp, or Arp2/3 loss of function also results in defective E-Cadherin endocytosis. CONCLUSION Altogether our results show that Cdc42 functions with Par6 and aPKC to regulate E-Cad endocytosis and define Cip4 and WASp as regulators of the early E-Cad endocytic events in epithelial tissue.

Epidermal growth factor receptor signaling.

Inhibitory signaling is an important way of fine tuning EGFR activity to enable a cell to discriminate between short and prolonged exposure to signaling molecules. Over the last few years a number of different mechanisms have been shown to abrogate receptor activity (Fig. 3Fig. 3). First, inhibitory signaling molecules are expressed in response to EGFR activation. The most prominent example is encoded by the Drosophila gene argos. Argos is a secreted molecule that has a single EGF-like domain with an expanded B-loop. Binding experiments showed that Argos binds directly to the EGFR presumably via its EGF-like domain and inhibits EGFR activity. Argos prevents Spitz binding and may also interfere with ligand-independent activation of the EGFR by preventing the formation of active EGFR dimers.Fig. 3Negative regulators of EGFR signaling. Several proteins were found to attenuate EGFR signaling. Argos, Decorin, and Kekkon bind to the extracellular domain of the EGFR. PKC is able to phosphorylate the EGFR at T654. Binding of Cbl at Y1045 results in ubiquitination and subsequent degradation. The recently identified Echinoid protein interferes with EGFR signaling downstream. LRR, leucine-rich repeat; Ig, immunoglobulin-like domain; FnIII, fibronectin type III repeat.View Large Image | View Hi-Res Image | Download PowerPoint SlideBesides Argos a number of other molecules attenuate EGFR activity. Again Drosophila has been a powerful system for identifying new components which can inhibit EGFR. Kekkon, which is expressed in response to EGFR activity, encodes a type I transmembrane protein and resembles cell adhesion molecules. The extracellular domain of Kekkon binds to the EGFR and provides a mechanism for negative-feedback. Two additional Kekkon-like proteins are encoded by the Drosophila genome which await characterisation.The Echinoid protein shows identity to the L1 adhesion protein and defines an additional pathway for antagonizing EGFR activity downstream of the receptor. EGFR activity can also be influenced by components of the extracellular matrix. One example is the leucine-rich proteoglycan Decorin that can bind EGFR and attenuate EGFR phosphorylation.sprouty and the cbl are genes which encode global inhibitors of EGFR signaling. Sprouty contains a conserved cysteine-rich domain and associates with components of the EGFR signaling cascade such as Grb2/Drk and GAP1. The function of Sprouty is, however, not restricted to the EGFR pathway. Cbl contains an unusual SH2 domain that is responsible for EGFR binding via Y1045. This interaction leads to the phosphorylation of Cbl, which may facilitate the ubiquitin ligase activity of the Cbl RING finger domain. Ubiquitination of the EGFR leads to its internalization via clathrin-coated vesicles and subsequent degradation.In summary, EGFR signaling appears to be integrated in a complex regulatory network linking extracellular signals to the cytoplasm and the nucleus. In the last decade the modulation of EGFR activity by negative regulators has become more and more evident. Now the picture is getting even more complicated by the fact that differential trafficking of activated EGFR may lead to different cellular responses. Further studies may not only help to increase our understanding of the role of EGFR signaling in the developing animal, they may ultimately lead to the development of efficient anti-cancer therapeutics.

The WAVE Regulatory Complex Links Diverse Receptors to the Actin Cytoskeleton

The WAVE regulatory complex (WRC) controls actin cytoskeletal dynamics throughout the cell by stimulating the actin-nucleating activity of the Arp2/3 complex at distinct membrane sites. However, the factors that recruit the WRC to specific locations remain poorly understood. Here, we have identified a large family of potential WRC ligands, consisting of ∼120 diverse membrane proteins, including protocadherins, ROBOs, netrin receptors, neuroligins, GPCRs, and channels. Structural, biochemical, and cellular studies reveal that a sequence motif that defines these ligands binds to a highly conserved interaction surface of the WRC formed by the Sra and Abi subunits. Mutating this binding surface in flies resulted in defects in actin cytoskeletal organization and egg morphology during oogenesis, leading to female sterility. Our findings directly link diverse membrane proteins to the WRC and actin cytoskeleton and have broad physiological and pathological ramifications in metazoans.

Drosophila Cip4/Toca-1 integrates membrane trafficking and actin dynamics through WASP and SCAR/WAVE.

BACKGROUND Developmental processes are intimately tied to signaling events that integrate the dynamic reorganization of the actin cytoskeleton and membrane dynamics. The F-BAR-domain-containing proteins are prime candidates to couple actin dynamics and membrane trafficking in different morphogenetic processes. RESULTS Here, we present the functional analysis of the Drosophila F-BAR protein Cip4/Toca1 (Cdc42-interacting protein 4/transducer of Cdc42-dependent actin assembly 1). Cip4 is able to form a complex with WASP and SCAR/WAVE and recruits both actin-nucleation-promoting factors to invaginating membranes and endocytic vesicles. Actin-comet-tail-based movement of these vesicles depends not only on WASP but largely on WAVE function. In vivo, loss of cip4 function causes multiple wing hairs. A similar phenotype is observed when vesicle scission is affected after Dynamin suppression. Gene dosage experiments show that Cip4 and WAVE functionally interact to restrict wing hair formation. Further rescue experiments confirm that Cip4 is able to act through WAVE and WASP in vivo. Biochemical and functional data support a model in which Cdc42 acts upstream of Cip4 and recruits not only WASP but also SCAR/WAVE via Abi to control Dynamin-dependent cell polarization in the wing. CONCLUSION Cip4 integrates membrane trafficking and actin dynamics through WASP and WAVE. First, Cip4 promotes membrane invaginations and triggers the vesicle scission by recruiting Dynamin to the neck of nascent vesicles. Second, Cip4 recruits WASP and WAVE proteins to induce actin polymerization, supporting vesicle scission and providing the force for vesicle movement.

Kette regulates actin dynamics and genetically interacts with Wave and Wasp

During development of the Drosophila nervous system, kette is required for axonal growth and pathfinding. It encodes a highly conserved homolog of the Nck-associated protein 1 (NAP1) that genetically interacts with the Drosophila homolog of Nck, dock. We show that in vivo as well as in tissue culture models most of the Kette protein is found in the cytoplasm where it colocalizes with F-actin to which it can bind via its N-terminal domain. Some Kette protein is localized at the membrane and accumulates at focal contact sites. Loss of Kette protein results in the accumulation of cytosolic F-actin. The kette mutant phenotype can be suppressed by reducing the wave gene dose, demonstrating that kette antagonizes wave function. Overexpression of the wild-type Kette protein does not interfere with normal development, whereas expression of an activated, membrane-tethered Kette protein induces the formation of large F-actin bundles in both, tissue culture cells and in vivo. This gain-of-function phenotype is independent of wave but can be suppressed by reducing the wasp gene dose, indicating that Kette is able to regulate Wasp, to which it is linked via the Abelson interactor (Abi). Our data suggest a model where Kette fulfils a novel role in regulating F-actin organization by antagonizing Wave and activating Wasp-dependent actin polymerization.

kette and blown fuse interact genetically during the second fusion step of myogenesis in Drosophila.

Drosophila myoblast fusion proceeds in two steps. The first one gives rise to small syncytia, the muscle precursor cells, which then recruit further fusion competent myoblasts to reach the final muscle size. We have identified Kette as an essential component for myoblast fusion. In kette mutants, founder cells and fusion-competent myoblasts are determined correctly and overcome the very first fusion. But then, at the precursor cell stage, fusion is interrupted. At the ultrastructural level, fusion is characterised by cell-cell recognition, alignment, formation of prefusion complexes, electron dense plaques and membrane breakdown. In kette mutants, electron dense plaques of aberrant length accumulate and fusion is interrupted owing to a complete failure of membrane breakdown. Furthermore, we show that kette interacts genetically with blown fuse (blow) which is known to be required to proceed from prefusion complexes to the formation of the electron dense plaques. Interestingly, a surplus of Kette can replace Blow function during myogenesis. We propose a model in which Dumbfounded/Sticks and stones-dependent cell adhesion is mediated over Rolling Pebbles, Myoblast city, Crk, Blown fuse and Kette, and thus induces membrane fusion.

Abi activates WASP to promote sensory organ development

Actin polymerization is a key process for many cellular events during development. To a large extent, the formation of filamentous actin is controlled by the WASP and WAVE proteins that activate the Arp2/3 complex in different developmental processes. WAVE function is regulated through a protein complex containing Sra1, Kette and Abi. Using biochemical, cell biological and genetic tools, we show here that the Abi protein also has a central role in activating WASP-mediated processes. Abi binds WASP through its carboxy-terminal domain and acts as a potent stimulator of WASP-dependent F-actin formation. To elucidate the biological function of abi in Drosophila melanogaster, we studied bristle development, a process known to require wasp function. Reduction of abi function leads to a loss of bristles similar to that observed in wasp mutants. Activation of Abi results in the formation of ectopic bristles, a phenotype that is suppressed by a reduction of wasp activity but is not affected by the reduction of wave function. Thus, in vivo Abi may set the balance between WASP and WAVE in different actin-based developmental processes.

Sra-1 interacts with Kette and Wasp and is required for neuronal and bristle development in Drosophila.

Regulation of growth cone and cell motility involves the coordinated control of F-actin dynamics. An important regulator of F-actin formation is the Arp2/3 complex, which in turn is activated by Wasp and Wave. A complex comprising Kette/Nap1, Sra-1/Pir121/CYFIP, Abi and HSPC300 modulates the activity of Wave and Wasp. We present the characterization of Drosophila Sra-1 (specifically Rac1-associated protein 1). sra-1 and kette are spatially and temporally co-expressed, and both encoded proteins interact in vivo. During late embryonic and larval development, the Sra-1 protein is found in the neuropile. Outgrowing photoreceptor neurons express high levels of Sra-1 also in growth cones. Expression of double stranded sra-1 RNA in photoreceptor neurons leads to a stalling of axonal growth. Following knockdown of sra-1 function in motoneurons, we noted abnormal neuromuscular junctions similar to what we determined for hypomorphic kette mutations. Similar mutant phenotypes were induced after expression of membrane-bound Sra-1 that lacks the Kette-binding domain, suggesting that sra-1 function is mediated through kette. Furthermore, we could show that both proteins stabilize each other and directly control the regulation of the F-actin cytoskeleton in a Wasp-dependent manner.

Syndapin Promotes Formation of a Postsynaptic Membrane System in Drosophila

Syndapins belong to the F-BAR domain protein family whose predicted functions in membrane tubulation remain poorly studied in vivo. At Drosophila neuromuscular junctions, syndapin is associated predominantly with a tubulolamellar postsynaptic membrane system known as the subsynaptic reticulum (SSR). We show that syndapin overexpression greatly expands this postsynaptic membrane system. Syndapin can expand the SSR in the absence of dPAK and Dlg, two known regulators of SSR development. Syndapins N-terminal F-BAR domain, required for membrane tubulation in cultured cells, is required for SSR expansion. Consistent with a model in which syndapin acts directly on postsynaptic membrane, SSR expansion requires conserved residues essential for membrane binding in vitro. However, syndapins Src homology (SH) 3 domain, which negatively regulates membrane tubulation in cultured cells, is required for synaptic targeting and strong SSR induction. Our observations advance knowledge of syndapin protein function by 1) demonstrating the in vivo relevance of membrane remodeling mechanisms suggested by previous in vitro and structural analyses, 2) showing that SH3 domains are necessary for membrane expansion observed in vivo, and 3) confirming that F-BAR proteins control complex membrane structures.

The F-BAR protein Cip4/Toca-1 antagonizes the formin Diaphanous in membrane stabilization and compartmentalization.

Summary During Drosophila embryogenesis, the first epithelium with defined cortical compartments is established during cellularization. Actin polymerization is required for the separation of lateral and basal domains as well as suppression of tubular extensions in the basal domain. The actin nucleator mediating this function is unknown. We found that the formin Diaphanous (Dia) is required for establishing and maintaining distinct lateral and basal domains during cellularization. In dia mutant embryos lateral marker proteins, such as Discs-large and Armadillo/&bgr;-Catenin spread into the basal compartment. Furthermore, high-resolution and live-imaging analysis of dia mutant embryos revealed an increased number of membrane extensions and endocytic activity at the basal domain, indicating a suppressing function of dia on membrane invaginations. Dia function might be based on an antagonistic interaction with the F-BAR protein Cip4/Toca-1, a known activator of the WASP/WAVE-Arp2/3 pathway. Dia and Cip4 physically and functionally interact and overexpression of Cip4 phenocopies dia loss-of-function. In vitro, Cip4 inhibits mainly actin nucleation by Dia. Thus, our data support a model in which linear actin filaments induced by Dia stabilize cortical compartmentalization by antagonizing membrane turnover induced by WASP/WAVE-Arp2/3.