To explore functional association of D-WIP with the myoblast-fusion machinery, attempts were made to determine its subcellular localization. Visualization of myoblast fusion-site components in wild-type embryos is difficult, due to the dynamic nature of the fusion process. A common solution is to examine localization in myoblast-fusion mutants, in which embryonic FCMs remain attached to precursor myotubes for an extended period of time. Therefore kette mutant embryos were examined; accumulation of Duf protein along the myotube surface next to FCM attachment sites was readily observed. D-WIP is similarly enriched at the attachment sites, but it is present on both sides of the myotube-FCM interface, an observation highlighted by the restriction of D-WIP to the FCM face of attachment sites in mbc mutant embryos (Massarwa, 2007).
A cell-culture model of myoblast attachment, in which S2 cells separately transfected with duf or sns are mixed and allowed to aggregate, was examined. Colocalization of Duf and SNS at points of cell-cell contact is readily observed, mimicking in vivo myoblast attachment. In cells in which D-WIP was cotransfected with sns prior to aggregation with duf-expressing cells, D-WIP was found to colocalize with SNS at the cell attachment sites. D-WIP similarly colocalizes with Duf at cell attachment sites when these proteins are coexpressed in S2 cells, followed by aggregation with sns-expressing cells. D-WIP appears to localize more avidly to the attachment sites when transfected into sns-expressing cells. In addition to the localization experiments, it was possible to demonstrate coimmunoprecipitation of D-WIP and Duf when coexpressed in S2 cells. In summary, this series of in vivo, cell-culture, and biochemical approaches strongly supports an association between D-WIP and the Duf/SNS cell-surface adhesion molecules, on both aspects of myoblast attachment sites (Massarwa, 2007).
An established function for Vrp/WIP proteins is to localize WASp to cortical sites at which Arp2/3 activity is required (Moreau, 2000; Sasahara, 2002). Attempts were made to determine whether a similar scenario operates during myoblast fusion. To visualize Wsp in these experiments, use was made of a Wsp-GFP fusion protein that is fully functional, as determined by in vivo rescue experiments. When expressed separately in S2 cells, Wsp-GFP displays a punctate, cytoplasmic distribution, while D-WIP localizes just beneath the surface of these cells. Upon coexpression, however, Wsp-GFP colocalizes with D-WIP and acquires its subcortical pattern. D-WIPΔC, which lacks the putative Wsp-binding domain, localizes to the cell cortex, but fails to recruit Wsp-GFP, which remains cytoplasmic. These results imply that D-WIP associates with Wsp through the conserved C-terminal domain and is capable of altering Wsp subcellular localization (Massarwa, 2007).
Duf/SNS-based S2 cell aggregation was used to test if D-WIP recruits Wsp to sites of myoblast attachment. Coexpression of Wsp-GFP and D-WIP together with duf in S2 cells, followed by aggregation with sns-expressing cells, results in the joint recruitment of Wsp-GFP and D-WIP to the Duf-SNS attachment sites. In a converse experiment, Wsp-GFP was expressed together with D-WIP and sns prior to aggregation with duf-expressing cells. Again, both Wsp-GFP and D-WIP are strongly recruited to the Duf-SNS attachment sites. In the absence of D-WIP, however, Wsp-GFP maintains its cytoplasmic distribution in either duf- or sns-expressing cells (Massarwa, 2007).
Is Wsp similarly recruited in vivo to sites of myoblast fusion? Toward this end, UAS-Wsp-GFP was expressed in kette mutant embryos with the twi-GAL4 driver. Wsp-GFP was found to localize with D-WIP to both aspects of myotube-FCM attachment sites in these embryos. In contrast, Wsp-GFP assumes a cytoplasmic distribution in both the myotubes and FCMs of D-WIPD30 mutant embryos (Massarwa, 2007).
If the primary role of D-WIP is to recruit Wsp to sites of myoblast fusion, it may be possible to bypass the requirement for D-WIP by localizing Wsp to the myoblast cell surface via alternative means. Indeed, expression of UAS-Wspmyr, which encodes a myristoylated, membrane-tethered form of Wsp in D-WIPD30 mutant embryos, resulted in substantial rescue of the D-WIPD30-fusion phenotype. Expression of the UAS-Wsp full-length construct in D-WIPD30 mutant embryos with the same driver has no rescuing effect, underscoring the significance of Wsp membrane localization to the fusion process. Thus, Wsp localization via D-WIP to the myotube-FCM attachment site is an essential feature of myoblast fusion (Massarwa, 2007).
Since WASp-family proteins commonly require activation by signaling molecules, the ability of Wsp variants lacking different effector-binding domains to rescue the myoblast-fusion defects in Wspmat/zyg embryos was examined. This analysis suggested an essential role for the N-terminal WH1/EVH1 domain, which includes the binding site for members of the evolutionarily conserved Vrp/WIP family of actin-binding proteins (Ramesh, 1997; Aspenstrom, 2005). A single Vrp/WIP homolog, which is referred here to as D-WIP, is encoded in the Drosophila genome by the previously uncharacterized gene CG13503. D-WIP displays all of the structural hallmarks of Vrp/WIP homologs, including a pair of N-terminal WH2 actin-binding domains and a signature WASp-binding domain at the extreme C terminus (Massarwa, 2007).
The expression pattern of D-WIP lends support to the notion that D-WIP mediates Wsp function in the embryonic musculature. D-WIP mRNA is not detected in early embryos, implying an absence of a maternal contribution, while zygotic expression is first observed at stage 11 in muscle precursor cells. Muscle-specific expression of D-WIP peaks at stage 14, corresponding to the height of myoblast fusion. D-WIP expression levels decrease and disappear as muscles differentiate during later stages of embryogenesis (Massarwa, 2007).
To ascertain the identity of D-WIP-expressing myoblasts, the expression of D-WIP was examined in lameduck (lmd) mutant embryos. lmd encodes a transcription factor that is expressed only in FCMs and acts as a cardinal regulator of FCM-specific genes. D-WIP mRNA cannot be detected in lmd mutant embryos, implying that D-WIP expression is restricted to FCMs (Massarwa, 2007).
The myoblast-subtype expression pattern of D-WIP was confirmed by using polyclonal rat antisera to the D-WIP protein. At stage 12 of embryonic development, prior to the onset of the myoblast-fusion process, D-WIP protein is distributed exclusively within the cytoplasm and subcortical regions of FCMs and is markedly absent from founder cells. At more advanced stages of myogenesis, however, D-WIP can be detected within growing syncytial myotubes. D-WIP protein, produced in FCMs, is therefore incorporated into maturing muscles after fusion of FCMs with founder cells and myotubes (Massarwa, 2007).
Muscle development was followed in Wspmat/zyg embryos, which completely lack Wsp function. Visualization of mature muscle markers, such as Myosin Heavy Chain (MHC), revealed that the muscle pattern in these mutant embryos is severely disrupted. A prominent feature of the Wspmat/zyg mutant phenotype is groups of mononucleated myoblasts clustered around thin, abnormally elongated fibers. This phenotype is highly characteristic of embryonic muscle-fusion mutants, suggesting that Wsp is required for myoblast fusion during embryonic myogenesis (Massarwa, 2007).
In order to quantitate the requirement for Wsp during myoblast fusion, Wspmat/zyg embryos were stained for Even-skipped (Eve), which accumulates specifically in the nuclei of the large DA1 muscle that forms on the dorsal aspect of all embryonic trunk segments. Wild-type DA1 muscles contain 9-11 Eve-expressing nuclei, while mutants in which the fusion process is blocked express Eve in a smaller number of nuclei, corresponding to the number of fusion events that occurred. The number of Eve-expressing DA1 nuclei thus serves as an established, sensitive assay for the degree of myoblast fusion. Wspmat/zyg embryos display 2–3 DA1 nuclei per segment, implying that fusion is arrested after a single round of founder cell-FCM fusion, generating a bi-/trinucleated myotube precursor (Massarwa, 2007).
To ascertain that the involvement of Wsp in muscle formation is carried out via the Arp2/3 complex, the mesodermal/muscle-specific driver twist-GAL4 (twi-GAL4) was used to express WspΔCA, encoding a Wsp variant lacking the extreme C-terminal Arp2/3-binding sequence, in wild-type embryos. A strong myoblast-fusion phenotype was observed, similar in severity to that observed in Wspmat/zyg embryos. In contrast, overexpression of full-length Wsp produces no deleterious effects, underscoring the functional significance of Wsp association with Arp2/3 via the CA domain. In addition, it was observed that embryos homozygous for ArpC1Q25st, a strong mutant allele of the ArpC1 subunit, commonly display unfused myoblasts, further implying a requirement for Arp2/3 activity during the fusion process. The relatively mild phenotype of ArpC1Q25st embryos is likely the result of maternal contribution of Arp2/3 gene products, which is essential for completion of oogenesis, and thus cannot be fully removed. These observations strongly imply that Wsp function during embryonic myoblast fusion involves an essential association with the Arp2/3-based actin-polymerization machinery (Massarwa, 2007).
The D-WIP locus was disrupted in order to study the functional requirements for D-WIP. Excision of EY02177, a P element inserted in the first intron of D-WIP, resulted in isolation of D-WIPD30, a small chromosomal deletion uncovering the D-WIP gene locus, as well as five additional proximal transcription units. Immunostaining of D-WIPD30 embryos with anti-MHC revealed dramatic disruption of the somatic muscle pattern. As in Wsp mutants, many individual, unfused myoblasts, which cluster next to mispositioned muscle fibers displaying a thin, abnormal morphology, are detected. Two lines of evidence verify that the severe myoblast-fusion phenotype results specifically from disruption of D-WIP. Incorporating Cos1-5, a cosmid-based insertion into the D-WIPD30 mutant background, restores all of the deleted genomic sequences apart from the D-WIP locus, but embryos of this genotype continue to exhibit severe myoblast-fusion abnormalities. A complementary approach employed expression of a UAS-D-WIP transgene in D-WIPD30 embryos by using the mesodermal twi-GAL4 driver, which resulted in complete phenotypic rescue (Massarwa, 2007).
FCM clustering near myotubes and formation of myotube precursors in D-WIPD30 mutant embryos indicate proper cell-surface localization and function of the molecular machinery governing recognition and adhesion between founder cells and FCMs. This conclusion is further supported by the localization of Duf and the Duf-binding protein Rols to myoblast attachment sites in D-WIPD30 mutant embryos (Massarwa, 2007).
To further characterize the D-WIP-fusion defect, Eve expression was monitored in DA1 muscles of D-WIPD30 embryos. On average, only 3.0 DA1 nuclei are observed in each segment. Thus, similar to Wsp, D-WIP appears to be dispensable for muscle precursor formation, but is required for subsequent rounds of fusion between growing myotubes and FCMs (Massarwa, 2007).
Vrp/WIP proteins bind WASp-family proteins via a conserved domain at their C terminus. A variant of D-WIP lacking the Wsp-binding domain (D-WIPΔC), completely fails to rescue the D-WIP mutant phenotype. Furthermore, this construct has a strong dominant-negative effect when expressed in muscles of wild-type embryos (Massarwa, 2007).
Several observations thus suggest a shared requirement for D-WIP and Wsp during embryonic myogenesis, including strong similarities in loss-of-function mutant phenotypes, and functional reliance on structural domains that mediate physical association between the two proteins. It is therefore proposed that D-WIP and Wsp function as a single module and act in concert to promote myoblast fusion (Massarwa, 2007).
Next, conditions were engineered in which D-WIP/Wsp gene function was restricted to one of the two myoblast cell types, and fusion was monitored. Supplying D-WIP exclusively in myotubes, by expressing UAS-D-WIP under control of the founder cell/myotube-specific duf-GAL4 driver, in a D-WIPD30 mutant background resulted in significant, although incomplete, rescue. Comparable rescue of myoblast fusion in Wspmat/zyg embryos is obtained when UAS-Wsp is expressed under duf-GAL4 (Massarwa, 2007).
Since FCM-specific GAL4 drivers are not available, an alternative approach was adopted to provide D-WIP and Wsp function exclusively in FCMs. Expression in wild-type embryos of the UAS-WIPΔC and UAS-WspΔCA dominant-negative constructs via duf-GAL4 is expected to eliminate D-WIP/Wsp activity, specifically in founder cells and myotubes. duf-GAL4-mediated expression of these constructs has no obvious effects on myogenesis, implying that expression of D-WIP and Wsp in FCMs is sufficient for normal levels of myoblast fusion. Taken together, these results suggest that the D-WIP/Wsp system can function in both myoblast cell types during myotube formation (Massarwa, 2007).
Transmission electron microscopy (TEM) analysis has established that myoblast fusion in Drosophila embryos proceeds as a sequence of defined morphological events. Adhesion and apposition of myoblast cell membranes are followed by the appearance of vesicular and plaque-shaped electron-dense structures on both sides of the apposed membranes. Initial cytoplasmic continuity is then obtained upon formation of small (<200 nm) fusion pores linking the two cells. Vesiculation and fragmentation of the aligned double membrane ensues, and the process is completed after removal of the residual membrane material (Massarwa, 2007).
All fusion mutants studied to date by TEM display an arrest in the fusion process prior to the formation of pores between fusing myoblasts. In contrast, TEM analysis of D-WIPD30 and Wspmat/zyg embryos reveals a common phenotype, consistent with an exceptionally advanced stage of myoblast-fusion arrest. Multiple discontinuities are apparent in the apposed myoblast membranes, suggesting that D-WIP and Wsp are not required until the final phases of double-membrane breakdown and removal. Furthermore, while the size of membrane discontinuities varies widely in wild-type myoblasts undergoing the final phase of fusion, with only a small minority (10%-20%) displaying small pores throughout the fusing membranes, the latter phenotype was observed in 50%-60% of fusing myoblasts of D-WIPD30 and Wspmat/zyg embryos, implying that disruption of D-WIP/Wsp module function results in arrest at a discrete phase of the fusion process (Massarwa, 2007).
It was reasoned that establishment of partial fusion between myoblasts in embryos lacking D-WIP and Wsp function would permit transfer of cytoplasmic material between the cells. A cytoplasmic form of GFP was expressed in D-WIPD30 embryos by using the founder cell/myotube duf-GAL4 driver. 'Leakage' of GFP into the attached FCMs was successfully monitored. In contrast, similar analysis of mbc mutant embryos, in which attached myoblast membranes remain intact, failed to detect any GFP in myotube-attached FCMs. These findings further substantiate the TEM analysis of the D-WIP and Wsp mutant phenotypes, and they demonstrate that D-WIP and Wsp function is required during the final stages of myoblast fusion (Massarwa, 2007).
Myoblast fusion takes place in two steps in mammals and in Drosophila. First, founder cells (FCs) and fusion-competent myoblasts (FCMs) fuse to form a trinucleated precursor, which then recruits further FCMs. This process depends on the formation of the fusion-restricted myogenic-adhesive structure (FuRMAS), which contains filamentous actin (F-actin) plugs at the sites of cell contact. Fusion relies on the HEM2 (NAP1) homolog Kette, as well as Blow and WASP, a member of the Wiskott-Aldrich-syndrome protein family. This study documents the identification and characterization of schwächling -- a new Arp3-null allele. Ultrastructural analyses demonstrate that Arp3schwächling mutants can form a fusion pore, but fail to integrate the fusing FCM. Double-mutant experiments revealed that fusion is blocked completely in Arp3 and wasp double mutants, suggesting the involvement of a further F-actin regulator. Indeed, double-mutant analyses with scar/WAVE and with the WASP-interacting partner vrp1 (sltr, wip)/WIP show that the F-actin regulator scar also controls F-actin formation during myoblast fusion. Furthermore, the synergistic phenotype observed in Arp3 wasp and in scar vrp1 double mutants suggests that WASP and SCAR have distinct roles in controlling F-actin formation. From these findingsa new model was derived for actin regulation during myoblast fusion (Berger, 2008).
During the fusion process, the actin cytoskeleton of myoblasts is dynamically organized. Consistent with this, the center of the adhesion ring in FCs and FCMs contains filamentous actin (F-actin). The actin regulators kette and wasp are essential for myoblast fusion. Kette is the Drosophila homolog of HEM2 (NAP1), and can be found in a complex with SCAR (homolog of the mammalian WAVE) and is a member of the Wiskott-Aldrich-syndrome protein (WASP) family. Moreover, kette interacts genetically with blow during myoblast fusion. WASP family members possess a conserved VCA (verprolin homologous, cofilin homologous and acidic) domain, which is involved in the binding of G-actin and Arp2/3. The wasp3D3-035 allele lacks the CA domain of the VCA domain and thus neutralizes the function of maternal WASP. Furthermore, genetic data indicate that Kette antagonizes the function of WASP. The WASP-interacting partner Verprolin1 [Vrp1, also known as Solitary (Sltr) and Wip; the Drosophila homolog of the mammalian WIP], however, acts in a complex with WASP to ensure successful myoblast fusion (Kim, 2007; Massarwa, 2007; Berger, 2008 and references therein).
The branching of F-actin is initiated by the de novo nucleation of actin, triggered through the activity of the Arp2/3 complex. Alone, the Arp2/3 complex is inactive and requires a set of binding partners, including members of the WASP family, to become active. The binding of these proteins has been proposed to lead to a conformational change in the position of the seven subunits of the Arp2/3 complex, especially in the relative position of the subunits Arp2 and Arp3 (Berger, 2008).
This study describes an EMS-induced Arp3-null allele, Arp3schwächling, having an abnormal myoblast-fusion phenotype. Ultrastructural analysis of Arp3schwächling and wasp3D3-035 revealed the formation of a fusion pore in both mutants. Whereas in Arp3schwächling mutants the membrane between fusing myoblasts is removed, in wasp3D3-035 embryos, membrane breakdown is not completed. The finding that fusion in Arp3schwächling embryos is disrupted despite the formation of a fusion pore indicates that the pore does not expand in Arp3schwächling mutants. Therefore, to gain a deeper insight into the F-actin regulation of myoblast fusion, Arp3 wasp double-mutant embryos were examined, that show a complete block of myoblast fusion. This phenotype suggests that WASP is not the only actin regulator controlling F-actin polymerization during fusion. Indeed, studies on scar single mutants and scar vrp1 double mutants strongly imply that the first and the second fusion steps require a different set of F-actin nucleation factors (Berger, 2008).
The genetic data presented in this study provide new insights into the regulation of branching F-actin during myoblast fusion. There are three classes of proteins that initiate the polymerization of new actin filaments: the actin-related protein complex Arp2/3, the Formins and Spire. These protein classes are evolutionary conserved in most eukaryotes and promote new actin assembly by a distinct mechanism. The Arp2/3 complex is the only known protein complex that initiates new actin filaments branching off an existing filament. Upon cell-cell contact, F-actin is mostly present in the tip of the FCM and in the area of the FC/growing myotube to which the FCM is attaching. Analyses of Arp3, wasp and scar mutants indicate that branching F-actin is essential for myoblast fusion. Based on double-mutant analyses, it is postulated that the WASP-Vrp1 complex promotes branched F-actin formation positively during the second step of myoblast fusion, thereby allowing proposal of a new model for actin regulation during myoblast fusion (Berger, 2008).
This work emanated from the identification of an Arp3-null allele. Fusion is severely disrupted in Arp3schwächling mutants. However, stainings with anti-Duf clearly show that this is not due to a failure in cell-cell recognition and adhesion. For example, FCMs still attach to the site of the growing myotubes where Duf is expressed in Arp3schwächling and wasp3D3-035 mutant embryos. Interestingly, myoblasts also adhere successfully in Arp3schwächling wasp3D3-035 double mutants that fail to fuse completely. Duf serves to attract FCMs, which can migrate towards the Duf-expressing source. Hence, the ability of FCMs to migrate is a prerequisite for myoblast fusion. The migration of cells, however, also depends on actin-cytoskeleton regulation. Observations clearly show that the nature of the fusion arrest in Arp3schwächling and wasp3D3-035 single mutants, and Arp3schwächling wasp3D3-035 double mutants, is not due to either the inability of FCMs to migrate nor to a failure of FCs/myotubes and FCMs to recognize and adhere, but rather to a specific defect in cell-cell fusion (Berger, 2008).
Electron microscopy analyses on Arp3schwächling and wasp3D3-035 mutants further assisted in dissection of the process in which F-actin formation is required during myoblast fusion. The WASP-Vrp1 complex is involved in the formation of a fusion pore (Massarwa, 2007). In line with that study, mutations in wasp3D3-035 and wipD30 stop fusion during membrane breakdown, but not after pre-fusion-complex formation. The pre-fusion complex, which has been described to consist of 1.4 vesicles per pre-fusion complex, looks identical in both of the mutants as well as in wild-type embryos (Berger, 2008).
A model is presented for myoblast fusion derived from the data presented in this study. Studies on Arp3 mutants indicate that the polymerization-based force of branching F-actin is required beyond the stage of membrane breakdown. After membranes have been removed between fusing myoblasts, the FCMs must become integrated into the FC/growing myotube. Because Arp3schwächling mutants fail to fuse after membrane removal, it is proposed that F-actin is required to allow integration into the growing myotubes. After the Duf- and Sns-mediated recognition of FCs and FCMs, further essential proteins for myoblast fusion, e.g. Blow, become recruited to the point of fusion in FCMs. Vrp1 localizes in the tip of the filopodia of FCMs. The localization of WASP to the membrane depends on Vrp1 activity. Thus, the recruitment of WASP presumably leads to an increase of F-actin at the site of fusion. As a result, the formation of a fusion pore is initiated and expands until the FCM becomes finally pulled into the FC/growing myotube (Berger, 2008).
Morphological and statistical analysis on the nuclei of the DA1 muscle suggested that no fusion takes place in Arp3 wasp double mutants. Because wasp3D3-035 mutants should lack the ability to promote F-actin formation and stop fusion, like Arp3schwächling mutants, after precursor formation, this was a surprising result. Therefore whether the actin regulator SCAR additionally controls F-actin branching during myoblast fusion was examined. Double-mutant and epistasis experiments of scar and wasp revealed that this was indeed the case. The complete disruption of myoblast fusion in Arp3 wasp double-mutant embryos -- despite the presence of maternal wasp and Arp3 -- in conjunction with the finding that SCAR is required for myoblast fusion, led to a proposal that SCAR and WASP control different steps of myoblast fusion. So far, genetic data suggest that WASP is required only during the second fusion step. This is in line with previous data (Massarwa, 2007). It further implies that SCAR is essential for the first fusion step. However, myoblast fusion is not disrupted completely in scarδ37-null mutant embryos. The maternally provided gene product of scar is probably able to compensate for the loss of zygotic scar during myoblast fusion. Nevertheless, scar germline clones with reduced levels of maternal and zygotic SCAR protein show an enhanced myoblast-fusion phenotype. Because the loss of maternal scar disrupts oogenesis, it is not possible to eliminate the maternal component of scar completely. Hence, further experiments are required to determine whether SCAR controls the first fusion step alone or acts in functional redundancy with an additional factor (Berger, 2008).
Myoblast fusion in scar vrp1 double mutants was blocked completely. This might indicate that SCAR regulates the first fusion step together with the WASP-interacting partner Vrp1. The Vrp1 protein is expressed from stage 10 onwards, shortly before the first fusion step is initiated. Members of the Verprolin/WIP family have been reported to influence actin polymerization in a WASP-independent manner. It remains to be investigated whether this is also the case during Drosophila myoblast fusion (Berger, 2008).
In addition to regulating the first fusion step, the activity of SCAR might also be required for the second fusion step. Support for this notion comes from findings that the vertebrate homolog of Kette, HEM2, which is present in a complex with SCAR, is essential for myoblast fusion. Kette and WASP have antagonistic functions during myoblast fusion. Because the expression of WASP in FCs failed to rescue the wasp mutant phenotype, one could assume that the activity of WASP is only required in FCMs. It is therefore predicted that the polymerization of branching actin filaments might be regulated in a myoblast-type-specific manner (Berger, 2008).
In summary, these observations suggest for the first time that the cellular machinery leading to the formation of F-actin is controlled by a different set of nucleation-promoting factors during the first and second fusion steps. Future studies should now focus on the mechanistical details to reveal how these factors become activated in FCs and FCMs (Berger, 2008).
Reference names in red indicate recommended papers.
Search PubMed for articles about Drosophila Vrp1
Anton, I. M., et al. (1998). The Wiskott-Aldrich syndrome protein-interacting protein (WIP) binds to the adaptor protein Nck. J. Biol. Chem. 273(33): 20992-5. 9694849
Anton, I. M. and Jones, G. E. (2006). WIP: a multifunctional protein involved in actin cytoskeleton regulation, Eur. J. Cell Biol. 85: 295-304. Medline abstract: 16546573
Aspenstrom, P. (2005). The verprolin family of proteins: regulators of cell morphogenesis and endocytosis. FEBS Lett. 579: 5253-5259. Medline abstract: 16182290
Berger, S., et al. (2008). WASP and SCAR have distinct roles in activating the Arp2/3 complex during myoblast fusion. J. Cell Sci. 121: 1303-1313. PubMed Citation: 18388318
Chou, H. C., et al. (2006). WIP regulates the stability and localization of WASP to podosomes in migrating dendritic cells. Curr. Biol. 16(23): 2337-44. Medline abstract: 17141616
de la Fuente, M. A., et al. (2007). WIP is a chaperone for Wiskott-Aldrich syndrome protein (WASP). Proc. Natl. Acad. Sci. 104(3): 926-31. Medline abstract: 17213309
Gargini R, Escoll M, García E, García-Escudero R, Wandosell F, Antón IM. (2016). WIP drives tumor progression through YAP/TAZ-dependent autonomous cell growth. Cell Rep. 17(8):1962-1977. PubMed ID: 27851961
Ho, H. Y., Rohatgi, R., Lebensohn, A. M., Le, Ma, Li, J., Gygi, S. P. and Kirschner, M. W. (2004). Toca-1 mediates Cdc42-dependent actin nucleation by activating the N-WASP-WIP complex. Cell 118(2): 203-16. 15260990
Kim, S., Shilagardi, K., Zhang, S., Hong, S. N., Sens, K. L., Bo, J., Gonzalez, G. A. and Chen, E. H. (2007). A critical function for the actin cytoskeleton in targeted exocytosis of prefusion vesicles during myoblast fusion. Dev. Cell 12: 571-586. PubMed Citation: 17419995
Kinley, A. W., et al. (2003). Cortactin interacts with WIP in regulating Arp2/3 activation and membrane protrusion. Curr. Biol. 13: 384-393. 12620186
Konno, A., et al. (2007). The expression of Wiskott-Aldrich syndrome protein (WASP) is dependent on WASP-interacting protein (WIP). Int. Immunol. 19(2): 185-92. Medline abstract: 17205972
Krzewski, K., Chen, X., Orange, J. S. and Strominger, J. L. (2006). Formation of a WIP-, WASp-, actin-, and myosin IIA-containing multiprotein complex in activated NK cells and its alteration by KIR inhibitory signaling. J. Cell Biol. 173(1): 121-32. Medline abstract: 16606694
Martinez-Quiles, N., et al. (2001). WIP regulates N-WASP-mediated actin polymerization and filopodium formation. Nat. Cell Biol. 3: 484-491. 11331876
Massarwa, R., Carmon, S., Shilo, B.-Z. and Schejter, E. D. (2007). WIP/WASp-based actin-polymerization machinery is essential for myoblast fusion in Drosophila. Dev. Cell 12: 557-569. Medline abstract: 17419994
Moreau, V., et al. (2000). A complex of N-WASP and WIP integrates signalling cascades that lead to actin polymerization. Nat. Cell Biol. 2(7): 441-8. 10878810
Myers, S. A., Leeper, L. R. and Chung, C. Y. (2006). WASP-interacting protein is important for actin filament elongation and prompt pseudopod formation in response to a dynamic chemoattractant gradient. Mol. Biol. Cell 17(10): 4564-75. Medline abstract: 16899512
Peterson, F. C., et al. (2007). Multiple WASP-interacting protein recognition motifs are required for a functional interaction with N-WASP. J. Biol. Chem. 282(11): 8446-53. Medline abstract: 17229736
Ramesh, N., et al. (1997). WIP, a protein associated with wiskott-aldrich syndrome protein, induces actin polymerization and redistribution in lymphoid cells. Proc. Natl. Acad. Sci. 94(26): 14671-6. 9405671
Sasahara, Y., et al. (2003). Mechanism of recruitment of WASP to the immunological synapse and of its activation following TCR ligation. Mol. Cell 10: 1269-1281. 12504004
Sawa, M. and Takenawa, T. (2006). Caenorhabditis elegans WASP-interacting protein homologue WIP-1 is involved in morphogenesis through maintenance of WSP-1 protein levels. Biochem. Biophys. Res. Commun. 340(2):709-17. Medline abstract: 16378591
Scott, M. P., Zappacosta, F., Kim, E. Y., Annan, R. S. and Miller, W. T. (2002). Identification of novel SH3 domain ligands for the Src family kinase Hck. Wiskott-Aldrich syndrome protein (WASP), WASP-interacting protein (WIP), and ELMO1. J. Biol. Chem. 277: 28238-28246. 12029088
Sun, Y., Martin, A. C. and Drubin, D. G. (2006). Endocytic internalization in budding yeast requires coordinated actin nucleation and myosin motor activity. Dev. Cell 11(1): 33-46. Medline abstract: 16824951
Tsuboi S. (2007). Requirement for a complex of Wiskott-Aldrich syndrome protein (WASP) with WASP interacting protein in podosome formation in macrophages. J. Immunol. 178(5): 2987-95. Medline abstract: 17312144
Vaduva, G., et al. (1999). The human WASP-interacting protein, WIP, activates the cell polarity pathway in yeast. J. Biol. Chem. 274(24): 17103-8. 10358064
Volkman, B. F., et al. (2002). Structure of the N-WASP EVH1 domain-WIP complex: insight into the molecular basis of Wiskott-Aldrich Syndrome. Cell 111: 565-576. Medline abstract: 12437929
Zett, M. and Way, M. (2002). The WH1 and EVH1 domains of WASP and Ena/VASP family members bind distinct sequence motifs. Curr. Biol. 12: 1617-1622. 12372256
date revised: 22 January 2017
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