The rearrangement of cytoskeletal elements is essential for many cellular processes. The tumor suppressor Adenomatous polyposis coli (APC) affects the function of microtubules and actin, but the mechanisms by which it does so are not well understood. This study reports that Drosophila syncytial embryos null for Apc2 display defects in the formation and extension of pseudocleavage furrows, which are cortical actin structures important for mitotic fidelity in early embryos. Furthermore, the formin Diaphanous (DIA) functions with APC2 in this process. Colocalization of APC2 and DIA peaks during furrow extension, and localization of APC2 to furrows is DIA-dependent. Furthermore, APC2 binds DIA directly through a region of APC2 not previously shown to interact with DIA-related formins. Consistent with these results, reduction of dia enhances actin defects in Apc2 mutant embryos. Thus, an APC2-DIA complex appears crucial for actin furrow extension in the syncytial embryo. Interestingly, EB1, a microtubule +TIP and reported partner of vertebrate APC and DIA1, may not function with APC2 and DIA in furrow extension. Finally, whereas DIA-related formins are activated by Rho family GTPases, these data suggest that the APC2-DIA complex might be independent of RHOGEF2 and RHO1. Furthermore, although microtubules play a role in furrow extension, this analysis suggests that APC2 and DIA function in a novel complex that affects actin directly, rather than through an effect on microtubules (Webb, 2009).
APC family proteins have many well-documented effects on the microtubule cytoskeleton, whereas APC functions with actin are much less well understood. The Drosophila syncytial embryo is an excellent in vivo system in which to study the role of APC2 and its partners in organizing the cytoskeleton. Drosophila APC2 localizes to actin in syncytial embryos, and a roles has been suggested role for APC2 in tethering cortical microtubules to actin (McCartney, 2001; Webb, 2009 and references therein).
This study report the cytoskeletal consequences of eliminating all APC2 in the syncytial embryo. Apc2-null mutants exhibit incomplete actin rings and a failure of actin furrow extension. Apc2δS mutants exhibit more nuclear loss than do null mutants, but have weaker actin defects, suggesting that the APC2δS protein might interfere with a tethering process for which APC2 is not essential. The presence of actin furrow defects in embryos that are mutant for multiple alleles of Apc2, including a null, strongly suggests that APC2 functions in the normal organization of actin furrows (Webb, 2009).
A novel role has been demonstrated for an APC2-DIA complex in the organization of the actin cytoskeleton. Formins such as DIA are best known for their ability to nucleate unbranched actin filaments and accelerate filament elongation. Drosophila DIA functions in actin-based furrow assembly during cellularization and conventional cytokinesis. dia mutant syncytial embryos have defects in the initiation and elongation of actin furrows, consistent with DIA subcellular localization and known roles for formins. This study shows that APC2 and DIA colocalize together and with actin specifically at times when furrows are elongating. The fact that APC2 and DIA bind directly in vitro, but their colocalization is cell cycle-dependent, suggests that the interaction is regulated in vivo (Webb, 2009).
The simplest model for the function of an APC2-DIA complex in actin furrow formation is that DIA-dependent nucleation and elongation of unbranched actin filaments is essential for furrow extension, and that APC2 promotes DIA activity. The fact that the dia-null phenotype is more severe than that of Apc2, coupled with the enhancement of the Apc2-null phenotype by a reduction of dia, support the model that APC2 is not essential for, but might enhance, DIA activity. The dependence of APC2 on DIA for localization indicates that DIA may directly affect the regulation of its own activity (Webb, 2009).
One mechanism regulating the activity of formins has been extensively studied. DIA-related formins (DRFs) are autoinhibited through the binding of the N-terminal DIA inhibitory domain (DID) to the C-terminal DIA autoregulatory domain (DAD). Binding of the GTPase-binding domain (GBD) by RHO-GTP relieves the autoinhibition and activates DRFs, which function as dimers. Although RHO1 and RHOGEF2 have been reported to act upstream of DIA during Drosophila cellularization and embryonic morphogenesis, other reports suggest that they are in a parallel pathway during these times. The distinct actin defects in Rho1 and RhoGEF2 mutants as compared with Apc2 and dia mutants suggest that RHO1 is not the GTPase that directly activates DIA during furrow formation in the syncytial embryo. However, because Rho1-null embryos cannot be generated genetically owing to requirements during oogenesis, it is possible that there is a role for RHO1 in activating DIA independent of RHOGEF2. In addition, although the activity of other GEFs and GTPases cannot be ruled out, these observations, along with those of formins in other systems, suggest the existence of alternative mechanisms for DIA-related formin activation (Webb, 2009).
This study shows that APC2 and DIA can bind directly to each other. Thus, APC2 could directly affect the function of DIA, perhaps by stabilizing the open conformation required for optimal DIA activity. Alternatively, APC2 could enhance the activity of DIA by binding to and recruiting other DIA-activating factors to the complex. Once DIA is activated it might dissociate from this complex, resulting in two pools of DIA. This notion is supported by the observation that DIA, but not APC2, is enriched at the furrow tip. It is proposed that in the absence of APC2, the efficiency of DIA activation is reduced, resulting in a decrease in the amount of unbranched actin filaments and a consequent production of shallow furrows. Consistent with this model for APC2 function, APC proteins are thought to play a scaffolding role in the Wnt regulatory 'destruction complex'. Furthermore, vertebrate APC binds ASEF and IQGAP, activators of RAC and RAC/CDC42, respectively, through its N-terminal Armadillo repeats. Thus, APC2 may promote the association of DIA with GEFs and GTPases, or with other proteins that promote the open conformation or otherwise enhance DIA activity (Webb, 2009).
EB1 regulates microtubule function in many organisms including Drosophila, in which its disruption affects spindle positioning and dynamics in the early embryo. Mouse EB1 can bind both DIA1 and APC1 and the binary interactions identified have suggested a ternary complex. This study tested the possibility that EB1 plays a role in an APC2-DIA complex. Although Eb1-null syncytial embryos exhibit weak defects in furrow extension, the lack of a genetic interaction with Apc2, and the lack of direct binding to APC2, argue that EB1 function in the syncytial embryo is independent of the APC2-DIA complex (Webb, 2009).
Taken together, the lack of post-translationally modified, stabilized microtubules in the syncytial embryo, the lack of discernable microtubule defects in the Apc2 mutant, and the lack of APC2 interactions with EB1, strongly suggest that the Drosophila APC2-DIA complex has a distinct cytoskeletal function from that of the mouse DIA1-APC-EB1 complex that stabilizes microtubules in cultured cells. In addition, the colocalization of APC2 and DIA specifically when furrows are extending during prophase and metaphase, rather than during the critical period of microtubule function in anaphase, supports the model that the APC2-DIA complex primarily affects actin. It is intriguing that interactions between APC proteins and formins have been conserved, and that such complexes can affect both the actin and microtubule networks (Webb, 2009).
Unlike conventional cytokinesis that uses actomyosin-based contraction to drive membrane invagination, Myosin II function is dispensable for pseudocleavage furrow formation. Many proteins are known to affect the dynamic organization of syncytial actin. Centrosomin, a core centrosome component, Sponge, a putative unconventional RacGEF, and Scrambled, a novel protein, all have roles in normal cap formation. Cap formation and expansion require the Arp2/3 complex and its activator SCAR. Well-known actin regulators such as RHO1, RHOGEF2 and Abelson (Abl tyrosine kinase) may not affect furrow extension directly, but rather regulate the overall balance of actin activity. The Myosin VI protein Jaguar appears to play a specific role in furrow extension, where it might stabilize actin filaments, as it does in the Drosophila testis (Webb, 2009).
The best-understood mechanism for pseudocleavage furrow extension is the NUF (Arfophilin)-RAB11-DAH (Dystrophin) pathway, which utilizes microtubule-based transport from the recycling endosome to move membrane and actin to the site of furrow extension. Once actin has been delivered to this site, APC2-DIA might promote the nucleation and elongation of unbranched filaments necessary for furrow extension. Interestingly, it has been shown that in the absence of dia, and in Apc2 mutant embryos reduced for dia, some actin remains cap-like. This intriguing observation suggests that there is a relationship between the dissolution of caps and the formation of furrows, and that these distinct pools of branched and unbranched actin might be in a balance. The APC2-DIA complex has emerged as a key factor affecting actin organization in the early embryo, and further study will reveal how the many regulatory pathways converge to influence dynamic changes in actin organization (Webb, 2009).
E-cadherin plays a pivotal role in epithelial morphogenesis. It controls the intercellular adhesion required for tissue cohesion and anchors the actomyosin-driven tension needed to change cell shape. In the early Drosophila embryo, Myosin-II (Myo-II) controls the planar polarized remodelling of cell junctions and tissue extension. The E-cadherin distribution is also planar polarized and complementary to the Myosin-II distribution. This study shows that E-cadherin polarity is controlled by the polarized regulation of clathrin- and dynamin-mediated endocytosis. Blocking E-cadherin endocytosis results in cell intercalation defects. A pathway is delineated that controls the initiation of E-cadherin endocytosis through the regulation of AP2 and clathrin coat recruitment by E-cadherin. This requires the concerted action of the formin Diaphanous (Dia) and Myosin-II. Their activity is controlled by the guanine exchange factor RhoGEF2, which is planar polarized and absent in non-intercalating regions. Finally, evidence is provided that Dia and Myo-II control the initiation of E-cadherin endocytosis by regulating the lateral clustering of E-cadherin (Levayer, 2011).
Epithelial tissues have a robust architecture that is essential for their barrier function. This barrier function depends on their ability to build adhesive contacts at adherens junctions through the recruitment and stabilization of E-cadherin (E-cad), β-catenin (β-cat) and α-catenin (α-cat) by actin filaments (F-actin). During development, epithelia are also extensively reshaped by remodelling of cell contacts. This plasticity is essential for morphogenesis during embryogenesis and organogenesis. Work in the past decade showed that this requires force generation by actomyosin networks and their anchoring at cell junctions by E-cad/β-cat/α-cat complexes. Thus, E-cad plays a pivotal role in junction robustness and plasticity by mediating both adhesion (cohesion) and tension transmission (remodelling). Understanding what controls the distribution and dynamics of E-cad/β-cat/α-cat complexes is therefore key to understanding cell packing and the mechanics of tissue morphogenesis. Disruption of this balance marks key steps in the progression of solid tumours. The loss of epithelial organization during the epithelial to mesenchymal transition is an extreme example in which E-cad endocytosis causes the loss of adhesion and tension transmission at the cell cortex (Levayer, 2011).
The early development of the Drosophila embryo is a powerful system to study epithelial morphogenesis. Spatial regulation of force generation by actomyosin networks and force transmission to adhesion by E-cad both contribute to apical cell constriction in the invaginating mesoderm1, and cell intercalation in the elongating ectoderm called the germ band. Germ-band extension (GBE) is driven by cell intercalation in the ventrolateral region, whereby cells exchange neighbours through planar polarized junction remodelling, namely shrinkage of 'vertical' junctions (that is, junctions oriented along the dorsoventral axis). Intercalation is powered by non-muscle Myosin-II (Myo-II): anisotropic actomyosin contractile flows from the medial apical region to 'vertical' junctions drive junction shrinkage. The shortening of vertical junctions is stabilized by Myo-II at the cortex. Actomyosin contractility is transmitted at the cortex by E-cad complexes through β-cat. Interestingly, E-cad/β-cat/α-cat complexes also exhibit a planar polarized distribution complementary to that of Myo-II: E-cad is less abundant in shrinking 'vertical' junctions. This E-cad polarity is also required to orient actomyosin flows to 'vertical' junctions. It is unknown what controls the planar polarized distribution of E-cad. This may depend on Rho kinase (ROCK), which is required for the polarized distribution of Par3 (Levayer, 2011).
This study shows that the planar polarized distribution of E-cad is also controlled by an upregulation of clathrin- and dynamin-mediated endocytosis at adherens junctions, in particular in 'vertical' junctions of intercalating cells. Blocking endocytosis causes the loss of E-cad planar polarization and a block of intercalation. This led to an investigation of the mechanisms that control planar polarized upregulation of clathrin-mediated endocytosis (CME) of E-cad at adherens junctions. Activation of WASP (Wiscott-Aldrich Syndrome Protein) and the Arp2/3 (Actin-Related Protein 2/3) complex by Cdc42 controls the branched actin polymerization that is required for vesicular scission. This study identified an additional pathway controlling the initiation of E-cad endocytosis through the recruitment of the AP2 (Adaptor Protein 2) complex and clathrin. This recruitment is driven by lateral clustering of E-cad that relies on unbranched actin polymerization induced by Dia, and the presence of Myo-II. Dia and Myo-II are both activated by the guanine exchange factor RhoGEF2 (Levayer, 2011).
This study has delineated two distinct roles for actin in E-cad endocytosis. Dia and Myo-II control the initiation of E-cad endocytosis by enrichment of clathrin and AP2 in an E-cad-dependent manner. This is tightly spatially regulated in the ventrolateral region and in 'vertical' junctions during cell intercalation by cortical RhoGEF2 localization, an activator of Dia and Myo-II in Drosophila embryos. This is distinct from the role of branched actin polymerization by Arp2/3, which promotes vesicular scission similarly to dynamin. At later stages of development, this depends on WASP and is controlled by Cdc42, aPKC (atypical protein kinase C) and Cip4 (Cdc42-interacting protein 4. In early embryos, as WASP is inhibited by the JAK/STAT pathway (Janus kinase/signal transducer and activator of transcription), Scar instead plays a critical role in vesicular scission. Inhibition of Arp2/3 in scar mutants and its constitutive activation (artificially induced by myrWASP) did not affect clathrin and AP2 concentration at adherens junctions, unlike Dia. The different tiers of regulation of E-cad endocytosis by Arp2/3 and Dia may reflect different roles for actin in constitutive versus regulated E-cad endocytosis. Certain situations require a rapid change in the rate of endocytosis, and may do so by tuning the rate of initiation by clathrin and AP2. It will be interesting to see whether rapid collapse of adherens junctions during epithelial to mesenchymal transition relies on a similar process (Levayer, 2011).
Crosslinking E-cad with an IgG is sufficient to promote dorsal endocytosis of E-cad by upregulating the concentration of clathrin, similarly to Dia activation, even following inhibition of Dia, Myo-II or RhoGEF2. Considering the highly correlated localizations of E-cad complexes with AP2 and RhoGEF2, it is proposed that Dia and Myo-II control the initiation of E-cad endocytosis by inducing lateral clustering of E-cad, similar to Fc receptor clustering during phagocytosis or nanoclusters of GPI (glycosylphosphatidylinositol)-anchored proteins. This may have been co-opted by the pathogen Listeria, whose entry into epithelial cells requires E-cad endocytosis. This mechanism may also require specific 'priming' of E-cad, by ubiquitylation as in mammals, although these tyrosines are not conserved in flies. Importantly, the mechanism of AP2 recruitment by E-cad remains unknown in all systems (Levayer, 2011).
Inhibition of E-cad endocytosis increased E-cad levels and disrupted its planar polarized distribution. Myo-II also accumulated in the medial apical region of cells. The GBE defects in shi-ts mutants or following clathrin inhibition are the result of the altered distribution of actomyosin tensile forces. E-cad/β-cat/α-cat complexes affect the lateral flow of medial actomyosin pulses and Myo-II polarized junctional accumulation, presumably through the regulation of tension transmission within the medial network and/or at the junctions. The medial accumulation of Myo-II when E-cad endocytosis is inhibited may thus reflect an inhibition of actomyosin flow towards the cortex. These results emphasize the interplay between actomyosin contractile dynamics and E-cad adhesive complexes during epithelial morphogenesis (Levayer, 2011).
Formins are key regulators of actin nucleation and elongation. Diaphanous-related formins, the best-known subclass, are activated by Rho and play essential roles in cytokinesis. In cultured cells, Diaphanous-related formins also regulate cell adhesion, polarity and microtubules, suggesting that they may be key regulators of cell shape change and migration during development. However, their essential roles in cytokinesis hamper testing of this hypothesis. Loss- and gain-of-function approaches were used to examine the role of Diaphanous in Drosophila morphogenesis. Diaphanous has a dynamic expression pattern consistent with a role in regulating cell shape change. Constitutively active Diaphanous was used to examine its roles in morphogenesis and its mechanisms of action. This revealed an unexpected role in regulating myosin levels and activity at adherens junctions during cell shape change, suggesting that Diaphanous helps coordinate adhesion and contractility of the underlying actomyosin ring. This hypothesis was tested by reducing Diaphanous function, revealing striking roles in stabilizing adherens junctions and inhibiting cell protrusiveness. These effects also are mediated through coordinated effects on myosin activity and adhesion, suggesting a common mechanism for Diaphanous action during morphogenesis (Homem, 2008).
Previous analysis of Diaphanous-related formins (DRF) function in morphogenesis was hampered by mammalian DRF redundancy and the key role of DRFs in cytokinesis. The genetic tools available in Drosophila were used to partially circumvent these difficulties. Dia is known to regulate cellularization. The analysis revealed new roles during morphogenesis. In embryos with severely reduced Dia function, gastrulation is disrupted. Apical constriction of central furrow cells is delayed or blocked in a subset of cells, suggesting a possible role for Dia in regulated apical constriction. Previous work suggested that an unknown Rho effector regulates actin during this process; perhaps this is Dia. Adjacent cells, which are stretched during invagination, become massively multinucleate in dia5M mutants. This may reflect the fact that the cells of the blastoderm undergo an incomplete form of cytokinesis, remaining connected to the underlying yolk by actin-lined yolk canals. These canals normally close at the onset of gastrulation in ventral furrow cells; this may prevent cell membrane rupture initiated at the yolk canal under stress. Dia may regulate yolk canal closure; it localizes there at the end of cellularization and similar defects are seen in mutants lacking the septin Peanut, a yolk canal component. Alternately, Dia may stabilize cortical actomyosin or its connections to AJs, with its absence weakening cortical integrity under stress (Homem, 2008).
Dia also plays a key role during germband retraction. The most striking effect of reduced Dia/Rho1 function is altered amnioserosal cell protrusiveness. They normally make stable AJs and form a tight tissue boundary with the epidermis; this is destabilized in dia/Rho1 and dia5M/Z mutants. Altered protrusive activity was seen in other contexts: dia5M/Z ectoderm exhibited cortical blebbing, and dia/Rho1 and dia5M/Z amnioserosal cells had altered protrusiveness during dorsal closure. Reduced Dia function destabilized AJs, and the striking increase in cell protrusiveness in dia/Rho1 mutants was substantially rescued by increasing Myosin phosphorylation. These data support the hypothesis that Dia normally helps restrict actomyosin contractility to AJs, and in its absence, AJs are destabilized and myosin activity outside AJs stimulates protrusiveness. Interestingly, in cultured cells, fully assembled AJs inhibit Rac1 and lamellipodial activity while increasing Rho and contractility, and myosin is required for mouse Dia1-induced AJ strengthening (Homem, 2008).
Given these morphogenetic roles, the mechanisms of action of Dia was explored, initially hypothesizing that it would primarily affect actin. DiaCA expression did elevate F-actin levels (reducing Dia did not dramatically decrease F-actin, but residual Dia may suffice). However, surprisingly, Dia activation also increased Myosin accumulation and cell contractility, and some effects of reduced Dia function were rescued by increasing Myosin activation. This suggests a surprising role for Dia in regulating Myosin levels/activity. The data also suggest that Dia stabilizes AJs, supporting earlier work in cultured cells. There are intimate connections between AJs and cortical actomyosin: AJs help organize the apicolateral actin ring while cortical actin stabilizes AJs. Interestingly, in cultured cells Myosin-mediated contraction of actin filaments is essential for cell-cell contact expansion, and mouse Dia1 AJ stabilization requires myosin activity, providing further evidence that contractility, AJ stability and Dia activity are mechanistically linked. The actomyosin network may also stabilize AJs by preventing endocytosis. Perhaps Dia, like formin 1, directly binds AJs. Linear actin filaments promoted by Dia at AJs might recruit α-catenin, which then might inhibit Arp2/3-induced actin branching, facilitating formation of a belt of unbranched actin. Thus, Dia is poised to act at the interface between AJs, cortical actin and contractility (Homem, 2008).
The Yin-Yang relationship between adhesion and protrusiveness is a very interesting one, underlying epithelial-mesenchymal transitions. Stable AJs probably actively inhibit protrusiveness, and Dia may assist this, acting in a reinforcing loop that concentrates actomyosin activity at AJs. There are interesting parallels with the role of Dia in cytokinesis. Dia stabilizes Myosin at the midzone. In its absence Myosin is delocalized around the cortex, leading to abnormal protrusiveness. Future work will reveal how Dia fits into the regulatory network coordinating adhesion and cytoskeletal regulation (Homem, 2008).
From these data, a mechanistic model was developed for the regulation of actin, Myosin and AJs by Dia during cell shape change, in which Dia activation stabilizes actin and active Myosin at AJs. In radially symmetric amnioserosal cells, Dia promotes organization/activation of an apical actomyosin network linked to AJs, inducing precocious apical cell constriction. Activation of endogenous Dia may regulate normal amnioserosal constriction: this now needs to be tested. Dia activation in cells where AJs are planar polarized, such as epidermal cells, promotes actomyosin organization preferentially at cell borders where AJs are enriched. This leads to cell widening or helps cells resist elongation, generating groove-cell-like morphology. Once again, Dia may be normally activated specifically in groove cells to modulate their shape (Homem, 2008).
How does Dia activation activate Myosin? It does not occur primarily through SRF, which triggers Myosin accumulation but not cell shape changes. Myosin activation via MLCK partially mimicked DiaCA, suggesting that Myosin activation is an important part of the process. However, MLCK did not precisely mimic DiaCA, suggesting that Dia acts preferentially at specific sites such as AJs rather than globally activating Myosin. The dual effects on actin and Myosin suggests the speculative possibility that feedback mechanisms exist to coordinate the Rho-regulated actin and Myosin pathways. Recent work revealed that active Dia1 can activate RhoA by binding the Rho-GEF LARG, and strong genetic interactions were seen between dia and the LARG relative RhoGEF2, making this idea more plausible. Future work is needed to test this hypothesis (Homem, 2008).
The process of myogenesis includes the recognition, adhesion, and fusion of committed myoblasts into multinucleate syncytia. In the larval body wall muscles of Drosophila, this elaborate process is initiated by Founder Cells and Fusion-Competent Myoblasts (FCMs), and cell adhesion molecules Kin-of-IrreC (Kirre) and Sticks-and-stones (Sns) on their respective surfaces. The FCMs appear to provide the driving force for fusion, via the assembly of protrusions associated with branched F-actin and the WASp, SCAR and Arp2/3 (see Drosophila Arp2/3 component Arpc1) pathways. This study utilized the dorsal pharyngeal musculature that forms in the Drosophila embryo as a model to explore myoblast fusion and visualize the fusion process in live embryos. These muscles rely on the same cell types and genes as the body wall muscles, but are amenable to live imaging since they do not undergo extensive morphogenetic movement during formation. Time-lapse imaging with F-actin and membrane markers revealed dynamic FCM-associated actin-enriched protrusions that rapidly extend and retract into the myotube from different sites within the actin focus. Ultrastructural analysis of this actin-enriched area showed that they have two morphologically distinct structures: wider invasions and/or narrow filopodia that contain long linear filaments. Consistent with this, formin Diaphanous (Dia) and branched actin nucleator, Arp3, are found decorating the filopodia or enriched at the actin focus, respectively, indicating that linear actin is present along with branched actin at sites of fusion in the FCM. Gain-of-function Dia and loss-of-function Arp3 both lead to fusion defects, a decrease of F-actin foci and prominent filopodia from the FCMs. Differential endocytosis of cell surface components was observed at sites of fusion, with actin reorganizing factors, WASp and SCAR, and Kirre remaining on the myotube surface and Sns preferentially taken up with other membrane proteins into early endosomes and lysosomes in the myotube (Haralalka, 2014: PubMed).
The formation of multinucleated muscle cells through cell-cell fusion is a conserved process from fruit flies to humans. Numerous studies have shown the importance of Arp2/3, its regulators, and branched actin for the formation of an actin structure, the F-actin focus, at the fusion site. This F-actin focus forms the core of an invasive podosome-like structure that is required for myoblast fusion. The formin Diaphanous (Dia), which nucleates and facilitates the elongation of actin filaments, was found to be essential for Drosophila myoblast fusion. Following cell recognition and adhesion, Dia is enriched at the myoblast fusion site, concomitant with, and having the same dynamics as, the F-actin focus. Through analysis of Dia loss-of-function conditions using mutant alleles but particularly a dominant negative Dia transgene, it was demonstrated that reduction in Dia activity in myoblasts leads to a fusion block. Significantly, no actin focus is detected, and neither branched actin regulators, SCAR or WASp, accumulate at the fusion site when Dia levels are reduced. Expression of constitutively active Dia also causes a fusion block that is associated with an increase in highly dynamic filopodia, altered actin turnover rates and F-actin distribution, and mislocalization of SCAR and WASp at the fusion site. Together these data indicate that Dia plays two roles during invasive podosome formation at the fusion site: it dictates the level of linear F-actin polymerization, and it is required for appropriate branched actin polymerization via localization of SCAR and WASp. These studies provide new insight to the mechanisms of cell-cell fusion, the relationship between different regulators of actin polymerization, and invasive podosome formation that occurs in normal development and in disease (Deng, 2015).
This study provides the first evidence that Dia is essential for Drosophila myoblast fusion. Dia is expressed in all myoblasts and is recruited to the myoblast fusion site. The spatial and temporal distribution of Dia at the fusion site parallels that of the F-actin focus, which forms the core of an invasive podosome. This actin rich podosome is critical for FCM invasion of the FC/Myotube during fusion. In keeping with its expression pattern, Dia is essential for myoblast fusion progression: both loss and gain of Dia function lead to a fusion block. Under both conditions, the integrity of the F-actin focus and hence the invasive podosome is compromised; myoblasts expressing DiaDN fail to form the focus, whereas myoblasts expressing DiaCA have many filopodia and have a diffuse organization of F-actin, both of which contribute to a failure in invasive podosome formation and fusion. Dia activity is required after FC/Myotube and FCM recognition and adhesion but upstream of Arp2/3 activity. It is required, in parallel with PI(4,5)P2 signaling, to build a functional F-actin focus at the fusion site. These experiments further indicate that Dia activity is critical for actin dynamics at the fusion site, which, in turn, regulate fusion progression. Moreover, the aberrant F-actin organization at the fusion site in both loss and gain of function is also due to altered localization of the Arp2/3 regulators, SCAR and WASp. Taken together, these data support a role for the formin Dia in a critical first step of actin polymerization at the fusion site, downstream of cell-cell recognition and adhesion, and link its activity to the formation of F-actin foci, required for myoblast fusion (Deng, 2015).
Actin remodeling is critical for myoblast fusion, but Arp2/3 was the only known actin polymerization factor that was shown to be necessary for myoblast fusion. This study now shows that the formin Dia is also required during myoblast fusion. Whereas Arp2/3 preferably binds to pre-existing actin filaments and generates uncapped F-actin, formins nucleate F-actin both de novo and from the barbed ends of pre-existing actin filaments. Thus, Dia can generate actin filaments de novo, which Arp2/3 can bind or elongate (Deng, 2015).
This study also shows that the level of Dia activity is critical for myoblast fusion. Too much actin polymerization leads to too many filopodia and absence of an invasive podosome with its characteristic F-actin core. Too little polymerization leads no actin focus and no podosome formation. FRAP data with DiaCA also hint at whether a limited pool of actin is available for the actin polymerization factors during myoblast fusion. Despite the high rates of actin turnover with expression of DiaCA, the final fluorescence levels of actin returns to the same value as in controls. Additional actin monomers are not recruited to the site, even with high levels of polymerization activity. Interestingly, the rate of actin turnover has also been measured in mutants that affect Arp2/3 activity: specifically, mutations in blow, which regulates the Arp2/3 NPF WASp, show lower rates of actin exchange than in controls, due to a reduced exchange rate for WASp on the barbed ends of actin at the fusion site. Together these data suggest future experiments aimed at examination of whether rates of actin polymerization regulated by both Dia and Arp2/3 are optimized for the available actin pool and tightly controlled for myoblast fusion to properly occur (Deng, 2015).
Both cooperative and antagonistic functions between Dia and Arp2/3 have been reported. This study demonstrates that the coordinated and cooperative activities of these two actin polymerization factors leads to the formation of the F-actin focus. With the exception of sltr/Dwip/vrp mutants that form a focus of wild-type size, single mutants in the Arp2/3 NPF pathways, WASp and SCAR, lead to enlarged foci; however, double mutants in WASp and SCAR pathways do not form foci. This is the same phenotype that is seen in myoblasts expressing the DiaDN. The data support Dia activity being upstream of WASp and SCAR activation of Arp2/3 at the fusion site. This suggests that, at the fusion site, Dia initially provides the necessary context upon which Arp2/3 can act and not vice versa, as has been suggested in other contexts in which linear actin filaments emerge from Arp2/3 based structures. Nevertheless, both sets of actin regulators are necessary for F-actin focus formation that provides the core of the invasive podosome. Neither Dia nor Arp2/3 alone are sufficient (Deng, 2015).
The interplay between Dia and Arp2/3 at the fusion site is also reflected by the localization studies. Too little or too much Dia activity resulted in improper localization and, by extension, improper activity of Arp2/3 NPFs. How could Dia regulate this localization? One possibility is that Dia indirectly regulates Arp2/3 localization. Dia could nucleate linear actin filaments, which then would provide the necessary substrate for recruitment, maintenance and /or activation of Arp2/3 and its regulators, such as the WASp-WIP complex. Another possibility is that Dia, through its interactions with members of the SCAR/WAVE complex such as Abi, may directly localize and/or maintain the localization of Arp2/3 regulators, which are then activated at the fusion site. Abi has been reported to bind directly with Dia in vitro, and this interaction is required for the formation and stabilization of cell-cell junctions. Dia likely changes the localization and integrity of the SCAR/WAVE complex by competitively binding to the N-terminal part of Abi, dissociating Kette/Nap1 from the complex, and thus changing the stability and localization of SCAR/WAVE. It has also been established that the recognition and adhesion receptor, Sns, is capable of recruiting the Arp2/3 NPFs, such as WASp, to the fusion site. While Sns is still clustered at the fusion site in DiaDN and DiaCA, its recruitment activity appears not sufficient for focus formation capable of supporting an invasive podosome (Deng, 2015).
This study has shown that localization of Arp2/3 NPFs is affected in Dia loss and gain of function. In addition to this spatial control, another important way of controlling Arp2/3 activity is through activation of the NPFs via small GTPases. SCAR is activated through Rac-dependent dissociation from SCAR inhibitory complex. WASp is activated by binding to Cdc42, which releases it from auto-inhibited state. This study did not examine the localization of these activated GTPases. However, previous work has shown that PI(4,5)P2 signaling is required for proper localization of activated Rac at the fusion site. How the localization and activity of small GTPases at the fusion site contribute to the spatial and temporal interplay between Dia and Arp2/3 regulation of actin polymerization requires further investigation (Deng, 2015).
It remains unresolved how Dia itself is recruited to the fusion site. The data suggest that the recognition and adhesion receptors Duf and Sns would be involved either directly or indirectly in recruiting Dia to the fusion site, as embryos that fail to express either of these adhesion receptors fail to recruit Dia to the fusion site. In addition, recent data from Drosophila epithelial tubes indicate that PI(4,5)P2 serves as a localization cue for Dia. Previous work has shown that PI(4,5)P2 accumulates at the fusion site after FC-FCM recognition and adhesion; sequestering of PI(4,5)P2 results in a significant fusion block. Therefore, whether PI(4,5)P2 regulates Dia localization at the fusion site was tested. Dia was found to be recruited to the fusion site in the PI(4,5)P2 sequestered myoblasts, suggesting that, in this context, PI(4,5)P2 signaling is not required for Dia localization. These data provide possible explanations for why in PI(4,5)P2 sequestering embryos, smaller actin foci are detected: the localized Dia may be sufficient to recruit low levels of Arp2/3 and its NPFs, which, upon activation, lead to the formation of small F-actin foci. Nevertheless, in the absence of PI(4,5)P2 signaling, Dia that is recruited to the fusion site is not sufficient to produce functional actin focus, capable of directing a fusion event. Recent work also indicates that charged residues in the N- and C-termini of mDia1 are sufficient both for mDia's clustering of PI(4,5)P2 and its own membrane anchorage. This interaction between mDia1 and PI(4,5)P2, in turn, regulates mDia1 activity. Whether such a mechanism is in play at the myoblast fusion site needs to be further investigated (Deng, 2015).
A working model is proposed for the interplay between the actin regulators during myoblast fusion. Dia is recruited to the fusion site upon engagement of the recognition and adhesion receptors by a yet-to-be determined mechanism. It is proposed that PI(4,5)P2 signaling at the fusion site regulates the localization and activation of downstream targets such as Rho-family of small GTPases. These small GTPases lead to the activation of Dia. Activated Dia, in turn, polymerizes linear actin filaments and, in combination with the recognition and adhesion receptors and PI(4,5)P2, recruits the Arp2/3 NPFs, SCAR and WASp. Activation of these Arp2/3 NPFs at the fusion site would be accomplished by small GTPases such as Rac. These, in turn, would activate Arp2/3, leading to branched actin and formation of the F-actin focus and the invasive podosome. Whether the Arp2/3 NPFs such as SCAR/WAVE would negatively regulate Dia to downregulate linear actin polymerization, as suggested for mDia2 in cell culture, or whether Dia competes with WASp for barbed end binding remains to be investigated. However, these mechanisms would underscore a switch from linear F-actin filopodium formation to the linear and branched F-actin invasive podosome-like structure that is necessary for fusion (Deng, 2015).
The actin focus formed at the fusion site is an F-actin rich, invasive podosome-like structure that has been suggested to provide a mechanical force for FCMs to invade the FC/Myotube. Similar invasive actin structures named invadosomes have been seen in different cell types, such as podosomes in macrophages and invadopodia in cancer cell. Arp2/3 is known to play a key role in invadosome formation, and recent studies have revealed the involvement of formins in developing invadosomes. The current data indicate that specific temporal and spatial interactions between the formin Dia and Arp2/3 are required for the actin focus and invasive podosome formation. The data thus provide new mechanistic insights for the interplay of Arp2/3 and Formins during invadosome formation in these contexts (Deng, 2015).
The Drosophila gene diaphanous is required for cytokinesis. Males homozygous for the dia1 mutation are sterile due to a defect in cytokinesis in the germline. Females trans-heterozygous for dia1 and a deficiency are sterile and lay eggs with defective eggshells; failure of cytokinesis is observed in the follicle cell layer. Null alleles are lethal. Death occurs at the onset of pupation due to the absence of imaginal discs. Mitotic figures in larval neuroblasts have been found to be polyploid, apparently due to a defect in cytokinesis (Castrillon, 1994).
The male-sterile dia1 allele has been identified in a P element screen for mutations affecting spermatogenesis. Although both spermatocytes and spermatids are initially present in dia1 testes, these cells degenerate and are not replenished. By 5 days after eclosion, most mutant testes are devoid of germinal contents. Female fertility is not significantly affected, and the viability of both males and females is normal (Castrillon, 1994).
During spermatogenesis, a spermatogonium (the product of a stem cell division) undergoes four rounds of mitotic division to give rise to a cyst of 16 spermatocytes; meiosis then produces a cyst of 64 haploid spermatids. Wild-type chromosomal segregation and cytokinesis result in spermatids that each contain two major cytological structures of identical size and shape: a pale round nucleus, and an adjacent dark nebenkern. The nebenkern results from the fusion of all the mitochondria in a spermatid; its size thus serves as a marker for the amount of cytoplasm inherited by a spermatid. The contents of testes from 50 newly eclosed dia1 males were examined. dia1 testes contain far fewer spermatids than normal, due to the reduced germinal content. The majority of spermatids were large and multinucleate. These abnormal spermatids contained either 2, 4 or 8 nuclei, with the size of the nebenkerne proportional to the number of nuclei. Of 159 unelongated spermatids identified, 51 (32%) contained one nucleus (phenotypically normal); 26 (16%) contained two nuclei; 81 (51%) contained four nuclei, and 1 (1%) contained 8 nuclei. The presence of spermatids containing 2 or 4 nuclei within a common cytoplasm can be explained by a failure in cytokinesis in one or both of the meiotic divisions. Likewise, the rare spermatid containing 8 nuclei can be explained by a failure of cytokinesis in three consecutive cell divisions, the first being the mitotic division preceding meiosis. The nuclei in defective spermatids are almost always of wild-type size, indicating that chromosome segregation is normal in spite of the failure of cytokinesis. In contrast, in mutants that cause nondisjunction during meiosis, nuclei are of variable size. The finding that the initial failure of cytokinesis can occur at distinct points along the spermatogenesis pathway suggests why the germline in dia1 testes is eventually depleted. Since the 5-9 stem cells present in each testis continually divide to give rise to spermatogonia, failure of cytokinesis during stem cell divisions should result in the permanent inactivation of these cells (Castrillon, 1994).
In trans to a chromosomal deficiency or a null allele such as dia2, the dia1 allele exhibits an oogenesis phenotype. Although the viability of such trans-heterozygous adults is normal and the male germline phenotype is similar to that of dia1 males, female fertility is dramatically decreased. The ovaries of dia1/dia2 females are smaller than wild type. Egg chambers of all stages are present, the great majority of which contain 15 nurse cells and one oocyte. However, the eggs laid by dia1/dia2 females are shorter than wild-type and have short, fused, or extra dorsal appendages. Only 10% of the eggs hatch. This eggshell phenotype suggested a defect in the somatically derived follicle cells, which surround each developing egg chamber and secrete the eggshell. Indeed, follicle cells in mutant ovaries have an abnormal appearance when viewed by differential interference contrast microscopy. Their nuclei vary considerably in size relative to wild-type controls. Some of the cells appear to contain two nuclei. To visualize the follicle cell layer better, ovaries were double labelled with a fluorescent Concanavalin A derivative (to stain plasma membranes) and with propidium iodide (to stain nuclei) and examined by confocal microscopy. Individual follicle cells containing two nuclei are frequently observed in egg chambers from mutant mothers; such cells are not found in wild-type egg chambers. Therefore, it appears that cytokinesis fails to occur in some follicle cells. The abnormally large nuclei seen are likely due to nuclear fusion following the failure of cytokinesis (Castrillon, 1994).
Lethal dia mutations, including null mutations, are generated by imprecise excision of the P element in the dia1 allele. Null alleles such as dia2 result in early pupal lethality and anabsence of imaginal discs. This phenotype is consistent with dia being an essential mitotic gene. Due to the presence of maternal gene products, an embryo with a null mutation in such a gene can develop into a larva. However, since imaginal disc cells divide during the larval stages, by which time zygotic gene expression is required, such larvae will have defective or absent discs and will die at the onset of pupation. Homozygotes for less severe alleles such as dia3 also die as early pupae, but third instar larvae contain imaginal discs, albeit somewhat smaller than normal. Homozygotes for the weakest lethal allele, dia9, have imaginal discs of normal appearance but die as late pupae or pharate adults. Very few (less than 1%) of dia9 flies eclose. These flies are sickly and have a weak rough eye phenotype, consistent with a mitotic defect that affects a small fraction of cells. Testes from these surviving adults are almost completely devoid of germinal content (Castrillon, 1994).
The larval central nervous system (brain and ventral ganglion) is a rich source of dividing cells (neuroblasts) and is the most suitable tissue for examining mitosis in larvae. Chromosome morphology and segregation can be examined in aceto-orcein-stained preparations of the larval CNS. In wild type, a mitotic figure consists of 3 pairs of major chromosomes. In larvae homozygous for a weak lethal allele, dia9, only a small fraction of neuroblasts are polyploid and the number of mitotic figures is not affected. Homozygotes for a stronger allele, dia3, exhibit ploidy ranging from 2n, 4n and 8n to extreme hyperploidy. In addition, significantly fewer mitotic figures are present in dia3 homozygotes than in wild type. However, the fraction of mitotic figures in anaphase is the same as in wild type, whereas mutations that disrupt spindle function result in a decrease in the number of anaphases. In addition, chromosome morphology is generally normal, although a small number of mitotic figures contain highly condensed chromosomes. Homozygotes for a null allele, dia2 , have very few mitotic figures, and all are enormously hyperploid. The morphology of anaphase figures provides direct evidence that cytokinesis is defective in dia3 cells. Cleavage furrows are sometimes evident in wild-type anaphase figures, especially in well-isolated cells. However, cleavage furrows have not been observed in any dia3 anaphases and the cells appear completely round even when the chromosomes have finished migrating to opposite poles. Despite the cytokinesis defect, chromosome segregation appears to be relatively normal in polyploid cells. In bipolar dia3 anaphases, the spindles are well organized and the chromosomes are equally segregated, with no lagging chromosomes or other abnormalities. This is true even in anaphases that are clearly hyperploid. In more extremely hyperploid cells, anaphases are typically multipolar. Such multipolar spindles have also been observed in other mutants that produce hyperploid cells. Taken together, the male-sterile, female-sterile and lethal phenotypes associated with dia mutations demonstrate that dia is required for cytokinesis in both the soma and germline and in mitosis as well as meiosis (Castrillon, 1994).
In animal cells, cytokinesis is accomplished by the contractile ring, a transient structure containing actin and myosin II (see Zipper) filaments, that is anchored to the equatorial cortex. Interactions between these filaments lead to the constriction of a ring that pinches the dividing cell in the middle like an ever tightening purse string until cleavage is completed. Male meiosis was examined in mutants of the chickadee (chic) locus, a Drosophila gene that encodes profilin, a low molecular weight actin-binding protein that modulates F-actin polymerization. These mutants are severely defective in meiotic cytokinesis. Difficulties in meiotic cytokinesis are immediately obvious because of the characteristic appearance of spermatids directly after their formation at the so-called onion stage. Wild-type onion stage spermatids contain a single phase-light nucleus and a similarly sized phase-dark Nebenkern (a mitochondrial derivative). Failures in cytokinesis result in abnormally large Nebenkern associated with multiple normal-sized nuclei. The resulting phenotypes fall into multiple groups: in testes of males homozygous for chic a large fraction of onion-stage spermatids contain a single Nebenkern of larger than normal size, associated with two or more normal-sized nuclei. A substantial proportion have two nuclei with an intermediate-size Nebekern, but most frequently, these aberrant spermatids contain four nuclei and a very large Nebenkern. These phenotypes reflect failures of cytokinesis at either one or the other or both meiotic divisions, respectively, that would prevent proper subdivision of mitochondria and nuclei into daughter spermatids (Giansanti, 1998).
To investigate the relationships between the central spindle and the contractile ring, meiosis was examined in the cytokinesis-defective mutants KLP3A and diaphanous. The KLP3A gene encodes a kinesin-like protein that accumulates in the central spindle midzone during anaphase and telophase of both meiotic divisions. Accordingly, mutations in this gene disrupt central spindle formation and cause frequent failures in meiotic cytokinesis. To check whether the defect in central spindle integrity observed in KLP3A mutants also affects actin ring assembly, KLP3A mutant testes were stained with rhodamine-labeled phalloidin. The results of this experiment clearly show that most mutant ana-telophases (90%) are completely devoid of actin rings. The rare ana-telophases that exhibit thin and incomplete actin rings also contain more densely packed central spindles than those of cells completely lacking contractile rings. Despite the absence of the contractile ring, KLP3A mutants do not exhibit aberrant actin accumulations or problems in aster migration like those described above for chic and tsr mutants (Giansanti, 1998).
The diaphanous gene encodes a protein that interacts with profilin through its proline-rich domain. All the ana-telophases present in testes homozygous for dia mutants are completely devoid of actin rings. There are severe defects in the central spindle, similar to those observed in chic and KLP3A. The effects on the actomyosin contractile ring and the central spindle observed in chic and dia mutants could be specific consequences of lesions in the corresponding gene products. Alternatively, these effects could result from a more general disruption of the actin cytoskeleton. To discriminate between these possibilities, wild-type testes were treated with cytochalasin B prior to fixation and staining. Cytochalasin B binds the barbed ends of actin filaments and promotes the conversion of ATP-actin monomers to ADP-actin, preventing proper assembly of the contractile ring in most cell types. Remarkably, incubation with this drug produces an almost exact phenocopy of strong chic alleles. No F-actin staining is observed in any contractile ring-like structures at the equator of ana-telophase cells (Giansanti, 1998).
In all cases examined, the central spindle and the contractile ring in meiotic ana-telophases were simultaneously absent. Together, these results suggest a cooperative interaction between elements of the actin-based contractile ring and the central spindle microtubules: when one of these structures is disrupted, the proper assembly of the other is also affected. In addition to effects on the central spindle and the cytokinetic apparatus, another consequence of chic mutations was observed: a large fraction of chic spermatocytes exhibit abnormal positioning and delayed migration of asters to the cell poles. A similar phenotype is seen in testes treated with cytochalasin B and has been noted previously in mutants at the twinstar locus. These observations all indicate that proper actin assembly is necessary for centrosome separation and migration, and that the central spindle and the contractile ring are interdependent structures (Giansanti, 1998).
The best candidate at present for mediating interactions between the central spindle and cortical actin, at least during male meiosis, is the KLP3A kinesin-like protein. This protein could interact directly with both the central spindle microtubules and components of the contractile ring. Alternatively, KLP3A could transport to the spindle midzone molecules that mediate F actin-microtubule interactions. At the moment, it is not possible to discriminate between these possibilities, nor is there any information on the proteins that bind to or might be transported by KLP3A. It is believed, however, that the isolation and characterization of additional mutations causing cytological phenotypes similar to those of KLP3A, chic, and dia, will eventually provide substantial insight into the mechanisms underlying microtubule-actin interaction during cytokinesis (Giansanti, 1998).
The Drosophila formin homology (FH) protein Diaphanous has an essential role during cytokinesis. To gain insight into the function of Diaphanous during cytokinesis and explore its role in other processes, embryos deficient for Diaphanous were generated and three cell-cycle-regulated actin-mediated events during embryogenesis were analyzed: formation of the metaphase furrow, cellularization, and formation of the pole cells. In dia embryos, all three processes are defective. Actin filaments do not organize properly to the metaphase and cellularization furrows and the actin ring is absent from the base of the presumptive pole cells. Furthermore, plasma membrane invaginations that initiate formation of the metaphase furrow and pole cells are missing. Immunolocalization studies of wild-type embryos reveal that Diaphanous localizes to the site where the metaphase furrow is anticipated to form, to the growing tip of cellularization furrows, and to contractile rings. In addition, the dia mutant phenotype reveals a role for Diaphanous in recruitment of myosin II, anillin and Peanut to the cortical region between actin caps. There findings thus indicate that Diaphanous has a role in actin cytoskeleton organization and is essential for many, if not all, actin-mediated events involving membrane invagination. Based on known biochemical functions of FH proteins, it is proposed that Diaphanous serves as a mediator between signaling molecules and actin organizers at specific phases of the cell cycle (Afshar, 2000).
The proto-oncogenic kinase Abelson (Abl) regulates actin in response to cell signaling. Drosophila Abl is required in the nervous system, and also in epithelial cells, where it regulates adherens junction stability and actin organization. Abl acts at least in part via the actin regulator Enabled (Ena), but the mechanism by which Abl regulates Ena is unknown. A novel role is described for Abl in early Drosophila development, where it regulates the site and type of actin structures produced. In Abl's absence, excess actin is polymerized in apical microvilli, whereas too little actin is assembled into pseudocleavage and cellularization furrows. These effects involve Ena misregulation. In abl mutants, Ena accumulates ectopically at the apical cortex where excess actin is observed, suggesting that Abl regulates Ena's subcellular localization. Other actin regulators were also examined. Loss of Abl leads to changes in the localization of the Arp2/3 complex and the formin Diaphanous, and mutations in diaphanous or capping protein beta enhance abl phenotypes (Grevengoed, 2003).
Different actin regulators play fundamentally different biochemical roles. Models often picture all of these regulators modulating actin assembly and disassembly at a single site, but of course individual cells target different actin regulators to distinct sites, creating actin structures with diverse functions. Syncytial embryos provide an excellent example. During interphase, they assemble actin-based microvillar caps above each nucleus. As they enter prophase, caps are disassembled and actin polymerization is redirected to the pseudocleavage furrows. This is likely to require new machinery: both Arp2/3 and the formin Dia are required for pseudocleavage furrow formation, but not for actin caps. Cellularization also requires distinct machinery to polymerize/disassemble apical microvilli and to recruit and modulate actin at the cellularization front. For transitions to occur smoothly, two fundamental changes have to occur: the location at which actin polymerization occurs must change, and a different constellation of actin regulators must be deployed to produce the distinct actin structures observed (Grevengoed, 2003).
The data support a hypothesis in which the balance of activity of different actin regulators at distinct sites is tightly regulated, influencing the nature of the actin structures produced. One regulator is Abl. In its absence, Ena localizes ectopically to the cortical region, upsetting the temporal and spatial balance of actin regulators. This leads to a change in both the location and nature of actin polymerization during mitosis. Excess actin is polymerized into microvillar projections that extend from the apical region of the furrows, whereas insufficient actin is directed to the pseudocleavage furrows. Similarly, during cellularization in ablM mutants, actin polymerization continues to be directed to apical microvilli, whereas in a wild-type embryo this ceases early in cellularization (Grevengoed, 2003).
The data also suggest that there is cross-talk between different modulators of actin polymerization, and that the balance of their activities determines the outcome. Although many actin modulators are unaffected in ablM mutants, both the Arp2/3 complex and Dia are recruited to sites of ectopic actin polymerization. However, genetic analysis suggests that although Ena mislocalization plays a critical role in the actin alterations seen in ablM mutants, Dia and Arp2/3 mislocalization may not. In fact, reduction of the dose of Dia enhanced the ablM phenotype. Dia normally promotes actin polymerization lining the furrows. In ablM mutants, the balance of actin polymerization is already shifted to the apical microvilli because of ectopic Ena localization. Reduction in the dose of Dia might further reduce actin polymerization in pseudocleavage furrows, resulting in the observed enhancement of the ablM phenotype. The abnormal recruitment of Dia to the apical regions in ablM mutants may also reduce pseudocleavage furrow formation (Grevengoed, 2003).
It will now be important to investigate how the cell regulates the distinct types of actin polymerization required for distinct cellular and developmental processes. One mechanism of cross-talk may involve direct or indirect recruitment of one type of actin modulator by another. Abl's ability to interact with both Ena and the Arp2/3 regulator WAVE1 is interesting in this regard. However, the recruitment of Arp3 and Dia to ectopic actin structures observed in ablM mutants may have a more simple explanation. Both are thought to have a higher affinity for newly polymerized, ATP-bound actin, which is likely to be increased where ectopic actin polymerization appears to occur (Grevengoed, 2003).
Drosophila Abl also functions in other contexts. It has a role in embryonic morphogenesis, where it also acts, at least in part, via Ena. However, in this context Abl also affects AJ stability. Since Ena is normally highly enriched in AJs, it is hypothesized that Abl helps restrict Ena localization to AJs, and thus helps initiate the proper organization of actin underlying AJs. In Abl's absence, Ena may localize to sites other than AJs, leading to ectopic actin polymerization at those sites and reduction in actin polymerization at AJs (analogous to the divergent effects on apical actin and pseudocleavage/cellularization furrows). Since the cortical actin belt underlying the AJ plays an important role in its stability, this could explain the phenotype of abl mutants. A similar model may help explain the roles of Abl and Ena in axon outgrowth. The network of actin filaments in the growth cone is complex, with different types of actin in filopodia and in the body of the growth cone. By regulating Ena localization, Abl may influence the balance of the different types of actin, thus influencing growth cone motility. Likewise, in fibroblasts, where Ena/VASP proteins regulate motility, the Arp2/3 regulators N-WASP and WAVE localize to sites at the leading edge distinct from those where Mena is found. Whether Abl or Arg regulate the localization of Ena/VASP family proteins in mammals remains to be determined. Likewise, it is possible that deregulation of Ena/VASP proteins underlie some of the alterations in cell behavior in Bcr-Abltransformed lymphocytes. Experiments to test whether Ena/VASP activity is important for either mammalian Abl's normal function or for the pathogenic effects of Bcr-Abl will help answer these questions (Grevengoed, 2003).
The steroid hormone 20-hydroxyecdysone (ecdysone) is the key regulator of postembryonic developmental transitions in insects and controls metamorphosis by triggering the morphogenesis of adult tissues from larvae. The Rho GTPase, which mediates cell shape change and migration, is also an essential regulator of tissue morphogenesis during development. Rho activity can modulate gene expression, in part, by activating LIM kinase (LIMK) and consequently affecting actin-induced SRF transcriptional activity. A link has been established between Rho-LIMK-SRF signaling and the ecdysone-induced transcriptional response during Drosophila development. Specifically, Rho GTPase, via LIMK, regulates the expression of several ecdysone-responsive genes, including those encoding the ecdysone receptor itself, a downstream transcription factor (Br-C), and Stubble, a transmembrane protease required for proper leg formation. Stubble and Br-C mutants exhibit strong genetic interactions with several Rho pathway components in the formation of adult structures, but not with Rac or Cdc42. In cultured SL2 cells, inhibition of Rho, F-actin assembly, or SRF blocks the transcriptional response to ecdysone. Together, these findings indicate a link between Rho-LIMK signaling and steroid hormone-induced gene expression in the context of metamorphosis and thereby establish a novel role for the Rho GTPase in development (Chen, 2004).
Metamorphosis in Drosophila is stringently controlled by pulses of the steroid hormone ecdysone at discrete developmental stages. During larval-pupal transition, ecdysone triggers coordinated changes in tissue morphology that involve histolysis of larval tissues and the initiation of adult structures. Rho GTPase-mediated signaling pathways have been implicated in several aspects of morphogenesis during Drosophila embryo formation. However, a role for Rho signaling in metamorphosis has not yet been reported. Among the downstream mediators of Rho signaling are the LIM kinases, and a closely related Drosophila ortholog of mammalian LIM kinases (designated Dlimk) is specifically expressed at relatively high levels in late larval and pupal stages, suggesting a potential role in Rho-LIMK signaling during this transition. In adult flies, Dlimk is expressed at substantially higher levels in males than in females, consistent with a potential evolutionarily conserved role in spermatogenesis, a process in which mammalian LIMK2 has been implicated. Dlimk mRNA is uniformly expressed throughout eye, wing, and leg imaginal discs (Chen, 2004).
The malformed legs in DlimkD522A flies closely resemble leg defects in flies in which Rho signaling is perturbed through genetic disruption of Rho1, DrhoGEF2 (a guanine nucleotide exchange factor for Rho1), sqh (myosin light chain), and zipper (nonmuscle myosin heavy chain). Sqh and zipper are downstream targets of Drok and regulate actomyosin contractility. Loss-of-function mutants of Rho1 or DrhoGEF2 strongly suppress the severity of wing defects associated with Dlimk expression. Reducing Rho activity by overexpressing the potent Rho inhibitor, p190 RhoGAP, also efficiently suppresses Dlimk-induced wing defects. Moreover, reducing levels of Diaphanous or Drok, two Rho targets that promote actin assembly, also substantially reduces the severity of Dlimk-induced wing defects. A loss-of-function allele of blistered, the Drosophila SRF ortholog, also suppresses the Dlimk-induced wing defects, suggesting that regulation of SRF-dependent transcription by Rho-LIMK signaling plays a role in wing morphogenesis. Significantly, in mammalian cells, LIMK and Diaphanous cooperate to regulate SRF activity (Geneste, 2002). Reducing levels of the Rho-related GTPases, Rac1, Rac2, and Cdc42, or the Rac activator, Myoblast city (Mbc), or the Rac/Cdc42 effector target, PAK, has very little effect on the Dlimk-induced wing phenotype. Thus, it appears that in the developing leg and wing, Dlimk specifically mediates a Rho-actin signaling pathway required for imaginal-disc morphogenesis (Chen, 2004).
Cells migrating through a tissue exert force via their cytoskeleton and are themselves subject to tension, but the effects of physical forces on cell behavior in vivo are poorly understood. Border cell migration during Drosophila oogenesis is a useful model for invasive cell movement. This migration requires the activity of the transcriptional factor Serum response factor (SRF) and its cofactor MAL-D and evidence is presented that nuclear accumulation of MAL-D is induced by cell stretching. Border cells that cannot migrate lack nuclear MAL-D but can accumulate it if they are pulled by other migrating cells. Like mammalian MAL, MAL-D also responds to activated Diaphanous, which affects actin dynamics. MAL-D/SRF activity is required to build a robust actin cytoskeleton in the migrating cells; mutant cells break apart when initiating migration. Thus, tension-induced MAL-D activity may provide a feedback mechanism for enhancing cytoskeletal strength during invasive migration (Somogyi, 2004).
The conserved protein structure, in particular the conserved RPEL motifs (MHD), as well as the functional interactions with SRF suggested that mammalian and fly MAL proteins might be regulated in similar ways. In a series of interesting experiments, activation of mammalian SRF and nuclear accumulation of MAL have shown to respond to changes in actin dynamics in NIH-3T3 cells. The N-terminal RPEL motifs of MAL were required for this regulation, which has also been called the Rho-actin pathway. One of the strongest activators of MAL/SRF was an activated form of Diaphanous, which acts downstream of Rho. To determine whether MAL-D could be subject to the same regulation, a corresponding activated form of Drosophila Diaphanous (HA-diaCA) was made and overexpressed in border cells. Border cell migration was blocked by HA-diaCA; however, nuclear accumulation of MAL-D was nevertheless stimulated. This effect was observed on endogenous MAL-D but was most obvious when looking at border cell clusters cooverexpressing MAL-D and HA-diaCA. In border cells, as in follicle cells, overexpressed MAL-D was predominantly cytoplasmic. In contrast, when HA-diaCA was present, MAL-D was predominantly nuclear. When both proteins were expressed at high levels, the nuclear pool of MAL-D was still detectable, but MAL-D was mainly cytoplasmic, suggesting that nuclear translocation was saturable. Thus, the ability of the Rho pathway to activate MAL proteins appears to be conserved in Drosophila (Somogyi, 2004).
What is the molecular mechanism for MAL-D regulation by tension? Given that the actin cytoskeleton and tension or cell shape changes are interdependent, it is likely that this regulation is related to the regulation of MAL/SRF by actin dynamics (the Rho pathway). Two models were proposed to explain the effect of actin on MAL and SRF. The simplest model is that free G-actin sequesters MAL in the cytoplasm, and depletion of this G-actin pool by actin polymerization results in MAL translocation/activation. Observations in border cells do not fit very well with this simple model. In normal cells, even very highly overexpressed MAL-D is almost exclusively cytoplasmic, indicating practically unlimited capacity in the cytoplasm. Expression of constitutively active Diaphanous, which should 'release' MAL-D by causing actin polymerization, did cause accumulation of MAL-D in the nucleus. But further overexpression of MAL-D led to more protein in the cytoplasm, not in the nucleus as would be expected if G-actin depletion in the cytoplasm (induced by active Diaphanous) were the trigger for nuclear translocation. Finally, even though endogenous MAL-D is expressed at low levels, overexpression of a nonpolymerizable form of actin in border cells did not appear to sequester MAL-D in the cytoplasm. These data seem more consistent with the alternative 'active' model of MAL activation, wherein a subpopulation of actin or an actin protein complex accumulates when actin polymerization is favored, leading to MAL nuclear translocation and activity (Somogyi, 2004).
There are two general ways in which regulation of MAL by actin and by tension might be related. Changes in actin dynamics, as induced by activated Diaphanous, may induce changes in tension, which could then affect MAL. For example, RhoA activation can induce formation of stress fibers, which are contractile structures. Conversely, changes in cell tension could affect RhoA, Diaphanous, and thereby actin dynamics, which then in turn directly regulate MAL. In fact, RhoA and Diaphanous, two of the most potent activators of SRF/MAL, have been shown to be important mediators of mechanosensitive changes at focal adhesions. The physical interaction observed between the conserved N-terminal domain of MAL and unpolymerized forms of actin suggests that regulation of MAL by actin is quite direct and thus supports this type of relationship. Tension applied to cell-matrix attachments or cell-cell interactions may also locally increase actin polymerization by other means and thereby activate MAL. A more speculative link to MAL regulation is offered by actin itself. A specific conformation of actin, or a specific protein complex containing actin, may be induced by tension and serve as the signal that is perceived by MAL. This would be consistent with the idea that a particular subpopulation of actin is responsible for the active regulation of MAL. It would be an elegant way for hard-working migratory cells to regulate strength as needed by the actin cytoskeleton. It is usually thought that actin-myosin supplies force and tension; the MAL/SRF system suggests a role for the complex actin cytoskeleton in force perception as well (Somogyi, 2004).
The physical interaction of the plasma membrane with the associated cortical cytoskeleton is important in many morphogenetic processes during development. At the end of the syncytial blastoderm of Drosophila the plasma membrane begins to fold in and forms the furrow canals in a regular hexagonal pattern. Every furrow canal leads the invagination of membrane between adjacent nuclei. Concomitant with furrow canal formation, actin filaments are assembled at the furrow canal. It is not known how the regular pattern of membrane invagination and the morphology of the furrow canal is determined and whether actin filaments are important for furrow canal formation. Both the guanyl-nucleotide exchange factor RhoGEF2 and the formin Diaphanous (Dia) are required for furrow canal formation. In embryos from RhoGEF2 or dia germline clones, furrow canals do not form at all or are considerably enlarged and contain cytoplasmic blebs. Both Dia and RhoGEF2 proteins are localised at the invagination site prior to formation of the furrow canal. Whereas they localise independently of F-actin, Dia localisation requires RhoGEF2. The amount of F-actin at the furrow canal is reduced in dia and RhoGEF2 mutants, suggesting that RhoGEF2 and Dia are necessary for the correct assembly of actin filaments at the forming furrow canal. Biochemical analysis shows that Rho1 interacts with both RhoGEF2 and Dia, and that Dia nucleates actin filaments. These results support a model in which RhoGEF2 and dia control position, shape and stability of the forming furrow canal by spatially restricted assembly of actin filaments required for the proper infolding of the plasma membrane (Grosshans, 2005).
This morphological analysis of the mutant phenotypes reveals a new function of RhoGEF2 and dia in the formation of the furrow canal. This function is consistent with the co-localisation of both proteins with F-actin at the furrow canal and the reduced amounts of F-actin in RhoGEF2 and dia mutants. Biochemical analysis demonstrates actin polymerisation by Dia and thus supports the model that RhoGEF2 and Dia organise actin filaments to control the formation of the furrow canals. Furthermore, evidence is provided that the previously characterised genes nullo and sry- alpha act in a genetic pathway in parallel to RhoGEF2 and dia, suggesting that they control two distinct aspects of furrow canal formation. This conclusion is based on the assumption that amorphic situtations were used in these experiment. The possibility cannot be excluded that RhoGEF2 and dia stabilise the furrow canal rather than control its initial formation. A function in the formation is supported by the observation that the proportion of nuclei in multinuclear cells does not increase in the course of cellularisation (Grosshans, 2005).
The following arguments support the hypothesis that RhoGEF2 and dia act in the same genetic pathway that controls spatially restricted assembly of actin filaments. In both dia and RhoGEF2 mutants the morphology of the furrow canal is disrupted. The furrow canals are much larger than normal and filled with cytoplasmic blebs. Both proteins are localised at the furrow canal and both precede the appearance of the cellularisation front. The localisation of both proteins does not depend on F-actin. However, they are directly or indirectly involved in the assembly of F-actin since the amount of F-actin is reduced at the furrow canal of the mutant embryos. The strongest argument for a functional connection is that Dia localisation at the furrow canal depends on RhoGEF2 during the early phase of cellularisation. Rho1 may mediate this functional link by direct interactions with RhoGEF2 and Dia. However, the findings do not show that RhoGEF2 exclusively functions via dia. Other targets of Rho1-GTP, like citron kinase, protein kinase N or Rho kinase may be activated in parallel to Dia. Although a reduction of MyoII at the furrow canal was observed during the first half of cellularisation in embryos from RhoGEF2 germline clones, correspondingly lower MyoII levels are also observed in embryos from dia germline clones, which indicates that the reduction of MyoII may be a consequence of reduced F-actin levels. Consistent with the reduction of F-actin at the furrow canal, levels of MyoII were also reduced in the mutant embryos. In contrast to the reduction at the furrow canal, cortical F-actin appeared to be increased in some embryos from dia germline clones. This increase was variable and not observed in all of the experiments, however (Grosshans, 2005).
The difference in the RhoGEF2 and dia mutant phenotypes clearly shows that dia has additional functions and may be controlled by other not yet identified factors besides RhoGEF2. Whereas RhoGEF2 mutants pass through the cleavage cycles without obvious defects, dia is involved in formation of pole cells and pseudo cleavage furrows. As a possible consequence of these additional functions, dia mutants in contrast to RhoGEF2 mutants often have a more disrupted F-actin array, larger furrow canals and a more disturbed cellularisation than RhoGEF2 mutants. Furthermore in the early phase of cellularisation Dia localisation depends on RhoGEF2, whereas later, after the furrow has formed, Dia becomes enriched to a certain degree at the cellularisation front independently of RhoGEF2. One gene that may act in parallel to RhoGEF2 to control Dia localisation is Abl. Embryos from Abl germline clones have reduced amounts of Dia at the furrow canal and show a disrupted F-actin array similar to that observed in dia and RhoGEF2 mutants. However, the molecular link between Abl and Dia is elusive and no abnormalities in the morphology of the furrow canal in Abl mutants have been described. Thus Dia may be controlled and activated by multiple pathways including RhoGEF2 among others (Grosshans, 2005).
It is not known how the position of the invaginating plasma membrane is determined. RhoGEF2 and Dia are not likely to be part of a pattern formation process, but their localisation reflects an early readout of this pattern, since the nuclei and centrosomes are properly arranged in RhoGEF2 and dia mutants. RhoGEF2 and Dia proteins are early markers for these sites and precede furrow canal formation because specific staining was detected for both Dia and RhoGEF2 when the nuclei were still spherical and when the cellularisation front was not yet visible. Other factors beside RhoGEF2 and Dia are also involved in furrow canal formation, because many furrow canals still form in RhoGEF2 and dia mutants, which indicates that there is genetic redundancy (Grosshans, 2005).
At present it can only be speculated about which factors and mechanisms are responsible for RhoGEF2 localisation. Candidates may be among the group of genes involved in furrow canal formation. However, for all of these mutations no ultrastructural analysis has been reported that would allow the morphological defect to be defined and their function for furrow canal formation to be compared with the function of RhoGEF2 and dia. Among this group are Rab11 and nuf, which encode a GTPase of the recycling endosome and its putative effector. Considering the assumed biochemical activities, it is conceivable that vesicle targeting is important for transporting factors to the site of membrane invagination. This raises the possibility that RhoGEF2 is transported by such vesicles to the sites of membrane infolding. Analysis of RhoGEF2 protein distribution in nuf and Rab11 mutants and the phenotype of double mutants may address this hypothesis. Alternatively, RhoGEF2 may be transported to the site of the future furrow canal along microtubules that form open baskets around the nuclei, or other recruiting factors may precede at the site of membrane invagination (Grosshans, 2005).
Furthermore, slow as molasses (slam) is required for timed formation of the furrow and invagination of the membrane in the first half of cellularisation. Like Dia and RhoGEF2 Slam protein localises to the furrow canal and localisation precedes furrow canal formation. Slam may act by recruiting MyoII to the furrow canal, but the biochemical activities of Slam have not been defined. Although the membrane does not invaginate initially in slam mutants, a complete F-actin array is visible. Thus despite the overlapping localisation of Slam, RhoGEF2 and Dia, their functions are clearly distinguishable (Grosshans, 2005).
How do RhoGEF2 and Dia act in furrow canal formation? If the biochemical activity of Dia is considered to nucleate actin filaments and the enlarged and labile furrow canals in the dia mutants, it is conceivable that Dia organises and assembles a coat of F-actin at the site of membrane invagination and furrow canal formation. The coat of F-actin may be important for the compactness and stability of the furrow canal to prevent infoldings of the cytoplasm. Such a function may be related to the function of F-actin in endocytic events. The subset of actin filaments controlled by RhoGEF2 would not significantly contribute to pulling in the plasma membrane, since membrane invagination proceeds with normal speed in RhoGEF2 mutants. Alternatively, RhoGEF2 and Dia may perform their function independently of actin polymerisation. Although the amount of F-actin is reduced in the mutants, the possibility that the polymerisation activity of Dia is not required for all or part of its function cannot be excluded. Dia may also influence the organisation of microtubules, as interactions of mDia1 with microtubules and EB1, a microtubule-associated protein, have been described (Grosshans, 2005).
The differences in protein localisation and mutant phenotypes of RhoGEF2 and nullo suggest that they have distinct activities. In contrast to the frequently missing furrow canals in single mutants, their complete absence in embryos lacking both gene functions clearly implies, however, that their functions are redundant from a genetic point of view. These results show that RhoGEF2 and dia are required for the formation of a compact and stable furrow canal. If one of the two pathways is disturbed, the furrow canal can still form, albeit with a lower and variable efficiency that depends on the conditions. For example the nullo phenotype is strongly temperature sensitive. However, if both pathways are affected, furrow canals do not form at all. Future studies will resolve how the actin filaments are involved in bending the plasma membrane that leads to the furrow canal and will further demonstrate how RhoGEF2 protein is expressed in the hexagonal array to serve as a template for local actin polymerisation (Grosshans, 2005).
The correct localization of myosin II to the equatorial cortex is crucial for proper cell division. A collection of genes was examined that causes defects in cytokinesis and revealed (with live cell imaging) two distinct phases of myosin II localization. Three genes in the rho1 signaling pathway, pebble (a Rho guanidine nucleotide exchange factor), rho1, and rho kinase, are required for the initial recruitment of myosin II to the equatorial cortex. This initial localization mechanism does not require F-actin or the two components of the centralspindlin complex, the mitotic kinesin pavarotti/MKLP1 and racGAP50c/CYK-4. However, F-actin, the centralspindlin complex, formin (diaphanous), and profilin (chickadee) are required to stably maintain myosin II at the furrow. In the absence of these latter genes, myosin II delocalizes from the equatorial cortex and undergoes highly dynamic appearances and disappearances around the entire cell cortex, sometimes associated with abnormal contractions or blebbing. These findings support a model in which a rho kinase-dependent event, possibly myosin II regulatory light chain phosphorylation, is required for the initial recruitment to the furrow, whereas the assembly of parallel, unbranched actin filaments, generated by formin-mediated actin nucleation, is required for maintaining myosin II exclusively at the equatorial cortex (Dean, 2005).
This study has discovered three steps in the myosin II localization/activation process that involve distinct groups of genes: (1) an initial recruitment of myosin II to the equatorial cortex that is independent of F-actin and centralspindlin but requires rho1 signaling; (2) a secondary stabilization of myosin II at the midzone that requires F-actin and a second set of genes that are likely involved in building a specific type of actin network, and (3) the activation of furrowing once myosin II is localized that depends on centralspindlin (Dean, 2005).
Rho1, its activating guanidine nucleotide exchange factor pebble, and rho kinase are each required for the initial recruitment of myosin II to the equatorial cortex. Rho1 has been implicated in two pathways that are important for cytokinesis. In the first pathway, rho1 signals to F-actin through the formin diaphanous. However, proteins on this F-actin pathway, including F-actin itself, are not essential for the initial myosin II recruitment to the equatorial cortex. However, rho kinase, another downstream target of rho1, is essential. Because rho kinase phosphorylates the myosin II RLC, it is possible that phosphorylation of the RLC is essential for myosin II recruitment to the furrow. This hypothesis could not be directly tested, because the myosin II heavy chain forms large aggregates when the RLC is depleted by RNAi (Dean, 2005).
Phosphorylation of the RLC both activates the motor domain and, in some myosins, increases bipolar thick filament formation. Because F-actin is not required for myosin II recruitment, activation of the motor is unlikely to be the mechanism by which phosphorylation of the RLC would cause recruitment of myosin II to the equatorial cortex. It is quite possible, however, that the rho kinase-mediated myosin II phosphorylation leads to thick filament assembly and that this assembly is important for localization of myosin to the equatorial cortex. Indeed, in Dictyostelium, it is clear that bipolar thick filament formation is sufficient for myosin II localization to the midzone of a mitotic cell. The nonactin-based mechanism of recruitment of myosin II filaments remains unknown (Dean, 2005).
In contrast to the lack of F-actin involvement in the early recruitment of myosin II to the equatorial cortex at anaphase, F-actin disruption by Latrunculin A results in a failure to maintain myosin II in the equatorial region. Interestingly, the downstream rho1 effectors diaphanous/formin and chickadee/profilin are also necessary for myosin II maintenance at the equatorial midzone. Although the loss of these genes could deplete F-actin, phalloidin staining has shown that F-actin is still present in all of the RNAi-treated cells. In addition, these RNAi-treated cells still contract, unlike when F-actin is completely disrupted with LatA. Thus, myosin II appears to be interacting with F-actin in the cortex as it disperses in dynamic patches throughout the cortex of these diaphanous- or chickadee-depleted cells (Dean, 2005).
It is suggested that the role of diaphanous/formin and chickadee/profilin in maintaining the myosin II contractile ring is through the creation of specific F-actin structures. In particular, formin- and profilin-mediated nucleation results in unbranched actin filaments because profilin promotes the barbed-end growth of formin-capped actin filaments. Indeed, electron microscopy has shown that F-actin in the cleavage furrow mainly consists of unbranched, bundled filaments. These parallel filaments contrast with Arp2/3-mediated nucleation, which creates a highly branched actin filament network. Indeed, Arp2/3, although essential for lamellipodia formation, is not required for cytokinesis in Drosophila cells. The hypothesis here is that once myosin II is recruited to the equatorial cortex of the cell by a rho kinase-dependent mechanism, possibly localized activation of RLC phosphorylation, it is retained there because of its higher affinity for parallel, unbranched actin filaments than to branched actin networks. Consistent with this hypothesis, myosin II is depleted from the lamellipodia in migrating cells where Arp2/3 is localized and branched F-actin networks are formed but is enriched in the lamella where F-actin filaments are more likely to be aligned in parallel bundles. Thus, it is proposed that high rho1 signaling to Diaphanous at the cleavage furrow maintains a higher concentration of parallel actin filaments in this region compared with the rest of the cortex, and these parallel filaments serve to selectively retain myosin II at the equator to form a stable contractile ring. In the absence of these parallel actin filaments, myosin II can bind branched F-actin throughout the cortex, perhaps occasionally organizing them into parallel bundles that cause increased myosin recruitment corresponding to the flashes of cortical myosin accumulation, but these interactions are unstable (Dean, 2005).
Live-cell imaging shows that when pavarotti or racGAP50c are depleted, the cells do not display significant contractions despite recruiting myosin II to the equatorial cortex. Although there is some modest membrane contractile activity in these cells, it is clear that significant contraction or furrowing requires both components of the centralspindlin complex. It is surprising that only these proteins were found to be necessary for cortical contraction at sites of myosin II localization. Data from fixed cells, as well as earlier studies, indicated that Drosophila cells do not undergo equatorial contractions during mitosis when Diaphanous or Chickadee is depleted. However, live-cell imaging shows that when either of these two genes is depleted in S2 cells, not only is myosin II transiently localized to the equatorial cortex before dispersing, but cells do indeed display transient equatorial contraction. It is difficult to recognize these events in fixed cells because of their transient nature and the somewhat irregular shapes of cells depleted of these proteins. This work highlights the importance of live-cell imaging in the study of dynamic processes such as cytokinesis (Dean, 2005).
In addition to the suppression of furrowing, depletion of centralspindlin also leads to an inability to retain F-actin exclusively at the equatorial cortex during cytokinesis. This similar phenotype of the centralspindlin complex and the F-actin affecting proteins suggests that centralspindlin may be an upstream regulator of F-actin filament formation. Indeed kinase-dead mutants of Pavarotti have been shown to accumulate at the spindle poles and are associated with an abnormal accumulation of F-actin near the centrosomes. Centralspindlin may be acting indirectly by helping to localize an important actin-affecting protein at the central spindle, or it may act more directly on the cortex. Because RacGAP50c has been shown to bind Pebble in vitro, it has been hypothesized that centralspindlin affects the F-actin cortex through rho1 signaling by the localization and/or activation of Pebble. However, RacGAP50c depletion does not lead to a lack of myosin II recruitment as does Pebble or Rho1 depletion, and, thus, centralspindlin must act in a rho1-independent manner. For instance, the racGAP activity of centralspindlin may itself be important for signaling to the F-actin cortex. Finally, centralspindlin cannot be the major actomyosin ring positioning signal because myosin II is properly recruited in its absence (Dean, 2005).
Morphogenesis of the Drosophila embryo is associated with dynamic rearrangement of the actin cytoskeleton mediated by small GTPases of the Rho family. These GTPases act as molecular switches that are activated by guanine nucleotide exchange factors. One of these factors, DRhoGEF2, plays an important role in the constriction of actin filaments during pole cell formation, blastoderm cellularization, and invagination of the germ layers. This study shows that DRhoGEF2 is equally important during morphogenesis of segmental grooves, which become apparent as tissue infoldings during mid-embryogenesis. Examination of DRhoGEF2-mutant embryos indicates a role for DRhoGEF2 in the control of cell shape changes during segmental groove morphogenesis. Overexpression of DRhoGEF2 in the ectoderm recruits myosin II to the cell cortex and induces cell contraction. At groove regression, DRhoGEF2 is enriched in cells posterior to the groove that undergo apical constriction, indicating that groove regression is an active process. The Formin Diaphanous is required for groove formation and strengthens cell junctions in the epidermis. Morphological analysis suggests that Dia regulates cell shape in a way distinct from DRhoGEF2. It is proposed that DRhoGEF2 acts through Rho1 to regulate acto-myosin constriction but not Diaphanous-mediated F-actin nucleation during segmental groove morphogenesis (Mulinari, 2008).
Analysis of segmental groove morphogenesis revealed that during invagination groove founder cells undergo two distinct phases of shape change. Initial apical constriction is associated with accumulation of F-Actin, DRhoGEF2 and Dia at the site of groove initiation. Together with the loss of apical constriction in DRhoGEF2 or dia mutants and the observation that DRhoGEF2 or constituatively active diaCA-overexpressing grooves form earlier, are deeper, and persist longer than wild-type grooves, this indicates that contractile forces in epidermal cells provide a driving force for groove morphogenesis (Mulinari, 2008).
Subsequent to apical constriction, groove founder cells and their immediate neighbors elongate their apical-basal axis and extend their basal sides inward. Apical-basal elongation does not occur when DRhoGEF2 or diaCA are overexpressed, suggesting that activation of these factors may counteract this specific change in cell shape. Inward movement of the epithelial baseline is not affected, and it occurs to a similar depth as in wild type, suggesting that it is not resulting from a pushing force generated by cell elongation. Expression of DRhoGEF2 in underlying mesodermal cells does not affect groove formation. Thus, cell autonomous bending of the epithelium rather than a pulling force originating in underlying tissues may be the main contributor to groove invagination (Mulinari, 2008).
It has been suggested that germband retraction that occurs in parallel to segmental groove formation may contribute to the forces that cause groove founder cells to invaginate. In DRhoGEF2 mutants, the posterior midgut primordium remains at the posterior pole due to failure of the endoderm to invaginate. Consequently, the germband is thrown into deep folds that illustrate the pressure exerted on the epithelial sheet. Yet, segmental grooves do not form in these embryos. Furthermore, local expression of DRhoGEF2 causes the formation of deep grooves in expressing cells but not in neighboring tissues. These observations do not exclude that global tissue rearrangements support segmental groove invagination, but they suggest that the major contributing force to groove formation is due to autonomous action of epidermal cells (Mulinari, 2008).
At the onset of stage 13 when germband retraction has completed, DRhoGEF2, F-actin, and myosin II accumulate apically in four to five cells posterior to the grooves. These cells subsequently constrict apically, which may bend the epithelium in an outward direction. Thus, groove regression may be actively driven by actomyosin-based constriction of epidermal cells. This is supported by previous findings showing that in zipper mutants, which partially lack nonmuscle myosin, grooves occasionally do not regress (Mulinari, 2008).
Groove regression coincides with the formation of denticles in ventral epidermal cells posterior to the groove. Denticle forming cells undergo cell shape changes that are followed by polarized accumulation of F-actin, myosin II, and Dia into condensations at the posterior cell boundary. DRhoGEF2 in contrast to Dia and myosin II, does not polarize to the F-Actin condensations and DRhoGEF2 mutants are able to form denticles. DRhoGEF2 is therefore unlikely to play a role in denticle formation. It is proposed that the narrowing of cells along the anterior-posterior axis that occurs throughout the ventral and lateral epidermis before hair formation may be due to DRhoGEF2-controlled cell contraction and may contribute to the regression of segmental grooves (Mulinari, 2008).
In conclusion, the data suggest that groove invagination and regression are active processes driven by acto-myosin-based contractile filaments in epidermal cells that are regulated by DRhoGEF2 and Dia in a concentration-dependent manner (Mulinari, 2008).
The small GTPase Rho1 regulates acto-myosin constriction through a pathway including Rho-kinase, the regulatory subunit of myosin light chain phosphatase and myosin II. DRhoGEF2 regulates cell contraction in different developmental contexts and DRhoGEF2-mediated recruitment of myosin II to the apical cortex and cell contraction require Rho-kinase, suggesting that DRhoGEF2 controls acto-myosin contraction through the Rho-kinase pathway (Mulinari, 2008).
Rho1 also regulates F-actin nucleation and polymerization through a pathway including Dia and the actin-binding protein Profilin. In cell culture systems, mammalian Dia1 has been shown to act as a positive feedback regulator of Rho-activity through direct interaction with the C terminus of the mammalian DRhoGEF2 homologue LARG. However, it is not clear whether Dia can act as an effector of DRhoGEF2 during morphogenesis in vivo. Drosophila Dia is required in processes such as metaphase furrow invagination, pole cell formation and cellularization that are also defective in DRhoGEF2 mutants consistent with the idea that both proteins may function in concert (Mulinari, 2008).
The data suggest that DRhoGEF2 and Dia are required for segmental groove morphogenesis. Interestingly, morphologic changes elicited by DiaCA at the epithelial and cellular level were distinct from those observed with DRhoGEF2. Whereas DRhoGEF2-overexpression caused cells to contract and take on a rounded shape, thereby reducing contact with each other, diaCA-expressing cells remained tightly packed and columnar. In addition, cells showed increased levels of the junctional proteins β-catenin and E-cadherin, suggesting a strengthening of cell-cell contacts. Consistent with this, mammalian Dia1 has been implicated in maintenance and stabilization of adherens junctions. In contrast, Rho-kinase-mediated generation of contractile force leads to the physical disruption of cell-cell contacts and cell rounding reminiscent of DRhoGEF2 overexpression. Another distinguishing feature between DRhoGEF2 and dia is the accumulation of F-actin in response to diaCA expression that is consistent with the role of Dia in nucleation and elongation of F-actin filaments. F-actin accumulation was not observed in response to DRhoGEF2-overexpression consistent with the view that DRhoGEF2 regulates F-actin contraction but not polymerization. This study found that DRhoGEF2 and Dia have qualitatively different effects on F-actin remodeling, supporting the view that they may be connected to the actin cytoskeleton by distinct Rho-effector pathways. Despite the differences, overexpression of either DRhoGEF2 or diaCA in some instances seems to result in increased contractility. In DRhoGEF2, this is likely due to direct regulation of acto-myosin contractility through the Rho-kinase pathway. In DiaCA, the increased levels of actin elicited by Dia activation might indirectly promote the formation of acto-myosin filaments. Alternatively, DiaCA could affect contractility through its effect on myosin (Mulinari, 2008).
Expression of either diaCA or DRhoGEF2 led to increased myosin II levels. In Schneider cells, DRok activity and myosin phosphorylation were found to be required for myosin II recruitment to contractile acto-myosin fibers, whereas Dia was required for maintenance of myosin at contractile rings. Similar to Rho-kinase, DRhoGEF2 is required for apical recruitment of myosin II in the ventral furrow during gastrulation. A role of Dia in this process has not been reported but it is possible that DRhoGEF2 and Dia may regulate different aspects of myosin function also in the Drosophila embryo (Mulinari, 2008).
In summary, DRhoGEF2 and Dia were found to be required for segmental groove formation, and they may act in concert to regulate a series of specific cell shape changes that lead to groove invagination. However, clear differences in the response of cells to activation of DRhoGEF2 or Dia at the morphologic and molecular level suggest that both genes may regulate the actin cytoskeleton through distinct effector pathways (Mulinari, 2008).
The Hippo tumour suppressor pathway is a conserved signalling pathway that controls organ size. The core of the Hpo pathway is a kinase cascade, which in Drosophila involves the Hpo and Warts kinases that negatively regulate the activity of the transcriptional coactivator Yorkie. Although several additional components of the Hippo pathway have been discovered, the inputs that regulate Hippo signalling are not fully understood. This study reports that induction of extra F-actin formation, by loss of Capping proteins A or B, or caused by overexpression of an activated version of the formin Diaphanous, induces strong overgrowth in Drosophila imaginal discs through modulating the activity of the Hippo pathway. Importantly, loss of Capping proteins and Diaphanous overexpression does not significantly affect cell polarity and other signalling pathways, including Hedgehog and Decapentaplegic signalling. The interaction between F-actin and Hpo signalling is evolutionarily conserved, as the activity of the mammalian Yorkie-orthologue Yap is modulated by changes in F-actin. Thus, regulators of F-actin, and in particular Capping proteins, are essential for proper growth control by affecting Hippo signalling (Sansores-Garcia, 2011).
This study investigated a role of actin Capping proteins and changes in actin organization on tissue growth. Changing the organization of the actin cytoskeleton affects growth by modulating the activity of the Hpo pathway. Several observations support this conclusion. First, loss of Capping proteins, or induction of extra F-actin by overexpression of DiaCA, induced strong overgrowth of Drosophila imaginal discs. Second, changes in actin organization lead to the upregulation of Hpo pathway target genes, which depended on normal Yki activity. Third, the effects of DiaCA or loss of Capping proteins on Hpo signalling are specific downstream effects and not the cause of general defects in cellular organization and signalling. Fourth, actin dynamics and the Hpo pathway interact with each other in evolutionary distant species. Therefore, F-actin regulates growth in different species through effects on the Hpo pathway (Sansores-Garcia, 2011). p>Several observations were striking. First, the data suggest that the effects on Hpo signalling are specific effects of F-actin accumulation. Given the crucial role for F-actin in numerous cellular processes, it might have been expected that imbalances in F-actin organization lead to defects in many different signalling pathways. Surprisingly, however, while changing F-actin organization had strong effects on Hpo signalling, it did not significantly affect epithelial cell polarity, or Hh and Dpp signalling, indicating a specific molecular effect. Second, given the pleiotropic functions of F-actin, it might have been expected that knockdown of Capping proteins would lead to reduced growth. On the contrary, loss of Capping proteins or higher levels of F-actin induced by DiaCA lead to increased proliferation and overgrowth, although mutant regions showed some dying cells. It is therefore concluded that Capping proteins act as tumour suppressors that affect growth through the Hpo pathway (Sansores-Garcia, 2011).
The observations that both loss of Cpa and Cpb, as well as overexpression of activated Dia-induced overgrowth indicate that their effects on growth are due to F-actin accumulation. It is currently not known whether the observed effects involve a specific pool of F-actin or whether any increase in F-actin induces growth. A screen in S2 cells identified several other genes involved in F-actin formation that modulated Yki activity. It remains to be seen whether these also modulate Hpo signalling in vivo (Sansores-Garcia, 2011).
The effect of modulating F-actin organization on the Hpo pathway may be evolutionary conserved as strong effects on Yap localization and activity is seen in mammalian cells. Therefore, proteins that restrict F-actin formation may be tumour suppressors in humans and associated with cancer. Indeed, one example of an inhibitor of F-actin polymerization that is downregulated in several cancers is Gelsolin. Gelsolin is known to sever F-actin filaments and to cap them, which inhibits F-actin polymerization. Thus, modulators of the F-actin cytoskeleton affect cell proliferation in mammals and may be involved in the development of cancer (Sansores-Garcia, 2011).
To gain insight into the mechanism by which F-actin affects the Hpo pathway, the localization of different Hpo pathway components was analyzed. Mer and Ex, which contain FERM (4.1 protein-ezrin-radixin-moesin) domains and are known to bind F-actin, localized normally in cells that lost Cpa function, and similarly Hpo localization was unaffected. However, Yki localization was affected such that more Yki protein localized to the nuclei in cells that lost Capping protein function. Therefore, the F-actin status affects growth upstream of Yki, but might not affect growth by regulating the localization of upstream components in the Hpo pathway. The in vivo data show that overexpression of Ex or Hpo did not significantly rescue DiaCA-induced phenotypes in contrast to their ability to rescue fat and ex;mer mutant phenotypes. Overexpression of Wts, however, significantly suppressed DiaCA-induced overgrowth and Hpo pathway target gene expression. Interestingly, ex mutant cells have increased levels of F-actin, although not as much as cells depleted for Capping proteins. Thus, Ex could regulate Hpo signalling indirectly through its effect on F-actin. However, two observations argue against this possibility. First, overexpression of Hpo can rescue ex mutant phenotypes, but not those caused by DiaCA. Second, Ex and Mer directly interact with the Hpo cofactor Sav. Altogether, these data suggest that F-actin affects growth in parallel to Ex and Hpo but upstream of Yki (Sansores-Garcia, 2011).
Recent work showed that a small fraction of the mammalian homologues of Hpo, MST1, and MST2, localize to apical actin filaments. Upon disruption of the actin filaments, MST1/2 were activated, although it is not known whether this involves a relocalization of MST1/2. Consistent with MST activation, it was found that under similar conditions in which sever F-actin bundles are severed, Yap is exported from the nucleus and its activity is downregulated. It is not known whether the same or different mechanisms are engaged to regulate Hpo signalling in response to severing or inducing actin filaments, but elucidation of the molecular mechanisms involved will answer this question (Sansores-Garcia, 2011).
The data reveal an interaction between F-actin organization and the Hpo pathway in the regulation of growth. A possible connection between F-actin and growth may involve the sensing of mechanical forces. In vitro, cells change their rate of proliferation in response to external mechanical forces, which requires an intact actin cytoskeleton. In vivo, the actin cytoskeleton might act as a sensor to couple mechanical forces to growth control. While it is not clear whether these effects depend on the Hpo pathway, it is an exciting possibility to be tested in the future (Sansores-Garcia, 2011).
Actin-based protrusions are important for signaling and migration during development and homeostasis. Defining how different tissues in vivo craft diverse protrusive behaviors using the same genomic toolkit of actin regulators is a current challenge. The actin elongation factors Diaphanous and Enabled both promote barbed-end actin polymerization, and can stimulate filopodia in cultured cells. However, redundancy in mammals and the Diaphanous role in cytokinesis limit analysis of whether and how they regulate protrusions during development. This study used two tissues driving Drosophila dorsal closure, migratory leading-edge (LE) and non-migratory amnioserosal (AS) cells, as models to define how cells shape distinct protrusions during morphogenesis. Non-migratory AS cells were found to produce filopodia that are morphologically and dynamically distinct from those of LE cells. It was hypothesized that differing Enabled and/or Diaphanous activity drive these differences. Combining gain- and loss-of-function with quantitative approaches revealed Diaphanous and Enabled each regulate filopodial behavior in vivo and defined a quantitative 'fingerprint', the protrusive profile, which the data suggest is characteristic of each actin regulator. The data suggest LE protrusiveness is primarily Enabled-driven, while Diaphanous plays the primary role in the AS, and reveal each has roles in dorsal closure, but its robustness ensures timely completion in their absence (Nowotarski, 2014).
Cell-cell intercalation is used in several developmental processes to shape the normal body plan. There is no clear evidence that intercalation is involved in pathologies. This study used the proto-oncogene myc to study a process analogous to early phase of tumour expansion: myc-induced cell competition. Cell competition is a conserved mechanism driving the elimination of slow-proliferating cells (so-called 'losers') by faster-proliferating neighbours (so-called 'winners') through apoptosis and is important in preventing developmental malformations and maintain tissue fitness. Using long-term live imaging of myc-driven competition in the Drosophila pupal notum and in the wing imaginal disc, this study showed that the probability of elimination of loser cells correlates with the surface of contact shared with winners. As such, modifying loser-winner interface morphology can modulate the strength of competition. Elimination of loser clones requires winner-loser cell mixing through cell-cell intercalation. Cell mixing is driven by differential growth and the high tension at winner-winner interfaces relative to winner-loser and loser-loser interfaces, which leads to a preferential stabilization of winner-loser contacts and reduction of clone compactness over time. Differences in tension are generated by a relative difference in F-actin levels between loser and winner junctions, induced by differential levels of the membrane lipid phosphatidylinositol (3,4,5)-trisphosphate. These results establish the first link between cell-cell intercalation induced by a proto-oncogene and how it promotes invasiveness and destruction of healthy tissues (Levayer, 2015).
To analyse quantitatively loser cell elimination, long-term live imaging was performed of clones showing a relative decrease of the proto-oncogene myc in the Drosophila pupal notum, a condition known to induce cell competition in the wing disc. Every loser cell delamination was counted over 10 h, and the probability of cell elimination was calculated for a given surface of contact shared with winner cells. A significant increase was observed of the proportion of delamination with winner-loser shared contact, whereas this proportion remained constant for control clones. The same correlation was observed in ex vivo culture of larval wing disc. Cell delamination in the notum was apoptosis dependent and expression of flowerlose (fwelose), a competition-specific marker for loser fate, was necessary and sufficient to drive contact-dependent delamination. Moreover it was confirmed that contact-dependent death is based on the computation of relative differences of fwelose between loser cells and their neighbours. Thus, cell delamination in the notum recapitulates features of cell competition (Levayer, 2015).
This suggests that winner-loser interface morphology could modulate the probability of eliminating loser clones. Using the wing imaginal disc, winner-loser contact was reduced by inducing adhesion- or tension-dependent cell sorting and observed a significant reduction of loser clone elimination. This rescue was not driven by a cell-autonomous effect of E-cadherin (E-cad) or active myosin II regulatory light chain (MRLC) on growth, death or cell fitness but rather by a general diminution of winner-loser contact. Competition is ineffective across the antero-posterior compartment boundary, a frontier that prevents cell mixing through high line tension. Accordingly, there was no increase in death at the antero-posterior boundary in wing discs overexpressing fweloseA in the anterior compartment. However, reducing tension by reducing levels of myosin II heavy chains was sufficient to increase the shared surface of contact between cells of the anterior and posterior compartments, and induced fwelose death at the boundary. Altogether, it is concluded that the reduction in surface contact between winners and losers is sufficient to block competition, which explains how compartment boundaries prevent competition (Levayer, 2015).
Loser clones have been reported to fragment more often than controls, whereas winner clones show convoluted morphology, suggesting that winner-loser mixing is increased during competition. This could affect the outcome of cell competition by increasing the surface shared between losers and winners. Clone splitting was used as a readout for loser–winner mixing. Two non-exclusive mechanisms can drive clone splitting: cell death followed by junction rearrangement, or junction remodelling and cell–cell intercalation independent of death. To assess the contribution of each phenomenon, the proportion of clones fragmented 48 h after clone induction (ACI) was systematically counted. A twofold increase was observed in the frequency of split clones in losers (wild type (WT) in tub-dmyc) versus WT in WT controls. Overexpressing E-cad or active myosin II was sufficient to prevent loser clone splitting, whereas blocking apoptosis or blocking loser fate by silencing fwelose did not reduce splitting. Finally, the proportion of split clones was also increased for winner clones either during myc-driven competition or during Minute-dependent competition. Altogether, this suggested that winner–loser mixing is increased independently of loser cell death or clone size by a factor upstream of fwe, and could be driven by cell–cell intercalation. Accordingly, junction remodelling events leading to disappearance of a loser–loser junction were three times more frequent at loser clone boundaries than control clone boundaries in the pupal notum. The rate of junction remodelling was higher in loser–loser junctions and in winner–winner junctions than in winner–loser junctions. The preferential stabilization of winner–loser interfaces should increase the surface of contact between winner and loser cells over time. Accordingly, loser clone compactness in the notum decreased over time whereas it remains constant on average for WT clones in WT background. Similarly, the compactness of clones in the notum also decreased over time for conditions showing high frequency of clone splitting in the wing disc, whereas clone compactness remained constant for conditions rescuing clone splitting. Altogether, it is concluded that both Minute- and myc-dependent competition increase loser–winner mixing through cell–cell intercalation (Levayer, 2015).
It was then asked what could modulate the rate of junction remodelling during competition. The rate of junction remodelling can be cell-autonomously increased by myc. Interestingly, downregulation of the tumour suppressor PTEN is also sufficient to increase the rate of junction remodelling through the upregulation of phosphatidylinositol (3,4,5)-trisphosphate (PIP3). It was reasoned that differences in PIP3 levels could also modulate junction remodelling during competition. Using a live reporter of PIP3 that could detect modulations of PIP3 in the notum, a significant increase of PIP3 was observed in the apico-lateral membrane of tub-dmyc–tub-dmyc interfaces compared with WT–WT and WT–tub-dmyc interfaces (Fig. 3a, b). Moreover, increasing/reducing Myc levels in a full compartment of the wing disc was sufficient to increase/decrease the levels of phospho-Akt (a downstream target of PIP3, whereas fweloseA overexpression had no effect. Similarly, levels of phospho-Akt were relatively higher in WT clones than in the surrounding M-/+ cells. Thus differences in PIP3 levels might be responsible for winner–loser mixing. Accordingly, reducing PIP3 levels by overexpressing a PI3 kinase dominant negative (PI3K-DN) or increasing PIP3 levels by knocking down PTEN (UAS-pten RNAi) were both sufficient to induce a high proportion of fragmented clones and to reduce clone compactness over time in the notum , whereas increasing PIP3 in loser clones was sufficient to prevent cell mixing. Moreover, abolishing winner–loser PIP3 differences through larval starvation prevented loser clone fragmentation, the reduction of clone compactness over time in the notum and could rescue WT clone elimination in tub-dmyc background. It is therefore concluded that differences in PIP3 levels are necessary and sufficient for loser–winner mixing and required for loser cell elimination (Levayer, 2015).
It was then asked which downstream effectors of PIP3 could affect junction stability. A relative growth decrease can generate mechanical stress that can be released by cell-cell intercalation. Accordingly, growth reduction through Akt downregulation is sufficient to increase clone splitting and could contribute to loser clone splitting. However, Akt is not sufficient to explain winner-loser mixing because, unlike PIP3, increasing Akt had no effect on clone splitting. PIP3 could also modulate junction remodelling through its effect on cytoskeleton and the modulation of intercellular adhesion or tension. No obvious modifications of E-cad, MRLC or Dachs (another regulator of tension) was detected in loser cells. However, a significant reduction of F-actin levels and a reduction of actin turnover/polymerization rate were observed in loser-loser and loser-winner junctions in the notum. Similarly, modifying Myc levels in a full wing disc compartment was sufficient to modify actin levels, and F-actin levels were higher in WT clones than M-/+ cells. This prompted a test of the role of actin organization in winner-loser mixing. Downregulating the formin Diaphanous (Dia, a filamentous actin polymerization factor) by RNA interference (RNAi) or by using a hypomorphic mutant was sufficient to obtain a high proportion of fragmented clones and to reduce clone compactness over time, whereas overexpressing Dia in loser clones prevented clone splitting (UAS-dia::GFP) and compactness reduction. This effect was specific to Dia as modulating Arp2/3 complex (a regulator of dendritic actin network) had no effect on clone splitting. Thus, impaired filamentous actin organization was necessary and sufficient to drive loser-winner mixing. These actin defects were driven by the differences in PIP3 levels between losers and winners. Thus Dia could be an important regulator of competition through its effect on cell mixing. Overexpression of Dia was indeed sufficient to reduce loser clone elimination significantly (Levayer, 2015).
Filamentous actin has been associated with tension regulation. It was therefore asked whether junction tension was modified in winner and loser junctions. The maximum speed of relaxation of junction after laser nanoablation (which is proportional to tension) was significantly reduced in loser-loser and winner-loser junctions compared with winner-winner junctions. This distribution of tension has been proposed to promote cell mixing. Accordingly, decreasing PIP3 in clones reduced tension both in low-PIP3-low-PIP3 and low-PIP3-normal-PIP3 junctions, whereas overexpressing Dia in loser clones or starvation were both sufficient to abolish differences in tension, in agreement with their effect on winner-loser mixing and the distribution of F-actin. Thus the lower tension at winner-loser and loser-loser junctions is responsible for winner-loser mixing. Altogether, it is concluded that the relative PIP3 decrease in losers increases winner-loser mixing through Akt-dependent differential growth and the modulation of tension through F-actin downregulation in winner-loser and loser-loser junctions (Levayer, 2015).
Several modes of tissue invasion by cancer cells have been described, most of them relying on the departure of the tumour cells from the epithelial layer. This study suggests that some oncogenes may also drive tissue destruction and invasion by inducing ectopic cell intercalation between cancerous and healthy cells, and subsequent healthy cell elimination. myc-dependent invasion could be enhanced by other mutations further promoting intercalation (such as PTEN). Stiffness is increased in many tumours, suggesting that healthy cell-cancer cell mixing by intercalation might be a general process (Levayer, 2015).
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date revised: 23 November 2015
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