InteractiveFly: GeneBrief

capping protein alpha & capping protein beta: Biological Overview | References

The conserved Hippo tumor suppressor pathway is a key kinase cascade that controls tissue growth by regulating the nuclear import and activity of the transcription co-activator Yorkie. This study reports that the actin-Capping Protein αβ heterodimer, which regulates actin polymerization, also functions to suppress inappropriate tissue growth by inhibiting Yorkie activity. Loss of Capping Protein activity results in abnormal accumulation of apical F-actin, reduced Hippo pathway activity and the ectopic expression of several Yorkie target genes that promote cell survival and proliferation. Reduction of two other actin-regulatory proteins, Cofilin and the cyclase-associated protein Capulet, cause abnormal F-actin accumulation, but only the loss of Capulet, like that of Capping Protein, induces ectopic Yorkie activity. Interestingly, F-actin also accumulates abnormally when Hippo pathway activity is reduced or abolished, independently of Yorkie activity, whereas overexpression of the Hippo pathway component expanded can partially reverse the abnormal accumulation of F-actin in cells depleted for Capping Protein. Taken together, these findings indicate a novel interplay between Hippo pathway activity and actin filament dynamics that is essential for normal growth control (Fernández, 2011).

The Hippo pathway has emerged as a crucial regulator of tissue size in both Drosophila and mammals. In Drosophila, the Hpo pathway comprises a kinase cascade in which the Hpo kinase binds the WW domain adaptor protein Salvador (Sav) to phosphorylate and activate the Warts (Wts) kinase. Wts, in turn, facilitated by Mats, phosphorylates and prevents nuclear translocation of the transcriptional co-activator Yorkie (Yki). This leads to transcriptional downregulation of target genes that positively regulate cell growth, survival and proliferation, including the Drosophila inhibitor of apoptosis protein 1 (Diap1; thread - FlyBase), expanded (ex), Merlin (Mer) and wingless (wg) in the inner distal ring, within the proximal wing imaginal disc. The upstream components Ex, Hpo and Wts are also thought to regulate Yki through a phosphorylation-independent mechanism, by directly binding to Yki, sequestering it in the cytosol and thereby abrogating its nuclear activity (Fernández, 2011).

Multiple upstream inputs are known to regulate the core Hpo kinase cassette at various levels. Thus, the atypical cadherin Fat was identified as an upstream component of the Hpo pathway and was proposed to transduce signals from the atypical cadherin Dachsous (Ds) and Four-jointed (Fj), a Golgi-resident kinase that phosphorylates Fat and Ds. Moreover, the two Ezrin-Radixin-Moesin (ERM) family members, Ex and Mer have been reported to lie upstream of the Hpo pathway. Mer and Ex can heterodimerize and are believed to exert their growth suppression activity by activating the Hpo kinase. However, how the different inputs that feed into the core kinase cassette are coordinated to regulate Yki activity is unknown (Fernández, 2011).

ERM proteins form a structural linkage between transmembrane components and actin filaments (F-actin). For instance, mammalian Mer binds numerous cytoskeletal factors, including actin, and appears to act as an inhibitor of actin polymerization. Interestingly, the Merlin-actin cytoskeleton association is required for growth suppression and inhibition of epidermal growth factor (EGFR) signaling. Moreover, F-actin depolymerization promotes activation of the Hpo orthologs MST1 and MST2 in mouse fibroblasts (Densham, 2009). These observations suggest a role for F-actin dynamics in modulating Hpo pathway activity (Fernández, 2011).

Actin filament growth, stability and disassembly are controlled by a plethora of actin-binding proteins. Among these, the Capping Protein (CP) heterodimer, composed of α (Cpa) and β (Cpb) subunits, acts as a functional heterodimer to restrict the accessibility of the filament barbed end, inhibiting addition or loss of actin monomers (Cooper, 2008). In Drosophila, mutations in either cpa or cpb, lead to accumulation of F-actin within the cell and give rise to identical developmental phenotypes that are tissue specific. In the wing blade (BL), the most distal domain of the imaginal disc, cpa and cpb prevent cell extrusion and death, but they are not required for this function in the most proximal domain, the prospective body wall and the hinge wing imaginal disc (Janody, 2006). The Cofilin homolog Twinstar (Tsr) and the Cyclase-associated protein Capulet (Capt) also restrict actin polymerization: Tsr severs filaments and enhances dissociation of actin monomers from the pointed end, whereas Capt sequesters actin monomers, preventing their incorporation into filaments (Fernández, 2011).

This study investigated the relationship between the control of the actin cytoskeleton and Hpo pathway activity. Actin-binding proteins CP and Capt, but not Tsr, were shown to enhance Hpo signaling activity. Moreover, a new relationship was uncovered between the Hpo pathway and the machinery that regulates F-actin, and it was revealed that Hpo signaling activity, like CP, limits F-actin accumulation at apical sites independently of Yki. Finally, it is proposed that regulation of an apical F-actin network by Hpo signaling activity and CP sustains Hpo pathway activity, thereby limiting Yki nuclear import and the activation of proliferation and survival genes (Fernández, 2011).

This report shows an interdependency between Hpo signaling activity and F-actin dynamics in which CP and Hpo pathway activities inhibit F-actin accumulation, and the reduction in F-actin in turn sustains Hpo pathway activity, preventing Yki nuclear translocation and upregulation of proliferation and survival genes (Fernández, 2011).

It is suggested that Ex prevents apical F-actin accumulation through Hpo signaling activity but independently of Yki. ERM proteins can form a structural linkage between transmembrane components and the actin cytoskeleton. Mammalian Mer appears to act as an inhibitor of actin polymerization. Moreover, the Mer-actin cytoskeleton association has a crucial role for growth suppression and inhibition of EGFR signaling. In Drosophila, Mer and Ex are structurally related and appear to have partially redundant functions but vary in their requirement depending on the tissue or developmental stage. In imaginal discs, loss of ex shows stronger phenotypes when compared with those of Mer. Ex might also have a stronger requirement on F-actin dynamics, as loss of ex, but not that of Mer, triggered F-actin accumulation. Surprisingly, loss of hpo, sav, mats or wts also triggered apical F-actin accumulation. Ex is likely to affect F-actin through activation of the Hpo kinase cassette because in most ex mutant clones, overexpressing hpo suppressed F-actin accumulation. Some clones seemed to contain increased F-actin. However, these clones also constricted apically, suggesting that the effect on F-actin levels results from a reduction of the apical cell surface and that in the absence of ex, differential activity of overexpressed hpo triggers cell shape changes. Together, these observations argue that Ex prevents apical F-actin accumulation through Hpo signaling activity but independently of Yki (Fernández, 2011).

Loss of Hpo pathway activity or CP triggers apical F-actin accumulation. Ex localizes to the sub-apical region of epithelial cells, and colocalizes with an HA-tagged form of Cpa, Ex, Hpo, Sav and Wts all interact with each other through WW and PPXY motifs (Oh, 2009; Reddy, 2008). Therefore, a pool of Hpo, Sav and Wts, localized at apical sites, could directly regulate an actin-regulatory protein. Hpo pathway activity might act downstream of CP on F-actin. In agreement with this, ex overexpression significantly suppresses F-actin accumulation in cells with reduced CP levels. The role of Hpo signaling activity might be to inhibit an actin-nucleating factor, which adds new actin monomers to filament barbed ends free of the capping activity of CP. However, it cannot be excluded that ex overexpression enhances the activity of residual Cpa in cells knocked down using RNAi, nor that Hpo pathway activity acts in parallel to CP on F-actin. Interestingly, although endogenous Ex is upregulated in cells lacking CP, mutant cells still accumulated F-actin. wts mutant clones also upregulated Ex, which, when overexpressed, suppresses growth of wts mutant clones. Therefore, the increased levels of endogenous Ex in cells lacking either CP or wts appears to be insufficient to fully suppress the effects of loss of CP or wts on F-actin and growth, respectively (Fernández, 2011).

The data indicate that CP inhibits Yki nuclear accumulation, activation of Yki target genes, and consequently overgrowth of the proximal wing epithelium. Interestingly, Yki was also found to accumulate in nuclei of wild-type cells adjacent to the clone border. Consistent with a non-autonomous effect of CP loss on Hpo pathway activity, ex-lacZ and diap1-lacZ were upregulated in wild-type cells adjacent to CP mutant clones. However, Ex levels were reduced in wild-type neighboring cells. Cells expressing different amounts of ds and fj also upregulate ex-lacZ, but show reduced levels of Ex. Therefore, loss of CP might affect Fj or Ds levels, creating a sharp boundary of their expression. However, in contrast to clones overexpressing ds or mutant for fj, cell lacking CP also upregulated Ex and Mer inside the mutant clones, indicating that CP also acts cell-autonomously to promote Hpo signaling activity. CP might facilitate Yki phosphorylation by the Hpo kinase cassette as cpa-depleted tissues contain decreased phospho-Yki levels. But, the possibility cannot be excluded that CP also favors the direct binding of non-phosphorylated Yki to Ex, Hpo or Wts (Oh, 2009). Further analysis will be required to elucidate the mechanisms by which CP restricts Yki activity cell autonomously and in wild-type neighboring cells (Fernández, 2011).

The results argue for a constitutive role of CP in Hpo pathway activity, since Yki target genes are upregulated in the whole wing and eye imaginal discs. However, loss of CP did not fully recapitulate the phenotype for core components of the hpo pathway. Despite that, on average, cpb mutant clones located in the proximal wing disc domain were 25% larger than wild-type twin spots; 60% of mutant clones failed to grow. Moreover, in the distal wing epithelium, reducing CP levels induces mislocalization of the adherens junction components Armadillo and DE-Cadherin, extrude and death. Furthermore, in Drosophila, CP also prevents retinal degeneration (Delalle, 2005; Johnson, 2008). This indicates that although loss of CP can, under certain conditions, result in tissue overgrowth due to inhibition of Hpo pathway activity, other factors such as the polarity status, also determine the survival and growth of the mutant tissue. Therefore, in addition to promoting Hpo pathway activity, CP has additional developmental functions in epithelia. However, the possibility cannot be excluded that, like most upstream inputs that feed into the Hpo pathway, CP has a tissue-specific requirement in Hpo pathway activity (Fernández, 2011).

CP, Capt and Tsr all restrict F-actin assembly directly. CP and Capt control F-actin formation near the apical surface and inhibit ectopic expression of Yki target genes, whereas Tsr acts around the entire cell cortex and has no effect on Yki target genes. This argues that Hpo signaling activity is not affected by the excess of F-actin per se but provides significant support to the view that stabilization of an apical F-actin network by CP, Capt and Hpo signaling activity feeds back on the Hpo pathway to sustain its activity (Fernández, 2011).

These findings do not lead to an understanding of where F-actin accumulation intersects Hpo signaling activity because both Hpo signaling activity and F-actin dynamics feedback to each other. For instance, hpo or ex overexpression suppressed growth of CP-depleted cells. But, overexpressed ex and possibly hpo also suppress F-actin accumulation of Cpa-depleted cells. The control of F-actin by Hpo signaling activity and CP might constitute a parallel input, which sustains Hpo pathway activity. Alternatively, F-actin could act upstream of the core kinase cascade, which in turn feeds back to F-actin, to maintain its activity. The identification of additional actin cytoskeletal components that either promote Hpo pathway activity or act downstream of Hpo pathway activity on F-actin would help to discriminate between these possibilities (Fernández, 2011).

How F-actin influences Hpo signaling activity remains to be determined. The apical F-actin network, which regulates the formation and movement of endocytic vesicles from the plasma membrane, might promote the recycling or degradation of Hpo pathway components. Increased F-actin at apical sites would, therefore, affect protein turnover. Alternatively, apical F-actin might act as a scaffold to tether Hpo pathway components apically. In support of this, Ex, Hpo, Sav, Wts and Yki could all interact between each other through WW and PPXY motifs at apical sites (Oh, 2009; Reddy, 2008). Moreover, expression of a membrane-targeted form of Mats enhances Hpo signaling (Ho, 2010). Although Ex and Mer are properly localized in CP mutant cells, other members of the pathway might be mislocalized in the presence of excess F-actin. Interestingly, in mouse fibroblasts, the Hpo orthologs MST1 and MST2 colocalize with F-actin structures and are activated upon F-actin depolymerization (Densham, 2009), suggesting that by tethering Hpo pathway components, F-actin dynamics modulates their activity. Finally, the F-actin network might act as a mechanical transducer. Most of the mechanosensitive responses require tethering to force-bearing actin filaments. Tissue surface tension has been proposed to be a stimulus for a feedback mechanism that could regulate tissue growth. The tension exerted by neighboring cells might be sensed at the cell membrane by the actin cytoskeleton and translated to the regulation of cell proliferation through the Hpo signaling pathway (Fernández, 2011).

Modulating F-actin organization induces organ growth by affecting the Hippo pathway

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 Dia^CA, 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 Dia^CA 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).

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 Dia^CA 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 Dia^CA-induced phenotypes in contrast to their ability to rescue fat and ex;mer mutant phenotypes. Overexpression of Wts, however, significantly suppressed Dia^CA-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 Dia^CA. 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 (Densham, 2009). 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).

Drosophila Wash and the Wash regulatory complex function in nuclear envelope budding

Nuclear envelope (NE) budding is a recently described phenomenon wherein large macromolecular complexes are packaged inside the nucleus and extruded through the nuclear membranes. Although a general outline of the cellular events occurring during NE budding is now in place, little is yet known about the molecular machinery and mechanisms underlying the physical aspects of NE bud formation. Using a multidisciplinary approach, this study identified Wash, its regulatory complex (SHRC), capping protein and Arp2/3 as new molecular components involved in the physical aspects of NE bud formation in a Drosophila model system. Interestingly, Wash affects NE budding in two ways: indirectly through general nuclear lamina disruption via an SHRC-independent interaction with Lamin B leading to inefficient NE bud formation, and directly by blocking NE bud formation along with its SHRC, capping protein and Arp2/3. In addition to NE budding emerging as an important cellular process, it shares many similarities with herpesvirus nuclear egress mechanisms, suggesting new avenues for exploration in both normal and disease biology (Verboon, 2020).

Transport of macromolecules from the nucleus to the cytoplasm is essential for all developmental processes, including the regulation of differentiation and aging, and, when mis-regulated, is associated with diseases and cancer. This indispensable process has been thought to occur exclusively through nuclear pore complexes (NPCs), channels that regulate what exits (and enters) the nucleus. Recently, nuclear envelope (NE) budding was identified as an alternative pathway for nuclear exit, particularly for large developmentally required ribonucleoprotein (megaRNP) complexes that would otherwise need to unfold/remodel to fit through the NPCs. In this pathway, large macromolecule complexes, such as megaRNPs, are encircled by the nuclear lamina (type-A and -B lamins) and the inner nuclear membrane (INM), pinched off from the INM, fuse with the outer nuclear membrane and release the megaRNPs into the cytoplasm. Strikingly, NE budding shares many features with the nuclear egress mechanism used by herpesviruses. As viruses often utilize pre-existing host pathways, the parallel between nuclear exit of herpesvirus nucleocapsids and that of megaRNPs and/or other large cargoes suggests that NE budding may be a general cellular mechanism. Indeed, this pathway has also been implicated in the removal of obsolete macromolecular complexes or other material (i.e. large protein aggregates or poly-ubiquitylated proteins) from the nucleus (Verboon, 2020).

NE budding was first demonstrated in Drosophila synapse development, proving to be essential for neuromuscular junction (NMJ) integrity. In this context, a C-terminal fragment of the Wingless receptor Fz2, Fz2C, was shown to associate with megaRNPs that formed foci at the nuclear periphery and exited the nucleus by budding through the nuclear envelope (Speese, 2012). Failure of this process resulted in aberrant synapse differentiation and impaired NMJ integrity (Speese, 2012). In a subsequent study, the NE budding pathway was shown to be necessary for the nuclear export of megaRNPs containing mitochondrial RNAs: disruption of NE budding led to deterioration of mitochondrial integrity and premature aging phenotypes that were similar to those associated with lamin mutations (i.e., laminopathies). Similar endogenous perinuclear foci/buds have been observed in plants and vertebrates, as well as other Drosophila tissues (i.e., larval salivary gland nuclei), suggesting that cellular NE budding is a widely conserved process (Verboon, 2020).

The spectrum of processes requiring this non-canonical nuclear exit pathway and the molecular machineries needed for this process, which encompasses membrane deformations, traversal across a membrane bilayer and nuclear envelope remodeling for a return to homeostasis, are largely unknown. One class of proteins that are involved in membrane-cytoskeletal interactions and organization is the Wiskott-Aldrich Syndrome (WAS) protein family. WAS protein subfamilies are involved in a wide variety of essential cellular and developmental processes, as well as in pathogen infection and disease. WAS family proteins polymerize branched actin through the Arp2/3 complex, and often function as downstream effectors of Rho family GTPases. This study identified Wash as a new WAS subfamily that is regulated in a context-dependent manner: Wash can bind directly to Rho1 GTPase (in Drosophila) or it can function along with the multi-protein WASH regulatory complex [SHRC; comprised of SWIP, Strumpellin, FAM21 and CCDC53 (also known as WASHC4, WASHC5, WASHC2 and WASHC3, respectively, in mammals)]. Wash regulation by Rho family GTPases outside of Drosophila has not yet been described; instead its regulation has been characterized in the context of its SHRC. WASH and its SHRC are evolutionarily conserved and their mis-regulation is linked to cancers and neurodegenerative disorders. Importantly, it has been shown that Wash is present in the nucleus where it interacts directly with B-type lamins and, when mutant, affects global nuclear organization/functions, as well as causing an abnormal wrinkled nucleus morphology reminiscent of that observed in diverse laminopathies (Verboon, 2015a and 2015b). Mammalian WASH proteins have also been shown to localize to the nucleus in developmental and cell-type specific manners (Verboon, 2020).

This study shows that Wash, its SHRC, capping protein and Arp2/3 are also involved in the NE budding pathway, as mutants for any of these components lack Fz2C foci/lamin buds and display the NMJ integrity and premature aging phenotypes previously associated with the loss of NE budding. In addition, this study found that CCDC53 and SWIP (SHRC subunits) colocalize with Fz2C foci/lamin buds. Wash is shown to be present in several independent nuclear complexes. The nuclear interactions of Wash with its SHRC are separate from those with B-type Lamin, leading to effects on different subsets of nuclear Wash functions. This study also found that Wash-dependent Arp2/3 actin nucleation activity is required for proper NE budding. It is proposed that Wash and its SHRC play a physical and/or regulatory role in the process of NE budding (Verboon, 2020).

NE budding is an increasingly appreciated pathway for nuclear export of large macromolecular machineries, such as megaRNPs involved in the co-regulation of major developmental pathways or unwanted RNA or protein aggregates. Previous work has shown that Drosophila Wash is present in the nucleus where it is likely involved in a number of different nuclear processes (Verboon, 2015a). This study shows that all members of the four-subunit Wash regulatory complex (SHRC), as well as the heterodimeric capping protein, are also present within the nucleus and, along with Wash, are necessary for NE budding. Wash and SHRC act early in the NE budding pathway, and this process requires actin nucleation activity of Wash achieved through its interaction with Arp2/3. Loss of Wash, any of its SHRC subunits or Arp2/3 leads to the loss of Fz2C foci/NE buds, and mutants for these factors exhibit phenotypes associated with the two cellular processes shown to require NE budding: aberrant synaptic 'ghost' bouton formation leading to disrupted NMJ integrity, and mitochondrial degeneration associated with premature aging phenotypes. This study also shows that the interaction of Wash with Lamin B results in a general disruption of the nuclear lamina and separation of the Lamin B/Lamin C homotypic meshes, leading to inefficient, rather than loss of, NE bud formation (Verboon, 2020).

While the spectrum of processes that require this alternate nuclear egress mechanism is not yet known, SHRC components are linked to neurodegenerative disorders, including hereditary spastic paraplegias, Parkinson disease, amyotrophic lateral sclerosis (ALS) and Hermansky-Pudlak syndrome. As an increasing number of neurodegenerative diseases and myopathies have been associated with the accumulation of RNA-protein aggregates in the nucleus, NE budding may be part of the endogenous cellular pathway for removing such aggregates/megaRNPs from the nucleus in normal cells (Verboon, 2020).

The parallels between the mechanism of NE budding and herpesvirus nuclear egress, as well as the presence of similar endogenous perinuclear foci/buds in other plant and animal nuclei, has suggested that NE budding is a conserved endogenous cellular pathway for nuclear export. Indeed, INM-encapsulated electron-dense granules have been identified in yeast and Torsin-deficient HeLa cells, and these show similarities to the Fz2C foci/NE buds observed in Drosophila muscle and salivary gland nuclei. While the full relationship between NPCs and NE buds is not yet known, one important difference is that the yeast and HeLa nuclear granules observed are much smaller (∼120 nm) than Fz2C foci/NE buds (∼500 nm). This identification of Wash and SHRC, proteins with the capability of remodeling cortical cytoskeleton and/or membranes, in the physical aspects of NE budding lend support for NE budding being an alternative endogenous nuclear exit pathway (Verboon, 2020).

NE budding has been proposed to occur at sites along the INM where the nuclear lamina is modified by aPKC phosphorylation. Both A- and B-type lamins play a role in NE budding and are thought to be the target of aPKC phosphorylation within the nuclear lamina, similar to the PKC-mediated phosphorylation of lamins that precedes lamina disassembly in mitotic NE breakdown (Güttinger, 2009), apoptosis (Cross, 2000) or during viral capsid nuclear egress. Viral NE budding requires a virus-encoded nuclear egress complex (NEC), which has been implicated in the recruitment of kinases to the INM. Cellular counterparts for these virally encoded NEC proteins have not yet been identified. It is also not yet known how this kinase activity is restricted to specific sites along the nuclear lamina or how those specific sites are selected (Verboon, 2020).

Previous work has shown that Wash interacts directly with Lamin B and that loss of nuclear Wash results in a wrinkled nuclear morphology reminiscent of that observed in laminopathies (Verboon, 2015a). It was reasoned that Wash-mediated disruption of the nuclear lamina may account for its NE-budding phenotypes. Consistent with this idea, it was found that Lamin B knockdown nuclei and nuclei from a wash point mutant that disrupts the interaction of Wash with Lamin B (washΔΔLamB) exhibit a wrinkled nuclear morphology, reduced Fz2C foci/NE buds and NE-budding-associated phenotypes, albeit not as strong as those observed in Wash or SHRC mutants (Verboon, 2020).

Lamin A/C and Lamin B isoforms form homotypic meshworks that interact among themselves (in as yet unknown ways), and that are somehow linked to integral membrane proteins of the INM and to the chromatin adjoining the INM. Intriguingly, the current data suggests that these lamin homotypic meshes are likely layered, rather than interwoven, and that Wash affects the anchoring of these lamin homotypic meshes to each other and/or the INM. Lamin knockdown or disruption of the Wash-Lamin B interaction leads to separated lamin isoform meshes and wrinkled nuclear morphology that are not observed in SHRC and Arp2/3 knockdown nuclei, suggesting that Wash can also affect NE budding by a means independent of disrupted global nuclear lamina integrity. Interestingly, the functions of Wash mediated with the SHRC and with Lamin B involve separate nuclear complexes. Consistent with this, it was shown previously that Drosophila Wash encodes several independent biochemical activities (actin nucleation, actin bundling, MT bundling and actin-MT crosslinking) and that the use of these activities is context dependent. In particular, when Wash interacts with Lamin, it does not require an association with SHRC or Arp2/3. It is suggested that the interaction of Wash with Lamin B is required for organizing the nuclear lamina and likely requires the actin bundling and/or cross-linking activities of Wash rather than its actin nucleation activity, such that wash mutants that cannot bind Lamin result in separation of the Lamin isoform meshes from each other. Taken together, the data suggest that loss of the interaction between Wash and Lamin B makes NE budding inefficient by generally disrupting the nuclear lamina, rather than directly disrupting NE bud formation. The role of aPKC in NE budding may also be somewhat indirect by generally disrupting the nuclear lamina thereby reducing the efficiency of NE bud formation. Alternatively, aPKC may target Wash: WASH phosphorylation by Src kinases has been shown to be necessary for regulating NK cell cytotoxicity (Verboon, 2020).

For bud formation/envelopment of a megaRNP or macromolecular cargo to occur, the INM must interact with its underlying cortical nucleoskeleton to allow the INM deformation/curvature necessary to form the physical NE bud. Force must also be generated that allows the bud to extend into the perinuclear space, as well as for the scission of the nascent bud. In the cytoplasm, WAS family proteins are often involved in membrane-cortical cytoskeleton-coupled processes, including both 'inward' membrane deformations (i.e., endocytosis) and 'outward' membrane deformations (i.e., exocytosis and cell protrusions), that are required for signal/environment sensing and cell movement during normal development, as well as during pathological conditions. Mammalian WASH, in particular, has been implicated in endosome biogenesis and/or sorting in the cytoplasm, where it, along with its SHRC, drives Arp2/3-dependent actin assembly to influence endosome trafficking, remodels membrane, and facilitates membrane scission. Thus, Wash encodes the biochemical properties needed to regulate the membrane deformation/curvature necessary to form the NE bud and/or play a role in generating the forces necessary to pinch off the NE bud from the INM. Consistent with Wash playing a role in the physical production of a NE bud, it was found that Wash acts prior to Torsin, a protein that is implicated in NE bud scission from the INM, and it requires its actin nucleation activity (Verboon, 2020).

This study has identified Wash and its SHRC as new players in the cellular machinery required for the newly described endogenous NE budding pathway. The data suggest that Wash is involved in two nuclear functions that can affect NE budding. (1) Wash is required to maintain the organization of Lamin isoforms relative to each other and the INM through its direct physical interaction with Lamin B. This Wash activity is SHRC and Arp2/3 independent, and is likely a non-specific mechanism because global disruption of the nuclear lamina/nuclear envelope would indirectly affect many nuclear processes, including NE budding. (2) Wash is specifically required for NE bud formation. This Wash activity is SHRC and Arp2/3 dependent. While the focus of NE budding research to date has centered on the composition of the megaRNPs and the spectrum of cellular/developmental processes requiring NE budding, Wash and the SHRC are likely involved in the physical aspects of NE budding. Thus, Wash and SHRC provide a molecular entry into the physical machinery that underlies NE budding. In the future, it will be exciting to further explore the roles of Wash in NE budding, and to determine how it functions to get macromolecular complexes through the INM, and how closely these nuclear roles parallel those in the cytoplasm (Verboon, 2020).

Zyxin antagonizes the FERM protein Expanded to couple F-actin and Yorkie-dependent organ growth

Coordinated multicellular growth during development is achieved by the sensing of spatial and nutritional boundaries. The conserved Hippo (Hpo) signaling pathway has been proposed to restrict tissue growth by perceiving mechanical constraints through actin cytoskeleton networks. The actin-associated LIM proteins Zyxin (Zyx) and Ajuba (Jub) have been linked to the control of tissue growth via regulation of Hpo signaling, but the study of Zyx has been hampered by a lack of genetic tools. A zyx mutant was generated in Drosophila using TALEN endonucleases, and this was used to show that Zyx antagonizes the FERM-domain protein Expanded (Ex) to control tissue growth, eye differentiation, and F-actin accumulation. Zyx membrane targeting promotes the interaction between the transcriptional co-activator Yorkie (Yki) and the transcription factor Scalloped (Sd), leading to activation of Yki target gene expression and promoting tissue growth. Finally, this study shows that Zyx's growth-promoting function is dependent on its interaction with the actin-associated protein Enabled (Ena) via a conserved LPPPP motif and is antagonized by Capping Protein (CP). These results show that Zyx is a functional antagonist of Ex in growth control and establish a link between actin filament polymerization and Yki activity (Gaspar, 2015).

The control of tissue size represents a major unsolved question in developmental biology. The conserved Hippo (Hpo) signaling pathway is thought to sense mechanical and nutritional cues to restrict tissue growth. Activation of the Ste20-like kinase Hpo (MST1/2 in mammals) and subsequent phosphorylation of the downstream Ndr-like kinase Warts (Wts-LATS1/2 in mammals) inhibits the transcriptional co-activator Yorkie (Yki-YAP/TAZ in mammals), via phosphorylation at S168. This prevents the interaction of Yki with transcription factor partners, such as Scalloped (Sd-TEAD1-4 in mammals), thereby inhibiting expression of pro-growth and survival genes (Gaspar, 2015).

The known upstream stimuli for Hpo signaling involve a number of regulatory proteins, many of which are associated with the actin cytoskeleton. In particular, the Drosophila proteins Expanded (Ex) and Merlin (Mer), which belong to the FERM (Four point one, Ezrin, Radixin, Moesin) domain family, and the protocadherins Fat (Ft) and Dachsous (Ds), were identified as tumor suppressors that prevent expression of Yki target genes. Whether Ex/Mer and Ft/Ds signaling represent entirely distinct branches of Hpo signaling remains unclear. For instance, Ft depletion leads to a reduction in apical Ex localization. However, Ft and Ex have been implicated in distinct functions: Ft/Ds are involved in the control of planar cell polarity (PCP), while Ex has strong effects on eye differentiation. The proposed mechanisms of Ft and Ex function are also distinct. In particular, Ex promotes cytoplasmic sequestration of Yki through direct binding and by promoting Hpo-Wts kinase activity, while Ft antagonizes the growth-promoting function of the atypical myosin Dachs (D), which, in turn, destabilizes Wts (Gaspar, 2015).

Several reports have highlighted the contribution of the actin cytoskeleton to Hpo signaling. The actin Capping Protein αβ heterodimer (CP), which prevents addition of actin monomers to F-actin barbed ends, antagonizes Yki activity, and thereby restricts tissue growth. Accordingly, in mammals, CapZ and other factors that restrict F-actin levels, have growth-restrictive effects via the control of YAP/TAZ subcellular localization, particularly in response to mechanical cues. Interestingly, YAP and TAZ respond to mechanical cues dependent on actomyosin networks and formin-dependent actin polymerization. Recently, the actin-associated LIM (Lin11, Isl-1, and Mec-3) domain protein Zyxin (Zyx) has been shown to mediate the effects of Ft-Ds signaling on Yki target genes, by promoting Wts destabilization via its interaction with D (Rauskolb, 2011). Importantly, Zyx provides a link to the actin polymerization machinery, since it directly interacts with the actin-binding proteins Enabled (Ena)/VASP via conserved F/LPPPP motifs, and promotes Ena function in barbed-end F-actin polymerization (Gaspar, 2015 and references therein).

The analysis of Drosophila zyx has been limited by the absence of a mutant. This study generated a zyx mutation and describe its effects on growth and Hpo signaling. Zyx is shown to strongly antagonize Ex function in growth control, eye differentiation and F-actin accumulation, while being largely dispensable for Ft-mediated tissue growth. Finally, this work suggests that Zyx's growth-promoting function requires its ability to bind the actin polymerization factor Ena (Gaspar, 2015).

Zyx was previously shown to promote Wts degradation in a mechanism based on a Zyx/Dachs interaction (Rauskolb, 2011). However, this study reports that zyx and dachs (d) have additive effects on tissue growth. In addition, zyx loss has a modest effect on ft growth phenotypes, which, in contrast, are highly sensitive to d mutations, highlighting the possibility of additional functions for Zyx in tissue growth (Gaspar, 2015).

Characterization of the zyx mutant shows that Zyx acts in the Ex branch of the Hpo pathway to control tissue growth. This is in contrast to a previous study using RNAi knockdown of zyx and ex, which concluded that zyx expression had only minor effects on the Ex branch (Rauskolb, 2011). The current results indicate that zyx loss can significantly reverse the lethality and growth defects of ex mutant animals. This antagonistic function of Ex and Zyx is not confined to growth regulation but extends to tissue differentiation. This study shows that Zyx restricts eye differentiation antagonistically to Ex and in parallel to Dachs but independently of Ft. Consistent with these observations, simultaneous loss of ex and ft leads to additive, and therefore apparently independent effects on eye differentiation. Therefore, it is proposed that Zyx is a key modulator of Ex function (Gaspar, 2015).

In growth control, Zyx function may be partially independent of Hpo-Wts signaling, as zyx is partially required for the overgrowth of hpo and wts mutant eye and wing but has no major effect on wts overexpression in the wing or Yki phosphorylation by Wts. Ex has been reported to sequester Yki in the cytoplasm through a direct interaction. However, since ex mutant overgrowth is suppressed by zyx loss, it is unlikely that Zyx directly antagonizes Ex protein. Instead, it is suggested that the interplay between Zyx and Ex in growth control is mediated through their antagonistic effects on F-actin (Gaspar, 2015).

This work links F-actin barbed-end polymerization with Zyx/Ex in the control of Yki activity and tissue growth. The Zyx domain encompassing the conserved LPPPP motif, which binds Ena, is required for Zyx to promote growth and to antagonize Ex function. Moreover, Zyx and Ena synergize to promote tissue growth. This supports the idea that Zyx promotes tissue growth via its interaction with Ena. Conversely, CP antagonizes Zyx-induced tissue growth and functions together with Ex in preventing F-actin polymerization. Therefore, an attractive possibility is that antagonistic effects on Yki activity between the activators Zyx/Ena on one hand and the inhibitors Ex and CP on the other hand is played out indirectly through their effects on F-actin polymerization. Consistent with this hypothesis, Zyx antagonizes the effect of Ex on apical F-actin accumulation (Gaspar, 2015).

Recent data suggest that the actin cytoskeleton acts in parallel to the core kinase cascade to control YAP/TAZ activity, with CapZ being proposed as one of the 'gatekeepers' restricting its nuclear translocation. Yki/YAP/TAZ may respond to the relative activities of Ena and CP, either by being sensitive to the presence of polymerizing actin barbed ends, or because Ena produces a specialized set of cortical actin filaments necessary for Yki/YAP/TAZ activation. The study of the mechanism(s) coupling F-actin and Yki/YAP/TAZ should resolve these issues. This study has shown that Zyx cortical localization is relevant for its function in promoting tissue growth. Since Zyx has been shown to rapidly relocalize to strained or severed actin filaments in cultured mammalian cells and Drosophila follicular epithelial cells, it is possible that Zyx may also link mechanical forces to growth control (Gaspar, 2015).

Finally, it is also interesting to note the possible redundancy in growth control between Zyx and other Ena-interacting proteins. Like Zyx, Pico/Lamellipodin contains an EVH1-interacting L/FPPPP motif, and its interaction with Ena promotes tissue growth in Drosophila. Since Ena localization is not strictly dependent on Zyx, it is tempting to speculate that Ena recruitment by multiple membrane-associated proteins, such as Zyx and Pico, is a common denominator in the regulation of growth by the actin cytoskeleton (Gaspar, 2015).

A dual function of Drosophila capping protein on DE-cadherin maintains epithelial integrity and prevents JNK-mediated apoptosis

E-cadherin plays a pivotal role in epithelial cell polarity, cell signalling and tumour suppression. However, how E-cadherin dysfunction promotes tumour progression is poorly understood. This study shows that the actin-capping protein heterodimer, which regulates actin filament polymerization, has a dual function on DE-cadherin in restricted Drosophila epithelia. Knocking down capping protein in the distal wing disc epithelium disrupts DE-cadherin and Armadillo localization at adherens junctions and upregulates DE-cadherin transcription. In turn, DE-cadherin provides an active signal, which prevents Wingless signalling and promotes JNK-mediated apoptosis. However, when cells are kept alive with the Caspase inhibitor P35, the activity of the JNK pathway and of the Yorkie oncogene trigger massive proliferation of cells that fail to stably retain associations with their neighbours. Moreover, loss of capping protein cooperates with the Ras oncogene to induce massive tissue overgrowth. Taken together, these findings argue that in some epithelia, the dual effect of capping protein loss on DE-cadherin triggers the elimination of mutant cells, preventing them from proliferating. However, the appearance of a second mutation that blocks cell death may allow for the development of some epithelial tumours (Jezowska, 2011).

Actin filament (F-actin) turnover and organization is a critical regulator of AJs assembly, maintenance, and remodelling (Cavey, 2009). F-actin growth, stability, disassembly and also their organization into functional higher-order networks are controlled by a plethora of actin-binding proteins (ABPs), strongly conserved between species. Capping protein (CP), composed of an α (Cpa) and β (Cpb) subunits, acts as a functional heterodimer by restricting accessibility of the filament barbed end, inhibiting addition or loss of actin monomers (Cooper, 2008). In Drosophila, removing either cpa or cpb, promotes accumulation of F-actin within the cell and gives rise to identical developmental phenotypes (Delalle, 2005; Gates, 2009; Janody, 2006). In the whole larval wing disc epithelium, loss of CP activity reduces Hpo pathway activity and leads to ectopic expression of several Yki target genes that promote cell survival and proliferation (Fernandez, 2011; Sansores-Garcia, 2011). However, inappropriate growth can only be observed in the proximal wing domain. In the distal wing primordium, cpa or cpb mutant cells mislocalize the AJs components DE-Cad and Arm, upregulate puc expression, extrude and die (Janody, 2006). This indicates that while loss of CP can under certain conditions result in tissue overgrowth due to inhibition of Hpo pathway activity, other factors such as the polarity status, also determine the survival and growth of the mutant tissue (Jezowska, 2011).

This study investigated the role of the actin-CP heterodimer in survival of cells in the distal wing disc epithelium. CP has a dual function in regulating DE-Cad: it stabilizes DE-Cad at cell-cell junctions, thereby preventing loss of epithelial integrity and inhibits upregulation of the DE-cad gene. DE-Cad would otherwise provide an active signal, which affects Wg signalling and promotes JNK-mediated apoptosis. However, when cells lacking CP are kept alive, JNK is converted into a potent inducer of proliferation (Jezowska, 2011).

This study demonstrates that in the distal wing disc epithelium, JNK signalling triggers apoptosis of cells with reduced CP expression but induces massive proliferation when apoptosis is blocked with P35. Yki activity is also required to allow overgrowth of 'undead' Cpa-depleted tissues. Induction of apoptosis has been shown to activate Yki through the JNK pathway and triggers compensatory cell proliferation. Thus, in CP-depleted cells kept alive with P35, Yki may act downstream of JNK signalling. Consistent with this, targeting Yki degradation in these tissues fully suppresses ectopic N-Cad expression but not MMP1 upregulation. Because CP also prevents Yki activity in the whole wing disc epithelium, independently of its effect on JNK signalling, (Fernandez, 2011; Sansores-Garcia, 2011), in the distal wing domain, excess Yki activity of 'undead' CP-depleted tissues may result from a dual effect, which involves a JNK-dependent and independent mechanisms (Jezowska, 2011).

JNK signalling has been reported to propagate from cell to cell in the wing disc, where it could trigger apoptosis or Yki-dependent compensatory proliferation. Neither non-autonomous apoptosis nor activation of JNK signalling was observed when patches of CP mutant cells were induced or dsRNA for CP was expressed with or without P35 (Janody, 2006). Therefore, the propagation of JNK activation might be impaired in tissues knocked down for CP. However, increase proliferation was observed of wild-type cells apposed to 'undead' Cpa-depleted tissues. This suggests that JNK propagation is not required to trigger compensatory cell proliferation (Jezowska, 2011).

Several observations argue that in cells lacking CP, a DE-Cad-dependent signal promotes JNK-mediated apoptosis by inhibiting Wg signalling. First, knocking down Cpa affects Wg signalling, which has been shown to prevent JNK-dependent cell death in this region. Second, removing one copy of DE-cad in Cpa-depleted cells partially suppresses apoptosis and ectopic MMP1 expression and restores Wg target genes expression. Third, loss of CP is associated with upregulation of the DE-cad gene and increased levels of the DE-Cad protein. One way by which DE-Cad may block Wg signalling is by tethering Arm. In agreement with this possibility, in the distal wing disc epithelium, overexpression of DE-cad compromises Wg signalling, while co-expression of Arm rescues the DE-cad overexpression phenotype. Moreover, in mouse, overexpression of E-Cad induces apoptosis and sequesters the transcriptionally competent pool of β-cat, effectively shutting off expression of Lef/TCF/β-cat-responsive genes. Interestingly, in Cpa-depleted tissues, the faster mobility form of Arm is enriched. Because this form was proposed to correspond to the cytoplasmic pool of Arm, following CP loss, increase DE-Cad levels might tether and stabilize Arm in the cytoplasm, preventing it to transduce Wg signalling. How a defect in Wg signalling triggers JNK-mediated cell death is not known. In cells lacking CP, JNK activation may occur in response to loss of DIAP1 since overexpressing DIAP1 strongly reduces ectopic MMP1 expression. However, it cannot be excluded that JNK signalling reduces DIAP1 levels since JNK signalling can also function upstream of DIAP1 (Jezowska, 2011).

In the distal wing domain, cells lacking CP mislocalize DE-Cad and Arm at AJs, upregulate expression of DE-cad and extrude from the epithelium (Janody, 2006). DE-cad appears to be a direct transcriptional target of the Hpo signalling pathway. CP inhibits Yki activity (Fernandez, 2011; Sansores-Garcia, 2011) and prevents shg-LacZ upregulation, even in mutant clones that maintain a polarized epithelial architecture in the proximal wing domain. Thus, increased DE-cad expression likely results from inhibition of Hpo pathway activity. However, while mutant clones for Hpo pathway components accumulate DE-Cad, mutant cells do not extrude from the wing disc epithelium. Therefore, the polarity defect of cells lacking CP is unlikely to result from increased DE-Cad levels. Different observations also argue that altered cell-cell adhesion does not result from a defect in Wg signalling or from ectopic activation of JNK signalling, as previously reported. First, reducing DE-cad levels do not restore Arm localization at AJs. Second, in Cpa-depleted tissues in which JNK signalling is blocked, dividing nuclei surrounded by dense F-actin patches are recovered on the basal surface of the distal wing disc epithelium. Third, unlike cells lacking CP, tissues expressing P35 and defective for Wg signalling or overexpressing DE-cad or in which high apoptotic levels were induced maintain a polarized epithelial architecture). Therefore, following loss of CP, the mislocalization of DE-Cad and Arm and the loss of cell-cell contacts are likely upstream or parallel events to DE-cad upregulation and JNK-mediated cell death. Because disruption of apical-basal polarity can trigger JNK activation, a model is favored by which CP prevents JNK-mediated cell death though a dual function on DE-Cad: it promotes DE-Cad-mediated cell adhesion and restricts DE-cad expression (Jezowska, 2011).

While the effect of CP loss on DE-cad transcription is not context dependent, the polarity defect is mainly observed in the distal wing domain. Different regions of the wing disc may have specific requirements in terms of AJs stability and remodelling. Because the distal wing disc is under higher mechanical stress, this epithelium may require higher dynamics of DE-Cad remobilization. CP might be critical to control this kinetic, making distal wing cells lacking CP more prone to lose cell-cell adhesion and extrude from the epithelium (Jezowska, 2011).

Interestingly, the proto-oncogene of the Src family kinases Src42A antagonizes DE-Cad-mediated cell adhesion and stimulates the transcription of DE-cad. Moreover, in the distal wing disc epithelium, the major inhibitor of Src family kinases C-terminal Src kinase (Csk), maintains AJs stability, prevents JNK-mediated apoptosis, whereas halving the genetic dose of DE-cad suppresses the apoptotic phenotype of dCsk-depleted cells. CP and mammalian c-Src both regulate F-actin. Conversely, the control of F-actin impacts on the kinase activity of c-Src. Thus, whether the main role of CP is to regulate Src activity in the distal wing disc is an exciting possibility to be tested in the future (Jezowska, 2011).

This study and others have previously shown that the CP heterodimer acts as tumour suppressor through its control of Hpo pathway activity (Fernandez, 2011; Sansores-Garcia, 2011). This study now shows that in specific epithelia, loss of CP also affects cell-cell adhesion, which is a fundamental step to an epithelial-to-mesenchymal transition (EMT), triggers MMP1 expression, which degrades the basal extracellular matrix, induces cell invasion and promotes massive proliferation of cells that fail to stably retain associations with their neighbours when cell death is blocked with P35. Moreover 'undead' CP-depleted cells show ectopic N-Cad expression, whose de novo expression promotes the transition from a benign to a malignant tumour phenotype. Finally, like other tumour suppressors, loss of CP cooperates with Ras^V12 in tissue overgrowth. These findings argue that in some epithelia in which CP activity is affected, the appearance of a second mutation that prevents apoptotic cell death may trigger the development of aggressive tumours in humans. However, in contrast to tumour progression, which correlates with loss of overall E-Cad expression and stimulation of canonical Wnt signalling, this study observed increase DE-Cad levels and inhibition of Wg signalling in tissues knocked down for CP. Interestingly, in flies, shg-LacZ expression is also enhanced in response to ectopic expression of the two oncogenes Src42A and Yki. This suggests the interesting hypothesis that transcriptional stimulation of DE-cad is an early mechanism of tumour suppression, which would promote the elimination of deleterious cells, possibly through inhibition of Wg signalling, rather than allowing them to proliferate and form tumours. Malignant cells that become resistant to cell death may compete successfully by losing the overall E-Cad expression and upregulating mesenchymal cadherins such as N-Cad to reinforce their fitness (Jezowska, 2011).

Enabled and Capping protein play important roles in shaping cell behavior during Drosophila oogenesis

During development, cells craft an impressive array of actin-based structures, mediating events as diverse as cytokinesis, apical constriction, and cell migration. One challenge is to determine how cells regulate actin assembly and disassembly to carry out these cell behaviors. During Drosophila oogenesis diverse cell behaviors are seen in the soma and germline. This study used oogenesis to explore developmental roles of two important actin regulators: Enabled/VASP proteins and Capping protein. Enabled was found to play an important role in cortical integrity of nurse cells, formation of robust bundled actin filaments in late nurse cells that facilitate nurse cell dumping, and migration of somatic border cells. During nurse cell dumping, Enabled localizes to barbed ends of the nurse cell actin filaments, suggesting its mechanism of action. This mechanism was further pursued using mutant Enabled proteins, each affecting one of its protein domains. These data suggest critical roles for the EVH2 domain and its tetramerization subdomain, while the EVH1 domain appears less critical. Enabled appears to be negatively regulated during oogenesis by Abelson kinase. The function of Capping protein was also explored. This revealed important roles in oocyte determination, nurse cell cortical integrity and nurse cell dumping, and support the idea that Capping protein and Enabled act antagonistically during dumping. Together these data reveal places that these actin regulators shape oogenesis (Gates, 2009).

The function of two key actin regulators were eliminated, and the effect on the diverse actin structures cells produce was examined during development. In the case of Ena, the results are surprising: cells use this tool to craft a diverse array of different actin assemblies that contribute to many different cell behaviors. In cultured fibroblasts, neurons and epithelial cells, Ena restrains migration by modulating actin dynamics at the leading edge, and generates filopodia by anti-capping and filament bundling. Ena also regulates axon outgrowth and guidance, manipulating actin assembly in growth cones, and plays roles in dendrite branching. During Drosophila morphogenesis Ena plays many roles. Some, like promoting leading edge filopodia and thus epithelial zippering during dorsal closure, fit well with Ena's anti-capping function. In other roles (segmental groove formation and head involution), the cell biological basis is less clear, but affected cell types exhibit striking patterns of Ena localization. Analysis of oogenesis further broadens the diversity of cell behaviors requiring Ena, highlighting roles in nurse cell cortical integrity, formation of nurse cell cytoplasmic filaments during dumping, and border cell migration (Gates, 2009).

In the case of the cytoplasmic filaments, this analysis combined with Ena's postulated biochemical functions provide interesting mechanistic insights into filament assembly. Ena is thought to promote filopodia by providing anti-capping activity, promoting filament elongation, and helping bundle filaments. Nurse cell cytoplasmic filaments form from bundled actin filaments projecting from the plasma membrane, with their barbed ends membrane-proximal. Ena localizes to these bundled barbed ends and filament formation is depressed in its absence, supporting the idea that it plays an important role in promoting filament elongation/bundling. Consistent with anti-capping being critical, reducing CP levels partially suppresses the effects of Ena inactivation (Gates, 2009).

Three ena mutants were used to begin to dissect mechanisms by which Ena acts. Ena²³ protein lacks the tetramerization domain. In nurse cells, Ena tetramerization may help collect individual actin filaments into the robust bundled cytoplasmic filaments, as has been suggested for filopodia in mammalian cells. Consistent with this, the Ena²³ mutant protein, which should be able to act in anti-capping but should be reduced in the ability to bundle capped filaments, displayed a significant decrease in its ability to mediate filament formation. Ena²³'s phenotype was similar to that of Ena⁴⁶, which completely lacks the EVH2 domain, suggesting tetramerization is a key part of EVH2 function. Both appeared to retain some residual function in filament assembly, however, as their phenotypes were less severe than that of FP4mito (which recruits Ena to the surface of mitochondria). Alternately, FP4mito sequestration of Ena may also sequester some protein partners, thereby increasing phenotypic severity. Ena²¹⁰ mutant protein, with a point mutation in the EVH1 domain impairing binding to EVH1 ligands, retained significant function. This suggests either that Ena's role in this process is largely independent of an EVH1 ligand (perhaps it is recruited to the cortex by other protein interactions), or that the point mutation in ena²¹⁰ does not fully inactivate EVH1 function (Gates, 2009).

None of the methods of disrupting Ena function fully eliminated cytoplasmic filaments. This may suggest either that none of the approaches completely eliminate all functional Ena (the data in embryos suggest that FP4mito produces a nearly null or null phenotype), or that Ena is not absolutely essential for filament assembly, although it does clearly regulate the rate/success of filament initiation or polymerization. It will be interesting to further explore how Ena's structure dictates its function in future experiments (Gates, 2009).

The mechanistic role of Ena in nurse cell cortical integrity must remain more speculative, as actin substructure at this position has not been closely investigated. Cortical integrity also requires Ena's partner Profilin and Ena's antagonist CP, as well as the actin-nucleating/branching Arp2/3 complex. Thus correct structural integrity of cortical actin appears to require balance between anti-capping and branching. Nurse cell cortical integrity also requires the cadherin-catenin complex; it may anchor cortical actin, or may have a more active role in regulating actin assembly through α-catenin and other actin regulators associated with AJs (Gates, 2009).

Ena also plays a role in border cell migration. This role has an interesting twist. While decreasing Ena/VASP function increases speed of fibroblast migration in cell culture, it slows migration of border cells. These two cell types migrate in quite different settings. Fibroblasts move over an extracellular matrix substrate by lamellipodial protrusion, adhesion, and tail retraction. In contrast, border cells migrate by squeezing in between nurse cells, and thus their substrate is cellular rather than matrix, and the shape of the leading process is constrained by the presence of other cells surrounding it. The shapes of border cell leading protrusions are quite different from fibroblast leading edge lamella, so perhaps the differential requirements for Ena are not so surprising. In fibroblasts, too much Ena activity increases lamellipodial dynamics but prevents production of a leading edge strong enough to promote stable protrusion. In border cells Ena inactivation may inhibit migration by several mechanisms. Ena inactivation could, in principle, affect ability of cells to protrude; however protrusions appeared relatively normal following Ena sequestration. Ena inactivation could reduce formation of finer protrusions like filopodia (as it did during Drosophila dorsal closure, and filopodia might serve a sensory function in migration. Finally, as Ena localizes to AJs in epithelia, Ena inactivation might affect DE-cadherin-mediated adhesion, which promotes border cell migration. It will be important to test these alternatives, for example by exploring the migration of wild-type border cells through a germline that is ena mutant (Gates, 2009).

Overexpressing Ena also causes border cell migration defects, as well as formation of excess filopodia. Neurons and leading edge cells during Drosophila dorsal closure also exhibit excess filopodia following Ena up-regulation, while fibroblasts do not. Interestingly, neurons and border cells express high levels of the actin bundler Fascin (singed in Drosophila), whereas fibroblasts do not. Since Fascin is thought to be a key regulator of filopodia, perhaps the explosive filopodia phenotype depends upon Fascin-mediated bundling. This remains to be tested. singed mutants do not have border cell migration defects, however, suggesting that it acts redundantly with other regulators of actin bundling under normal circumstances. Excess filopodia may slow migration several ways, from reducing the ability to produce a single leading process to altering chemosensory cues received via filopodia. More detailed analysis wild-type and mutant border cell protrusions will help address these issues (Gates, 2009).

The simplest model of Ena/VASP protein function suggests it acts as a CP antagonist, promoting filament elongation while CP prevents this. Studies in cultured cells support this basic hypothesis: in both mammalian B16F1 melanoma cells and Drosophila D16 cells, CP depletion triggers explosive formation of filopodia, and in melanoma cells this largely depends on Ena/VASP activity. In contrast Ena depletion prevents filopodial formation in both mammalian MVD7 cells and Drosophila D16 cells. In vivo, it is likely that cell behavior is regulated by differences in relative ratios of CP and Ena/VASP activity in the context of other actin regulators (Gates, 2009).

Nurse cell cytoplasmic filaments provide an interesting system in which to examine this balance. Ena plays an important though possibly not essential role in filament initiation, and this role appears to be restrained by negative regulation by Abl kinase. Naively, it was thought that CP depletion might have the opposite phenotype, producing more or more robust filaments. However, reducing CP function produced a more complex phenotype. Filaments were produced, but they were not uniformly distributed around the cortex, and were not effective at anchoring nuclei during dumping. The number of filaments extending to the nuclei did not seem increased, but instead the entire nurse cell cortex became 'furry' with actin (Gates, 2009).

What mechanism might explain this phenotype? Perhaps depleting CP produces so many elongating, 'anti-capped' actin filaments that they exceed the available Ena and/or other tip complex proteins. This may consume much of the available G-actin in producing numerous relatively short filaments, giving the cortex its furry appearance. Recent work supports this idea of 'monomer channeling' in vitro. Another speculative possibility, which is not mutually exclusive, is that in the absence of CP, the individual 'units' from which the cytoplasmic actin filaments are assembled grow longer than usual, compromising their mechanical strength and leading to the disorganization observed. Similar models were offered for the reduced lamellipodial persistence in fibroblasts with too much Ena activity. Reducing CP levels suppressed the effects of Ena inactivation on dumping, consistent with an antagonistic relationship. Future experiments, including further exploring epistatic relationships between Ena and CP, are required to test these hypotheses and further explore Ena, CP and their joint mechanisms of action (Gates, 2009).

When CP null germlines were generated, it was hypothesized this would dramatically increase cortical actin, as was observed in imaginal discs (Delalle, 2005; Janody, 2006) and follicle cells. However, this was not the case -- there may be a modest increase in cortical actin, but it is not dramatic. This suggests that other factors limit actin accumulation at the cortex -- these may include activity of nucleating factors like the Arp2/3 complex and formins (Gates, 2009).

However, loss of CP function in the germline did have one dramatic and surprising consequence: oocyte determination was often disrupted, and the oocyte determinant Orb was no longer enriched in the presumptive oocyte. Two other genes have very similar phenotypes: BicaudalD and Egalitarian. Both are thought to be co-factors for Dynein, modulating microtubule organization and mediating transport of cargos into the oocyte. Consistent with this, microtubule depolymerization also disrupts oocyte specification. It remains unclear why loss of CP impairs this process. Eliminating CP may impair function of the dynein-dynactin complex, of which it is a part (Cooper, 2008). Alternately, cross-regulatory interactions between actin and microtubules may be important for proper cytoskeletal structure and transport in the germarium, as is true later in oogenesis. Finally, CP may play a more direct role in transporting or anchoring Orb, or in some other step important for oocyte determination. This can now be examined in more detail (Gates, 2009).

Together these data provide insights into the developmental mechanisms that regulate the diverse actin structures critical for oogenesis. Future explorations of the detailed mechanisms of action of Ena and CP during these dynamic events and how they cooperate with other actin regulators like formins and the Arp2/3 complex will help further extend understanding of this important topic (Gates, 2009).

The Drosophila CD2AP/CIN85 orthologue Cindr regulates junctions and cytoskeleton dynamics during tissue patterning

Developing tissues require cells to undergo intricate processes to shift into appropriate niches. This requires a functional connection between adhesion-mediating events at the cell surface and a cytoskeletal reorganization to permit directed movement. A small number of proteins are proposed to link these processes. This study identifies one candidate, Cindr, the sole Drosophila melanogaster member of the CD2AP/CIN85 family (this family has been previously implicated in a variety of processes). Using Drosophila retina, it was demonstrated that Cindr links cell surface junctions (E-cadherin) and adhesion (Roughest) with multiple components of the actin cytoskeleton. Reducing cindr activity leads to defects in local cell movement and, consequently, tissue patterning and cell death. Cindr activity is required for normal localization of Drosophila E-cadherin and Roughest, and this study shows additional physical and functional links to multiple components of the actin cytoskeleton, including the actin-capping proteins capping protein alpha and capping protein beta. Together, these data demonstrate that Cindr is involved in dynamic cell rearrangement in an emerging epithelium (Johnson, 2008).

The evidence indicates that Cindr provides a functional link between dynamically regulated surface adhesion and the cytoskeletal changes required for normal pupal eye patterning. Loss of cindr activity leads to misplacement of retinal support cells, which adopt shapes uncharacteristic for their niche in the retinal field. The reasons for this became apparent when cell behavior was examined in live tissue. Reducing cindr prevents 1° cells from maintaining enwrapment of the cone cells; instead, cindr 1° cells are unable to firmly establish this niche and frequently retract, allowing neighboring interommatidial precursor cells (IPCs) to have direct contact with the cone cells. Similar instability was observed in the remaining IPCs fated to establish the 2°/3° hexagonal lattice. Histology demonstrated further changes both to AJ components and the actin cytoskeleton. Although additional roles for Cindr such as regulation of endocytosis cannot be ruled out, no evidence was observed for such a role during cell rearrangements (Johnson, 2008).

In wild-type tissue, Hbs and Rst are localized exclusively to 1°-IPC interfaces during IPC patterning; heterophilic interactions between these molecules are thought to direct the rearrangement of IPCs into single rows around each ommatidium. In cindr-IR tissue (an RNAi inverted repeat knockout), a mislocalization was observed of Rst to the entire IPC circumference. Such mislocalization would impede the generation of a preferential adhesive force, disrupting the direction or flow of cell movement and subsequent patterning. Irregularities were detected in the localization of DE-Cad around the circumference of retinal cells when Cindr was reduced. This presumably resulted in uneven or unreliable junctional stability that further destabilized the dynamic switching of cell positions. Genetic interactions with the loci for rst, hbs, and shg confirmed that these loci cooperate with cindr during patterning (Johnson, 2008).

It was also demonstrated that the actin cytoskeleton is dynamically remodeled during pupal eye patterning and that reducing cindr activity leads to a change in the details of cytoskeletal dynamics. In wild-type tissue, polymerized actin was initially detected almost exclusively at AJs of pre-1° cells and IPCs. These actin rings intensify as cells became rearranged and then, remarkably, once patterning is established, membrane-associated F-actin strongly diminishes. The functional significance of these changes is likely linked to concurrent modification of Rst- and AJ-mediated adhesion. For example, the levels of both DE-Cad and Armadillo (β-catenin) decrease between IPCs as they are rearranged, which would serve to facilitate Rst-mediated IPC movements toward 1° cells. Data indicate that the actin cytoskeleton is coordinately reinforced at AJs as adhesion is weakened. This may serve to maintain the surface integrity of the retinal cells while junctional strength decreases and the tissue is remodeled. Once patterning is achieved, the BMP receptor Thickvein then acts to restrengthen AJs; the reduction of F-actin may reflect the reduced need for a dense actin ring. Throughout this process, Cindr acts as a pivotal regulator to coordinate AJ modification and actin polymerization (Johnson, 2008).

Several data presented in this paper suggest that Cindr acts directly to regulate actin. First, the intensity and distribution of cytoplasmic Cindr puncta in retinal cells tracks that of F-actin in IPCs during development. Second, the striking dynamics of F-actin polymerization are lost, presumably helping account for their abnormal cell movements. Third, strong genetic interactions were observed between cindr-IR and multiple components of the actin regulatory machinery. Fourth, the two capping protein subunits Cpa and Cpb coimmunoprecipitated together with Cindr from Drosophila embryos. And, finally, several phenotypes are shared between tissue mutant for cindr-IR and tissue mutant for cpa or cpb: pupal eye mispatterning, gaps in the distribution of DE-Cad around the circumference of retinal cells, bristle malformation, and tissue degeneration (Johnson, 2008).

These results emphasize the important role of the actin cytoskeleton in regulating or maintaining AJ integrity. However, the data also argue that Cindr regulates the localization of transmembrane adhesion proteins at least in part independently of the cytoskeleton: reducing the genetic component of actin regulators enhances the patterning defects of cindr-IR but not the disruption to DE-Cad, and ectopic Arp66B rescues cindr-IR mispatterning but does not rescue aberrant localization of Rst. In the absence of Cindr, miscoordination of the actin machinery together with aberrant localization of junctional complexes is likely the underlying cause of tissue mispatterning during development. Similarly, deregulation of actin and junction instability is apparently cell lethal in more mature tissue, eventually leading to degeneration of mutant tissue. This may be analogous to the degeneration of mammalian podocytes that has been associated with mutations in CD2AP (Schiffer, 2004; Peters, 2006; Woroniecki, 2006). How Cindr itself is regulated during development remains an open question (Johnson, 2008).

Actin capping protein α maintains vestigial-expressing cells within the Drosophila wing disc epithelium

Tissue patterning must be translated into morphogenesis through cell shape changes mediated by remodeling of the actin cytoskeleton. Capping protein α (Cpa) and Capping protein β (Cpb), which prevent extension of the barbed ends of actin filaments, are specifically required in the wing blade primordium of the Drosophila wing disc. cpa or cpb mutant cells in this region, but not in the remainder of the wing disc, are extruded from the epithelium and undergo apoptosis. Excessive actin filament polymerization is not sufficient to explain this phenotype, since loss of Cofilin or Cyclase-associated protein does not cause cell extrusion or death. Misexpression of Vestigial, the transcription factor that specifies the wing blade, both increases cpa transcription and makes cells dependent on cpa for their maintenance in the epithelium. These results suggest that Vestigial specifies the cytoskeletal changes that lead to morphogenesis of the adult wing (Janody, 2006; full text of article).

Functional CPs are a highly conserved αβ heterodimer that bind the barbed ends of actin filaments through the C-terminal regions of both subunits. CPs and the Arp2/3 complex, which promotes filament branching, favor formation of the short highly branched actin filaments required to generate protrusive force at the leading edge of migrating cells. Ena/VASP proteins have the opposite activity, promoting formation of long unbranched parallel bundles of actin filaments. In mouse or Dictyostelium cells, depletion of CPs can cause extensive formation of filopodia and increase the length and bundling of actin filaments, reducing cell motility. Another function of CPs is to cap a short filament of the actin-related protein Arp1 in the Dynactin complex, which is required for Dynein-mediated transport along microtubules (Janody, 2006 and references therein).

Wing blade cells lacking either cpa or cpb are extruded from the epithelium and subsequently die. A number of possible mechanisms might account for this loss of CP mutant cells. As extrusion of cpa mutant cells still occurs in the presence of the apoptotic inhibitors p35 or th, apoptosis is likely to be a secondary consequence of extrusion; extruded cells might undergo apoptosis because they are deprived of anti-apoptotic signals present in their normal niche. In addition, JNK activity is not essential for extrusion; cpa mutant clones were not rescued by expression of a dominant-negative form of basket (bsk), which encodes JNK. However, the possibility cannot be exclude that the p35 or Th inhibitors block apoptosis too late to prevent release of an extrusion signal (Janody, 2006).

The function of capping proteins (CPs) in organelle or vesicle transport is unlikely to explain the extrusion phenotype. CPs are thought to stabilize the barbed end of the Arp1 microfilament in the Dynactin complex, which is required for transport along microtubules. cpa and cpb, like other Dynactin complex subunits, are required to maintain the position of nuclei in Drosophila photoreceptor neurons (Whited, 2004). However, removal of kinesin heavy chain (khc), which counteracts Dynein/Dynactin-based transport, failed to rescue extrusion of cpa mutant cells in the wing disc (Janody, 2006).

The possibility is considered that the cpa phenotype is due to its effect on monomeric G-actin levels rather than on the filamentous actin cytoskeleton. G-actin has been shown to negatively regulate the nuclear localization and activity of Mal, a transcriptional cofactor for SRF, and overexpression of Mal or of its activator diaphanous can cause extrusion and death of wing epithelial cells. However, overactivity of the MAL/dSRF pathway is unlikely to be responsible for extrusion of cpa mutant cells in the wing blade; clones mutant for both cpa and blistered (bs), which encodes Drosophila SRF, are still extruded from the wing epithelium (Janody, 2006).

Extrusion of cpa or cpb mutant cells might be a direct result of defects in the actin cytoskeleton. Consistent with the requirement for CPs to inhibit addition of actin monomers to the fast-growing end of actin filaments, a strong accumulation of actin filaments was observed in cpa mutant clones. However, tsr and capt mutations also induce excessive actin filament polymerization but do not cause cell extrusion. The major function of Tsr (Cofilin) is to promote dissociation of ADP-actin from the pointed end of the filament, while Cpa prevents elongation of the barbed end of each branch and Capt sequesters actin monomers. The phenotypic differences between cpa, tsr and capt might therefore be due to different degrees of branching of the actin network formed in mutant cells. Possibly long unbranched filaments do not provide a framework of sufficient strength to withstand forces that place tension on the cell within the epithelium (Janody, 2006).

Extrusion is associated with dispersion of the adherens junction components Arm and DE-Cad along the lateral membranes. However, this defect is also observed in tsr mutant clones, and mislocalization of adherens junction components caused by overexpression of a dominant form of the polarity gene crumbs does not lead to cell extrusion. Therefore, mislocalization of AJ components is unlikely to be sufficient to cause extrusion of cpa mutant cells. By contrast, expression of dominant-negative Rac1, which prevents actin localization to adherens junctions, induces cell extrusion and death. Thus, another possibility is that CPs may be crucial for linking actin filaments to the membrane. Loss of cpb displaces actin bundles from the cell membrane in Drosophila bristles by increasing the concentration of non-bundle actin snarls and CPs may specify actin filament position in the sarcomere. In the Drosophila wing blade epithelium, loss of CPs might disrupt attachment of the actin cytoskeleton to the adherens junctions, breaking the connection between cells and inducing cell extrusion. The localization of HA-Cpa to apical junctions and the mislocalization of actin filaments throughout cpa mutant cells in the wing blade are consistent with this possibility. Such a role would be restricted to the wing blade, as cpa mutant cells within the notum epithelium accumulate actin filaments only at the apical cell membrane (Janody, 2006).

Surprisingly, it was found that cpa and cpb are required to prevent cell extrusion and death only in the region of the wing disc giving rise to the wing blade, but not in the primordia of the hinge or notum, or in the eye or leg discs. The requirement for cpa depends on the wing blade selector gene Vg; expression of Vg in notum cells is sufficient to induce their extrusion in the absence of cpa. Vg also enhances the transcription of cpa in the wing blade primordium. Taken together, these results imply that patterning genes regulate cytoskeletal properties in order to achieve distinct morphological outcomes (Janody, 2006).

The molecular mechanism that makes Vg-expressing cells dependent on CPs for their maintenance in the epithelium is unknown, although the data support a cell-autonomous target of Vg. One possibility is that Vg promotes the expression or recruitment of an actin filament polymerizing factor. The role of CPs might be to restrict its activity at barbed ends, preventing the formation of a specific actin-based structure that induces loss of cell-cell contacts and extrusion. For example, Vg activates the expression of the type II transmembrane protein Four jointed (Fj), which regulates the activity of the cadherin Fat. Mammalian Fat1 can recruit Ena/VASP proteins, which promote actin polymerization at cell-cell contacts by antagonizing CPs. However, misexpression of fj does not induce extrusion of either wild-type or cpa mutant cells in the notum. DE-cadherin levels are also higher in the wing pouch, but increasing them in the hinge or notum by activating Wg signaling does not cause extrusion of cpa mutant cells. Alternatively, Vg might control the expression of factors that promote the remodeling of cell junctions required for morphogenesis of the wing. Cpa could be required to maintain the connection between cells in the epithelium during these morphogenetic movements (Janody, 2006).

The non-uniform distribution of and requirement for cpa suggests that cytoskeletal organization varies in different regions of the wing disc. It has been observed that lateral wing disc cells had moderately reduced levels of basolateral cortical F-actin. In addition, filopodial extensions called cytonemes are oriented towards the AP and/or DV boundary within the wing pouch, while hinge cells do not extend cytonemes and notum cells radiate short cytonemes in all directions. Changes in cytoskeletal organization have been shown to establish cell affinity boundaries, to control the subcellular localization of transcription factors and to modulate the transport of signaling molecules. Investigating the control of cpa by Vg may help understanding of how and why patterning genes regulate cell architecture. In addition, identifying additional target genes of Vg may illuminate how actin dynamics and changes in intercellular adhesion control the formation of the wing blade (Janody, 2006).

Capping protein and the Arp2/3 complex regulate nonbundle actin filament assembly to indirectly control actin bundle positioning during Drosophila melanogaster bristle development

Drosophila bristle development is dependent on actin assembly, and prominent actin bundles form against the elongating cell membrane, giving the adult bristle its characteristic grooved pattern. Previous work has demonstrated that several actin-regulating proteins are required to generate normal actin bundles. This study addresses how two actin regulators, capping protein, a barbed end binding protein, and the Arp2/3 complex, a potent actin assembly nucleator that acts downstream of WASp, function to generate properly organized bundles. As predicted from studies in motile cells, it was found that capping protein and the Arp2/3 complex act antagonistically to one another during bristle development. However, these proteins do not primarily act directly on bundles, but rather on a dynamic population of actin filaments that are not part of the bundles. These nonbundle filaments, termed snarls, play an important role in determining the number and spacing of the actin bundles. Reduction of capping protein leads to an increase in snarls, which prevents actin bundles from properly attaching to the membrane. Conversely, loss of an Arp2/3 complex component leads to a loss of snarls and accumulation of excess membrane-attached bundles. These results indicate that in nonmotile cells dynamic actin filaments can function to regulate the positioning of stable actin structures. In addition, the results suggest that the Arpc1 subunit may have an additional function, independent of the rest of the Arp2/3 complex (Frank, 2006).

The actin cytoskeleton is of vital importance in many cellular processes: it is an integral part of the contractile ring during cell division, its nucleation at the leading edge drives cell locomotion, its polymerization is important for endocytosis, and it provides a structural framework for specialized cell shapes. The last few years have seen an explosion in knowledge about how actin assembly is both initiated and limited. Two important players are the Arp2/3 complex and capping protein. The Arp2/3 complex consists of actin related proteins 2 and 3 along with five other subunits, Arpc1-Arpc5. On activation by a member of the Wiscott Aldrich Syndrome protein (WASp) family, this complex can nucleate new actin filament assembly as well as create branched networks of filaments through side binding to existing filaments. In addition to its actin nucleation activity, the Arp2/3 complex can cap the slow growing, or pointed, ends of actin filaments. Capping protein, a heterodimer of alpha and beta subunits, binds the fast growing, or barbed, ends of actin filaments and prevents both depolymerization and the addition of new monomers. The importance of capping protein and the Arp2/3 complex is evidenced by the fact that they are two of the five components absolutely required for formation of actin comet tails that drive intracellular movement of the pathogenic bacteria Listeria monocytogenes (Frank, 2006).

Although much has been learned in the last several years about the proteins and mechanisms regulating actin filament assembly during motility and in vitro, much less is understood about the processes governing the assembly of more stable actin structures in cells that do not move. Many questions remain unanswered about such structures. For example, how are stable actin structures positioned within the cell? Do the same proteins control filamentous actin assembly in stationary cells as in the leading edge of moving cells? Do these proteins function in the same way in these very different contexts (Frank, 2006)?

To investigate these questions, the large bristles, or macrochaetes, of the fruit fly Drosophila melanogaster are being used. The long bristle shaft (up to 400 μm in length) is generated from a single cell that undergoes extension during pupal development. Bristle cell elongation is dependent on actin assembly, and bundles of actin filaments lie against the membrane, parallel to the long axis of the bristle. These cortical bundles give the adult bristle its grooved appearance; the bundles lie at the positions of the valleys between the tall ridges of cuticle. Although it is apparent that the barbed ends of the actin filaments in bundles are oriented toward the bristle tips, it is not clear that extension of actin filaments in the bundles provides the pushing force of membrane protrusion. Rather, the bundles might serve as structural girders that maintain the cellular extension. Between the bundles are very dynamic masses of actin filaments termed snarls. Snarls were proposed to be transient structures that are not stabilized by cross-linking to other filaments or attachment to the membrane, perhaps byproducts or aberrantly formed structures that are eliminated. Whether snarls have any function is unknown (Frank, 2006).

Mutations in genes encoding many actin-regulating proteins, such as the cross-linkers forked and singed (encoding fascin), chickadee (encoding profilin, an actin monomer-binding protein that promotes filament assembly), the actin monomer sequesterer twinfilin, and capping protein (Hopmann, 1996), result in deformed bristles. Close examination of bristle development reveals aberrant actin bundle morphologies in these mutants. For example, capping protein beta mutant pupal bristles have irregularly sized actin bundles that are displaced from the membrane. It is hypothesized that this may be due to unregulated assembly of the filaments within the actin bundles, though the precise mechanism remained unclear (Hopmann, 1996). Previous work used the bristle system to elucidate the antagonistic interaction between capping protein and profilin: a chickadee mutation was able to suppress the bristle phenotype of a transheterozygous combination of capping protein beta mutant alleles (Hopmann, 2003). Reduction of capping protein leads to an increase in actin filament assembly that is abrogated by loss of profilin. Thus these proteins appear to act in a very similar manner to their function at the leading edge of motile cells (for review see Pollard, 2003; Frank, 2006 and references therein).

This article reports that, as predicted from studies in motile cells, capping protein and the Arp2/3 complex act antagonistically to one another during bristle development. However, the role of capping protein in actin bundle organization is quite different from previously thought. Instead of primarily acting directly on actin filaments within bundles, the results indicate that capping protein and the Arp2/3 complex regulate actin snarls. Actin bundle positioning is affected by the quantity and persistence of these dynamic actin filaments since their overabundance displaces bundles from the membrane, whereas their absence results in excess actin bundles attached to the membrane (Frank, 2006).

The results establish that the Arp2/3 complex and capping protein have opposing actions on dynamic actin structures, called snarls, in Drosophila bristle cells. In the absence of the Arp2/3 complex, no snarls are apparent, whereas when capping protein is reduced, snarls persist longer during development. Combinations of mutations that affect both proteins are more similar to wild type in number and persistence of snarls, as would be expected if the two activities need to be balanced for proper regulation of actin assembly. Because most other studies of capping protein and the Arp2/3 complex have utilized in vitro assays or motile cells in culture, the observation that these regulators function similarly in nonmotile cells in vivo is important, albeit not surprising. What is surprising, however, is that the main structural features important for the morphology of these cells, the large cortical actin bundles, are not the direct target of these activities, despite the fact that their organization and positioning is strongly affected by altered amount of these regulators (Frank, 2006).

This study presents evidence that early in bristle development capping protein and the Arp2/3 complex control actin assembly not in actin bundles, but rather in the actin snarls that are present between these bundles. It is hypothesized that snarls influence the proper positioning of the cortical actin bundles by competing with them for binding sites on the membrane. When the level of snarls is severely reduced (in homozygous arp3 mutant pupae), excess actin bundles associate with the membrane. This results in adult bristles that are shorter than wild type and have more, smaller grooves in their cuticle. In the presence of too many snarls (in transheterozygous cpb mutant pupae), actin bundles are unable to form against the membrane and instead develop internally. Because they are not stabilized by attachment to the membrane, they become irregularly sized and highly variable in number. This results in a cell membrane that is inadequately supported by actin bundles. Adult bristles that have numerous defects, such as irregular groove patterns and smooth regions in the cuticle, as well as bends, splits, and protrusions in the shaft are produced (Frank, 2006).

Actin snarls are dynamic and it has been suggested that their 2-min half-life is due to their failure to be stabilized either by cross-linking to other filaments or by forming stable attachments to the membrane. It has been argued that those filaments that do not become stabilized are turned over, and thus the actin bundles develop in a Darwinian 'survival of the fittest' manner. The snarls have been proposed to be nonproductive actin structures. This study proposes that the snarls have a function in forming properly shaped bristles: they are important for proper positioning of bundles. Snarls may have additional functions as well. They cause protrusions in the membrane between the bundles and lie beneath the regions that will become the ridges in the cuticle of the adult bristle (the actin bundles lie below the grooves). Thus the Arp2/3 complex–directed actin assembly in snarls may push the membrane out, much in the way that this complex causes membrane protrusion at the leading edge of motile cells. This region of the bristle membrane is then kept free from actin bundles so that the proper number and size of bundles are formed. When too many bundles form, cuticle is secreted in a disorganized manner. Thus it may be important that some regions of the membrane are kept clear of actin bundles to allow for proper secretion of chitin. Alternatively, actin filaments in the snarls could be directly involved in membrane deposition that occurs during cell extension and/or in the secretion of cuticle (patches of cuticulin are evident on developing bristles as early as 36 h APF). Snarls may be analogous to actin patches in Saccharomyces cerevisiae that are turned over very rapidly, contain the Arp2/3 complex and capping protein, and are involved in endocytosis and cell wall synthesis (Frank, 2006).

The interplay seen between actin bundles and snarls in bristles is reminiscent of that seen between actin cables and patches in S. cerevisiae. Yeast cells lacking functional capping protein have diminished actin cables and excess patches. Conversely, long-term lack of Arp3 results in cells that have lost patches and accumulate actin bundles. The antagonistic relationship between capping protein and the Arp2/3 complex, as well as their opposing effects on actin patches and bundles, might thus be a common theme in the regulation of the actin cytoskeleton (Frank, 2006).

Mutations in arp3 and wasp, as well as deficiencies that remove arpc4 and arpc5, all suppress the cpb bristle phenotype as would be expected from the opposing biochemical functions of the Arp2/3 complex (filament assembly nucleator) and capping protein (prevents further assembly of actin filaments). So it is surprising to find that mutations in arpc1 have the opposite phenotype. This is an especially unexpected result given that previous work has demonstrated that adult bristles completely lacking Arpc1 or Arp3 have an identical mild phenotype of more, smaller grooves. It is tempting to suggest that there is a genetic background issue at play here. It is believed that this is unlikely, however, because two different arpc1 alleles isolated in independent labs in different genetic backgrounds gave the same result (Frank, 2006).

One explanation for the enhancement seen by mutations in arpc1 is that in the absence of this subunit, a complex forms that in some way acts as a neomorph. Arpc1 contains the WASp interaction capability of the complex, so complexes lacking this subunit might be unactivatable, and thus might block the interaction between actin filaments and other important binding proteins. Although this possibility out cannot be ruled out, it is thought unlikely because of the following: Arpc1 (also referred to as p41) and Arpc5 (p16) subunits form an interacting pair. They find that when complexes are formed in the absence of Arpc5, Arpc1 also fails to assemble into the complex. If this neomorph model were correct (and assuming fly Arp2/3 complex components behave in the same way as their human homologues), it would be expected that reduction of Arpc5 would lead to loss of Arpc1 from the complex and cause enhancement of the cpb phenotype. However, this is not what was observed. A deficiency chromosome lacking the arpc5 region in fact suppressed the cpb bristle phenotype (Frank, 2006).

To explain arpc1 enhancement of bundle displacement observed in cpb mutants, it is noted that the difference in phenotype between cpb^6.15/cpb^F19 and cpb^6.15 +/cpb^F19 arpc1^Q25st is only apparent at late developmental times. In both genotypes early in development, bundles are displaced from the membrane. However, in cpb^6.15/cpb^F19 at late times, bundles often are associated with the membrane, whereas in cpb^6.15 +/cpb^F19 arpc1^Q25st they rarely are. Interestingly, in cpb null epithelial clones, bundles also remain displaced. These similar phenotypes suggest that capping protein and Arpc1 both participate in a late membrane-attachment process. Thus, it is suggested that in cases where bundles form not associated with the membrane initially, capping protein and Arpc1 promote bundle attachment late in development. The ability of some bundles to associate with the membrane in cpb^6.15/cpb^F19 bristles is likely due to the fact that in this genotype capping protein is not completely eliminated but rather is reduced to 48% of the wild-type level. When capping protein is completely absent or when both capping protein and Arpc1 are reduced in amount, then this late membrane attachment does not occur. Interestingly, capping protein localization on bundles starts at ~42 h APF, around the time when the effect of capping protein and Arpc1 on bundle membrane association is manifest. Under normal circumstances, this late bundle attachment activity is not essential, because bundles are already associated with the membrane. Similarly, in cases where snarls are reduced and excess bundles form (arp3, and presumably, arpc1 homozygous) this late attachment activity is not needed because the bundles form initially against the membrane. Obviously, other as yet unidentified proteins must be important for membrane association at early times (Frank, 2006).

Capping protein has previously been observed to be required for attachment of actin filaments to the Z disks during myofibrillogenesis; a role in membrane attachment in bristles would be consistent with this function. It has been suggested that Arpc1 (therein referred to as p41-Arc) might serve as a link to the membrane through its WD repeats binding to pleckstrin homology domains, which could in turn bind to phosphatidyl inositol 4,5-bisphosphate. Additionally, Arpc1 binds to the activating domain of WASp, which is itself activated at the membrane (Frank, 2006).

It is suggested that Arpc1 has a function outside the Arp 2/3 complex. Although work in yeast demonstrating an unique function for Arpc1 was later shown to be an experimental artifact, recent studies in the moss Physcomitrella patens has revealed that removal of Arpc1 results in different phenotypes than removal of Arpc4. Thus, it will be important to explore this phenomenon of potential differential Arp2/3 complex subunit functions further in the future (Frank, 2006).

Mutations in the Drosophila orthologs of the F-actin capping protein alpha- and beta-subunits cause actin accumulation and subsequent retinal degeneration

The progression of several human neurodegenerative diseases is characterized by the appearance of intracellular inclusions or cytoskeletal abnormalities. An important question is whether these abnormalities actually contribute to the degenerative process or whether they are merely manifestations of cells that are already destined for degeneration. A large screen was conducted in Drosophila for mutations that alter the growth or differentiation of cells during eye development. Mitotic recombination was used to generate patches of homozygous mutant cells. In the entire screen, mutations in only two different loci, burned (bnd) and scorched (scrd), resulted in eyes in which the mutant patches appeared black and the mutant tissue appeared to have undergone degeneration. In larval imaginal discs, growth and cell fate specification occur normally in mutant cells, but there is an accumulation of F-actin. Mutant cells degenerate much later during the pupal phase of development. burned mutations are allelic to mutations in the previously described cpb locus that encodes the beta-subunit of the F-actin capping protein, while scorched mutations disrupt the gene encoding its alpha-subunit (cpa). The alpha/beta-heterodimer caps the barbed ends of an actin filament and restricts its growth. In its absence, cells progressively accumulate actin filaments and eventually die. A possible role for their human orthologs in neurodegenerative disease merits further investigation (Delalle, 2005).

This study has shown that mutations in the Drosophila orthologs of the α- and β-subunits of the F-actin capping protein result in tissue degeneration. Mutation of either gene does not appear to interfere with tissue growth or cell fate determination. Rather, mutant tissue undergoes degenerative changes at a later stage of development. The earliest abnormality that was observed in mutant clones is an accumulation of actin, consistent with the known function of the α/β-heterodimer in capping actin filaments and arresting their growth. Indeed, others have previously described disorganized actin filaments in bristles of cpb mutants (Hopmann, 1996). Also, reducing the level of either the α- or β-subunit by RNAi in Drosophila cell lines induced cytoskeletal abnormalities. The current experiments show that the cytoskeletal abnormalities observed in mutant cells are not sufficiently deleterious to result in their immediate death or even to interfere with their proliferation or with cell fate determination in vivo. Rather, these cytoskeletal perturbations manifest as tissue degeneration at a later stage of development. Intriguingly, in the entire screen of the four main autosomal arms, mutations in only two genes, cpa and cpb, gave rise to a phenotype characterized by large clones of degenerating tissue (Delalle, 2005).

In addition to the actin-capping proteins, a number of other proteins also regulate the actin cytoskeleton in vivo. They do so by a variety of mechanisms including the regulation of bundling, cross-linking, severing, polymerizing-depolymerizing, and by sequestering actin monomers. Therefore the phenotype of mutations in tsr, a Drosophila cofilin/actin depolymerizing factor homolog that also inhibits actin polymerization, was tested. Mutations in tsr have previously been shown to result in defects in centrosome migration and cytokinesis accompanied by abnormal accumulations of F-actin. In addition to the strong accumulation of F-actin in tsr clones, high levels of apoptosis were also observed. In contrast, no increased apoptosis was seen in cpa or cpb clones in the developing eye discs. A likely explanation is that the disruption of the cytoskeleton in tsr clones is severe enough to cause cell death almost immediately, whereas the less severe abnormalities in cpa or cpb clones are not. An alternate possibility is that the mechanism of cell death that occurs in tsr clones differs from that which is activated in cpa and cpb clones. In either case, the delayed cell death observed in cpa and cpb mutant clones is more similar to the type of death that occurs in degenerative diseases (Delalle, 2005).

How do the cytoskeletal abnormalities found in cpa and cpb mutations eventually result in cell death? The accumulation of F-actin could cause the mislocalization or altered regulation of a number of actin-binding activities within the cell. In addition, the uncontrolled and undirected accumulation of actin could interfere with directed cell movement, cell polarity, or cell protrusions, thereby disrupting crucial signaling events. Alternatively, physical stresses due to uncontrolled actin polymerization may simply cause the cells to break apart and undergo lysis. Indeed, cells may be particularly susceptible to such stresses when they undergo significant shape changes during the later stages of pupal development (Delalle, 2005).

These findings raise the possibility that mutations in genes encoding actin-capping proteins could cause degenerative diseases in humans. Indeed, mutations that result in alterations in the actin cytoskeleton have been implicated in two types of progressive hearing loss. The autosomal dominant deafness DFNA1 syndrome results from mutations in the human ortholog of Drosophila diaphanous, which is a member of the formin gene family and is involved in regulating actin polymerization. The DFNA 20/26 syndrome results from mutations in the gamma actin 1 (ACTG1) gene. Recently, weakening of the nuclear actin network have been suggested to underlie X-linked Emery-Dreifuss muscular dystrophy characterized by loss of emerin, an LEM-domain protein of the nuclear inner membrane. Furthermore, the aggregation of actin and cofilin has been reported in the brains of identical twins with DYT1-negative dystonia. There is a single ortholog of cpb and three cpa orthologs in the human genome. Their role in either causing or modifying degenerative disease phenotypes may warrant further investigation (Delalle, 2005).

Ena/VASP proteins enhance actin polymerization in the presence of barbed end capping proteins

Ena/VASP proteins influence the organization of actin filament networks within lamellipodia and filopodia of migrating cells and in actin comet tails. The molecular mechanisms by which Ena/VASP proteins control actin dynamics are unknown. This study investigated how Ena/VASP proteins regulate actin polymerization at actin filament barbed ends in vitro in the presence and absence of barbed end capping proteins. Recombinant His-tagged VASP increased the rate of actin polymerization in the presence of the barbed end cappers, heterodimeric capping protein (CP), CapG, and gelsolin-actin complex. Profilin enhanced the ability of VASP to protect barbed ends from capping by CP, and this required interactions of profilin with G-actin and VASP. The VASP EVH2 domain was sufficient to protect barbed ends from capping, and the F-actin and G-actin binding motifs within EVH2 were required. Phosphorylation by protein kinase A at sites within the VASP EVH2 domain regulated anti-capping and F-actin bundling by VASP. It is proposed that Ena/VASP proteins associate at or near actin filament barbed ends, promote actin assembly, and restrict the access of barbed end capping proteins (Barzik, 2005).

A balance of capping protein and profilin functions is required to regulate actin polymerization in Drosophila bristle

Profilin is a well-characterized protein known to be important for regulating actin filament assembly. Relatively few studies have addressed how profilin interacts with other actin-binding proteins in vivo to regulate assembly of complex actin structures. To investigate the function of profilin in the context of a differentiating cell, an instructive genetic interaction between mutations in profilin (chickadee) and capping protein beta (cpb) was studied. Capping protein is the principal protein in cells that caps actin filament barbed ends. When its function is reduced in the Drosophila bristle, F-actin levels increase and the actin cytoskeleton becomes disorganized, causing abnormal bristle morphology. chickadee mutations suppress the abnormal bristle phenotype and associated abnormalities of the actin cytoskeleton seen in cpb mutants. Furthermore, overexpression of profilin in the bristle mimics many features of the cpb loss-of-function phenotype. The interaction between cpb and chickadee suggests that profilin promotes actin assembly in the bristle and that a balance between capping protein and profilin activities is important for the proper regulation of F-actin levels. Furthermore, this balance of activities affects the association of actin structures with the membrane, suggesting a link between actin filament dynamics and localization of actin structures within the cell (Hopmann, 2003).

Capping protein loss of function leads to dramatic increases in F-actin in the fly bristle, resulting in aberrant organization of the actin cytoskeleton. Reduction of profilin suppresses the disorganized actin phenotype caused by reduction of capping protein function, suggesting that profilin promotes actin assembly in the elongating bristle. These results emphasize the idea that the balance of activities of actin-binding proteins is critical for assembling actin structures that are organized and positioned properly (Hopmann, 2003).

Numerous studies have demonstrated the importance of the actin cytoskeleton for the normal elongation and morphogenesis of the fly bristle. Inhibitors of actin polymerization significantly decreases the elongation rates of bristles whereas inhibitors of microtubule polymerization have little effect. The morphology of bristle actin bundles is affected by changes in the amount of cross-linking proteins as well as mutations in genes that encode regulators of actin dynamics, including ADF/cofilin (twinstar, twinfilin), and ADF/cofilin phosphatase (slingshot). Yet many of these alterations do not cause severely displaced and disoriented actin bundles. In contrast, mutations in capping protein strongly affect not only the amount of F-actin but also the position and orientation of actin structures. In this regard, the phenotype of twinfilin (twf) mutants is particularly noteworthy. Twinfilin is a monomer-sequestering protein that is structurally related to ADF/cofilin. In twf mutant bristles, F-actin levels are increased and the actin bundles are very disorganized, as they are in cpb mutants. Furthermore, the actin bundles show the same dramatic displacement from the membrane in twf as they do in cpb. This contrasts with the phenotype of chic bristles, which do not show displacement of bundles, and underscores the fact that although twinfilin and profilin both have sequestering activity in vitro, they clearly have different roles in vivo (Hopmann, 2003).

What the analysis of individual mutant phenotypes does not reveal is how the different actin regulatory proteins work together to generate normal actin bundles. Analysis of cpb chic double mutants demonstrates this clearly. Because the original phenotypic characterization of cpb and chic single mutants suggested that they both led to increased levels of F-actin, the original expectation was that chic loss of function would enhance cpb loss of function. Instead, the opposite effect was observed. This approach has yielded valuable insights regarding the importance of the balance of capping protein and profilin activities in normal cells. In other cases, mutant combinations do exhibit predictable phenotypes. For example, double heterozygous combinations of twf and tsr, which encode ADF/cofilin, exhibit a moderate bristle phenotype even though the single mutant heterozygotes show little or no bristle phenotype. This is consistent with the proposed function of both proteins: reduction of twinfilin leads to increases in F-actin assembly due to reduced sequestering activity, and reduction of ADF/cofilin leads to a decreased rate of actin depolymerization. Thus, it is expected that the two mutations behave synergistically and cause an increase in F-actin. It is anticipated that additional mutant combinations will be equally informative about the complex interplay of activities required to construct normal actin bundles. At present, formulating a model that incorporates the many different actin regulators is difficult because there is limited data of this type available (Hopmann, 2003).

The results support the idea that profilin has polymerization-promoting activity. Expression of vertebrate or plant profilins in mammalian tissue culture cells leads to increases in F-actin and profilin null clones in the developing Drosophila eye exhibit greatly reduced levels of F-actin (Hopmann, 2003 and references therein).

However, the observation that profilin acts in an opposite manner as that of capping protein, seeming to stimulate actin polymerization in the fly bristle, seems at first difficult to reconcile with the original characterization of the chic bristle phenotype. In chic mutants, the elongating bristle seemed to have an increased number of actin bundles that are thinner than wild-type bundles. This phenotype was thought to reflect an overall increase in the amount of F-actin, which is consistent with a monomer-sequestering role for profilin. Two possible explanations are suggested for this seeming paradox. First, biochemical data on profilin activity have shown that its activity is dependent on the state of the barbed ends. Profilin-actin can add to free barbed ends but not to capped ones. Thus, in wild-type bristles, barbed ends may be maximally capped (except at the growing tip) and profilin's primary function would be to sequester monomer. In a chic mutant bristle, reduction in profilin-mediated sequestering activity might lead to the observed increase in F-actin. It would then be predicted that when capping protein is reduced, barbed ends are not maximally capped and thus, profilin's polymerization-promoting activity would predominate, which is consistent with current observations (Hopmann, 2003).

Another interpretation of the chic bristle phenotype is suggested by the results of inhibitor studies performed on cultured Drosophila pupae. Exposure of cultured pupae to cytochalasin D, an inhibitor of actin polymerization, causes the actin bundles in elongating bristles to fall apart by splitting into thinner subbundles, reminiscent of chic mutant bristles that exhibit an increased number of thinner bundles. The similarity of these two phenotypes suggests that continued actin polymerization is required to maintain the integrity of actin bundles, and reductions in actin polymerization cause the actin bundles to 'unravel'. Although it is clear that profilin can promote actin polymerization, the mechanism by which it does this is less well understood. Studies in yeast have demonstrated that profilin's nucleotide exchange activity is required for its function. Because ATP-actin is more readily incorporated onto barbed ends of filaments, this activity can explain profilin's effects on actin assembly. However, there is reason to believe Drosophila profilin may not work this way. Plant profilins do not catalyze nucleotide exchange, and some even seem to repress it. A comprehensive mutational analysis of profilin in fission yeast has identified tyrosine79 as critical to its ability to stimulate nucleotide exchange. When tyrosine79 is mutated to arginine, S. pombe profilin loses its exchange activity. Notably, the majority of plant profilins naturally contain arginine at the comparable position, whereas all characterized vertebrate profilins, which tend to have very high exchange activity, contain aspartate. Thus, there is a correlation between arginine at position 79 and low activity, tyrosine and moderate activity, and aspartate and high activity. Interestingly, Drosophila has arginine: it is the only nonplant profilin, besides that of shrimp, known to have arginine at this position. The exchange activity of Drosophila profilin is unknown, but it seems reasonable to predict that Drosophila profilin has low activity (Hopmann, 2003).

Although plant profilins do not enhance nucleotide exchange by actin monomers, some stimulate actin polymerization in vitro in thymosin-ß4/actin solutions. Thymosin-ß4 is a true monomer sequestering protein in that T-ß4-actin cannot add to a growing filament, whereas profilin-actin adds readily to the barbed ends of actin filaments. Profilin is thought to shuttle monomer out of the T-ß4 pool, and this may be the relevant mechanism in other cell types. Studies in Drosophila may prove useful in elucidating the details as well as the physiological relevance of alternate mechanisms of profilin activity (Hopmann, 2003).

This article, as well as previous work, has demonstrated that a reduction of capping protein function leads to increased F-actin and abnormal actin organization. It is likely that the aberrant actin cytoskeleton underlies all of the defects observed in the adult bristle such as decreased length, bending, branching, and abnormal groove patterns. Although some of the correlations between the actin abnormalities and adult phenotypes are fairly obvious, it may seem counterintuitive that increases in F-actin levels would lead to shorter bristles. One might expect increased F-actin polymerization to give rise to longer bristles. Indeed, treatment of cultured pupae with jasplakinolide, a drug that stabilizes F-actin, increases the growth rate of the bristle shaft. However, these experiments were done for 6-7 h, whereas bristle elongation takes ~16 h at 25°C. Perhaps the increased growth rate would not be maintained were it possible to expose the growing bristle to drug for the entire elongation period. It is hypothesized that in cpb mutants, actin is overpolymerized at the beginning of bristle elongation. Some component required for actin bundle assembly may be limiting in the bristle; therefore, in a cpb mutant bristle, the limiting component would be prematurely depleted due to the increase in F-actin. Comparing the growth rates of wild-type and mutant bristles can test this idea (Hopmann, 2003 and references therein).

Although the data demonstrate that reduction of capping protein function leads to increases in F-actin, these changes were not quantified. It would have been desirable to measure the concentrations of F-actin in the various mutant genotypes directly, but technical limitations prevented doing so in a controlled manner. Phalloidin staining often varies greatly between experiments, so the subtle differences that were expected between different genotypes could be obscured. Quantitative methods are currently being developed for measuring actin in situ (Hopmann, 2003).

One of the most puzzling features of the cpb mutant phenotype is the displacement of actin bundles from the membrane. An increase in the amount of F-actin in the bristle does not, by itself, seem to explain this phenotype. In bristles where the cross-linking protein fascin is overexpressed, F-actin amounts are increased and bundles are considerably larger, but they do not show significant displacement from the membrane. A structural function of capping protein in physically linking the bundles to the plasma membrane would explain this phenotype. Previous studies in chicken myoblasts have uncovered a structural requirement for capping protein in organizing actin filaments within the sarcomere. However, a structural role seems unlikely given that the displacement of bundles is suppressed when profilin dosage is reduced. Instead, the proper regulation of actin assembly may be important for the positioning of actin bundles. twf mutant bristles also exhibit this displacement phenotype. Because capping protein and twinfilin are known to associate in yeast, this raises the interesting possibility that these two proteins work together in regulating actin assembly such that the association of bundles with the membrane is established and/or maintained. Intriguingly, treatment of cultured pupae with okadaic acid, an inhibitor of protein phosphatases, causes a similar displacement of actin bundles, suggesting the phosphorylation status of one or more proteins may be relevant (Hopmann, 2003).

This article shows that the balanced activities of capping protein and profilin are essential in the regulation of actin dynamics and organization in the elongating Drosophila bristle. The data are consistent with the emerging idea that the activity of profilin is context dependent, and that in many cells, profilin promotes actin assembly. The data also suggest that perturbations of actin dynamics in the bristle lead to a striking displacement of actin bundles from the membrane. In the future, it is hoped the role of capping protein in the bristle will be clarified, resulting in a better understand of how capping protein is integrated with the many other actin regulators functioning in the bristle, such that actin bundles are correctly assembled and positioned (Hopmann, 2003).

Cofilin and Aip1 are essential for actin remodeling during lamella formation

Cell migration occurs through the protrusion of the actin-enriched lamella. The effects of RNAi depletion of approximately 90 proteins implicated in actin function on lamella formation have been investigated in Drosophila S2 cells. Similar to in vitro reconstitution studies of actin-based Listeria movement, it has been found that lamellae formation requires a relatively small set of proteins that participate in actin nucleation (Arp2/3 and SCAR), barbed end capping (capping protein), filament depolymerization (cofilin and Aip1), and actin monomer binding (profilin and cyclase-associated protein). Lamellae are initiated by parallel and partially redundant signaling pathways involving Rac GTPases and the adaptor protein Nck, which stimulate SCAR, an Arp2/3 activator. RNAi of three proteins (kette, Abi, and Sra-1) known to copurify with and inhibit SCAR in vitro leads to SCAR degradation, revealing a novel function of this protein complex in SCAR stability. These results have identified an essential set of proteins involved in actin dynamics during lamella formation in Drosophila S2 cells (Rogers, 2003).

The actin-binding protein cofilin/Twinstar is essential for actin-based functions in many cell types, and in vitro and in vivo studies indicate a role for cofilin in actin filament severing and turnover. Inhibition of cofilin by RNAi prevented S2 cell spreading on con A in >95% of treated cells. These cells retained their spherical morphology, and phalloidin staining revealed a dramatic cortical accumulation of filamentous actin as well as a wrinkled 'raisin-like' texture to the surface of the cell. The abnormal accumulation of filamentous actin within the cells suggests that actin turnover is inhibited in S2 cells depleted of either of these two proteins. Cofilin-inhibited S2 cells exhibited a high incidence of multinucleate cells, implicating a role in cytokinesis. This morphology and actin distribution was mimicked by RNAi inhibition of Aip1, a protein that acts cooperatively with cofilin in disassembling actin in Xenopus and budding yeast. Aip1 also produced a cytokinesis defect. These results indicate that both cofilin and Aip1 are essential for actin remodeling during lamella formation and that, despite the similarities in cell morphology produced by RNAi against either of them, these two proteins have distinct roles in actin regulation (Rogers, 2003).

Balancing different types of actin polymerization at distinct sites: roles for Abelson kinase and Enabled

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).

Genetic analysis suggests that in the early Drosophila embryo, the primary means by which Abl influences the cytoskeleton is through Ena. Reducing the Ena dose by half profoundly suppresses abl^M; it is as effective as adding back a wild-type abl transgene. Ena is also a critical target of Abl during embryonic morphogenesis. Although the data suggest that the primary effect of loss of Abl is Ena deregulation, they do not rule out Abl acting on the cytoskeleton by other mechanisms (Grevengoed, 2003).

The mechanism by which Abl regulates Ena has remained mysterious. This study demonstrates that Abl regulates Ena by regulating its intracellular localization. In the absence of Abl, Ena localizes to ectopic sites. Alterations in Ena and actin localization have been observed at the leading edge of migrating epidermal cells in abl mutants during dorsal closure. This suggests that regulation of Ena localization by Abl may be a more general mechanism. It is hypothesized that Abl targets Ena to places where it is needed to modulate actin dynamics, perhaps by excluding it from other sites where Ena activity would be detrimental (Grevengoed, 2003).

There are many ways in which Abl could restrict Ena localization. Abl's kinase activity is essential, and thus Abl phosphorylation of Ena may restrict its localization by preventing Ena binding to partners that localize to particular cortical sites, or by promoting Ena binding to partners that sequester it in the cytoplasm. Phosphorylation of Ena by Abl in vitro inhibits binding of Ena to SH3 domains, whereas Mena/VASP phosphorylation by PKA alters binding to SH3 domains and actin. However, if direct phosphorylation were the only mechanism by which Abl regulated Ena, mutating Ena's phosphorylation sites should create a protein that can no longer be regulated and thus would localize to ectopic sites. Instead, mutation of all of the Abl phosphorylation sites in Ena modestly reduced Ena function, rather than making it ectopically active as is seen in abl mutants (Grevengoed, 2003).

Thus, Abl may regulate Ena by additional mechanisms. Abl may modulate Ena localization and restrict Ena activity by direct binding (this could still require Abl kinase activity, since auto-phosphorylation or phosphorylation of other partners may regulate protein-protein interactions). Abl might sequester Ena in the cytoplasm in an inactive state, or it might recruit Ena to appropriate sites. Alternately, binding of Abl's SH3 domain to the Ena proline-rich region might prevent Ena from binding to other partners, such as profilin, which might in turn modulate both Ena localization and activity. In thinking about these different possible mechanisms, it is interesting to note that Abl localizes to the actin caps and apical pseudocleavage furrows during syncytial stages and the apical portion of the cellularization furrow, the precise places where ectopic actin accumulation occurs in its absence. Thus, it is poised to act at this location. Working out the details of the mechanism by which Abl regulates Ena localization will be one of the next challenges (Grevengoed, 2003).

This work provides an in vivo test of the current model for Ena function, and allows extension of this model. The excess growth of microvilli seen when Ena is ectopically localized in early embryos fits well with work on Ena/VASP function in mammalian fibroblasts, where forced localization of Ena/VASP proteins to the leading edge promotes the formation of long, unbranched filaments. Ena also localizes to the ends of filopodia and microspikes, suggesting that Ena's role in promoting long unbranched actin structures is broadly conserved. Earlier experiments in fibroblasts artificially altered Ena localization. This study demonstrates that Ena localization is a normal regulatory point in vivo, and that Abl is a critical player in this process. Finally, in vitro experiments have suggested that Ena promotes filament elongation by antagonizing capping protein. Mutations in cpb enhance the effects of mutations in abl in the CNS and probably during oogenesis. These data are consistent with Ena and capping protein playing antagonistic roles in vivo, with Abl potentially influencing the outcome of this antagonism. However, Abl and capping protein may also work together independently of Ena in the regulation of actin dynamics (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 abl^M 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 abl^M 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 abl^M mutants, Dia and Arp2/3 mislocalization may not. In fact, reduction of the dose of Dia enhanced the abl^M phenotype. Dia normally promotes actin polymerization lining the furrows. In abl^M 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 abl^M phenotype. The abnormal recruitment of Dia to the apical regions in abl^M 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 abl^M 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-Abl–transformed 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).

Antagonism between Ena/VASP proteins and actin filament capping regulates fibroblast motility

In fibroblasts, Ena/VASP proteins are most highly concentrated in focal adhesions, the leading edge of lamellipodia, and at the tips of filopodia. Lamellipodial protrusion rate has been directly correlated with the accumulation of Ena/VASP proteins at leading edges, suggesting that Ena/VASP proteins promote leading edge protrusion. In addition, Ena/VASP proteins are required for full motility of the actin-dependent intracellular pathogen Listeria monocytogenes. Paradoxically, Ena/VASP negatively regulates cell translocation. To resolve this paradox, the function of Ena/VASP during lamellipodial protrusion was analyzed. Ena/VASP-deficient lamellipodia protrude slower but more persistently, consistent with their increased cell translocation rates. Actin networks in Ena/VASP-deficient lamellipodia contain shorter, more highly branched filaments compared to controls. Lamellipodia with excess Ena/VASP contain longer, less branched filaments. In vitro, Ena/VASP promotes actin filament elongation by interacting with barbed ends, shielding them from capping protein. It is concluded that Ena/VASP regulates cell motility by controlling the geometry of actin filament networks within lamellipodia (Bear, 2002).

It is proposed that Ena/VASP proteins interact with actin filaments at or near their barbed ends and prevent or delay binding of capping protein to filaments while permitting continued elongation. Ena/VASP proteins associate with free barbed ends of actin filaments in vitro and, based on cytochalasin D displacement, free barbed ends are required for leading edge localization of ena/vasp proteins in vivo. the Ena/VASP-dependent changes in actin filament length in lamellipodia are consistent with VASP's antagonism of capping protein seen by in vitro polymerization assays. the results suggest that, in cells, cytochalasin D treatment largely counteracts the effects of Ena/VASP proteins on cell motility, lamellipodial dynamics, and the architecture of the actin network. Given the known effects of cytochalasin D on barbed end elongation and the reported effects on filament length in vivo, these observations are consistent with the proposed model for Ena/VASP proteins as 'anticapping' proteins. while ena/vasp proteins may represent the first known proteins with this activity, there may be other proteins with similar functions just as there are multiple types of filament-capping proteins. The inhibition of capping need not be particularly long lasting to produce the observed effects in cells. A transient inhibition of capping would still produce the long filaments observed in the lamellipodia of the FPPPP-CAAX cells (cells in which Ena/VASP is artificially recruited to the membrane). Since the dissociation rate of capping protein from filaments is slow (t1/2 = 28 min), capping is essentially a terminal event. Ena/VASP proteins may simply slow the association of capping protein with barbed ends (Bear, 2002).

Depolymerization of actin filaments by profilin: effects of profilin on capping protein function

Profilin interacts with the barbed ends of actin filaments and is thought to facilitate in vivo actin polymerization. This conclusion is based primarily on in vitro kinetic experiments using relatively low concentrations of profilin (1-5 microM). However, the cell contains actin regulatory proteins with multiple profilin binding sites that potentially can attract millimolar concentrations of profilin to areas requiring rapid actin filament turnover. The effects were examined of higher concentrations of profilin (10-100 microM) on actin monomer kinetics at the barbed end. Prior work indicated that profilin might augment actin filament depolymerization in this range of profilin concentration. At barbed-end saturating concentrations (final concentration, 40 microM), profilin accelerated the off-rate of actin monomers by a factor of four to six. Comparable concentrations of latrunculin has no detectable effect on the depolymerization rate, indicating that profilin-mediated acceleration is independent of monomer sequestration. Furthermore, it was found that high concentrations of profilin can successfully compete with CapG for the barbed end and uncap actin filaments, and a simple equilibrium model of competitive binding could explain these effects. In contrast, neither gelsolin nor CapZ could be dissociated from actin filaments under the same conditions. These differences in the ability of profilin to dissociate capping proteins may explain earlier in vivo data showing selective depolymerization of actin filaments after microinjection of profilin. The finding that profilin can uncap actin filaments has not been appreciated, and this newly discovered function may have important implications for filament elongation as well as depolymerization (Bubb, 2003).

Search PubMed for articles about Drosophila Capping protein

Barzik, M., Kotova, T. I., Higgs, H. N., Hazelwood, L., Hanein, D., Gertler, F. B. and Schafer, D. A. (2005). Ena/VASP proteins enhance actin polymerization in the presence of barbed end capping proteins. J Biol Chem 280: 28653-28662. PubMed ID: 15939738

Bear, J. E., et al. (2002). Antagonism between Ena/VASP proteins and actin filament capping regulates fibroblast motility. Cell 109: 509-521. PubMed ID: 12086607

Bubb, M. R., Yarmola, E. G., Gibson, B. G. and Southwick, F. S. (2003). Depolymerization of actin filaments by profilin. Effects of profilin on capping protein function. J Biol Chem 278: 24629-24635. PubMed ID: 12730212

Cavey, M. and Lecuit, T. (2009). Molecular bases of cell-cell junctions stability and dynamics. Cold Spring Harb Perspect Biol 1: a002998. PubMed ID: 20066121

Cooper, J. A. and Sept, D. (2008). New insights into mechanism and regulation of actin capping protein. Int Rev Cell Mol Biol 267: 183-206. PubMed ID: 18544499

Delalle, I., Pfleger, C. M., Buff, E., Lueras, P. and Hariharan, I. K. (2005). Mutations in the Drosophila orthologs of the F-actin capping protein alpha- and beta-subunits cause actin accumulation and subsequent retinal degeneration. Genetics 171: 1757-1765. PubMed ID: 16143599

Densham, R. M., O'Neill, E., Munro, J., Konig, I., Anderson, K., Kolch, W. and Olson, M. F. (2009). MST kinases monitor actin cytoskeletal integrity and signal via c-Jun N-terminal kinase stress-activated kinase to regulate p21Waf1/Cip1 stability. Mol Cell Biol 29: 6380-6390. PubMed ID: 19822666

Fernandez, B. G., Gaspar, P., Bras-Pereira, C., Jezowska, B., Rebelo, S. R. and Janody, F. (2011). Actin-Capping Protein and the Hippo pathway regulate F-actin and tissue growth in Drosophila. Development 138: 2337-2346. PubMed ID: 21525075

Frank, D. J., Hopmann, R., Lenartowska, M. and Miller, K. G. (2006). Capping protein and the Arp2/3 complex regulate nonbundle actin filament assembly to indirectly control actin bundle positioning during Drosophila melanogaster bristle development. Mol Biol Cell 17: 3930-3939. PubMed ID: 16822838

Gaspar, P., Holder, M. V., Aerne, B. L., Janody, F. and Tapon, N. (2015). Zyxin antagonizes the FERM protein Expanded to couple F-actin and Yorkie-dependent organ growth. Curr Biol 25: 679-689. PubMed ID: 25728696

Gates, J., Nowotarski, S. H., Yin, H., Mahaffey, J. P., Bridges, T., Herrera, C., Homem, C. C., Janody, F., Montell, D. J. and Peifer, M. (2009). Enabled and Capping protein play important roles in shaping cell behavior during Drosophila oogenesis. Dev Biol 333: 90-107. PubMed ID: 19576200

Grevengoed, E. E., Fox, D. T., Gates, J. and Peifer, M. (2003). Balancing different types of actin polymerization at distinct sites: roles for Abelson kinase and Enabled. J. Cell Biol. 163: 1267-1279. 14676307

Hopmann R. Cooper J. A., Miller K. G. (1996) Actin organization, bristle morphology, and viability are affected by actin capping protein mutations in Drosophila. J. Cell Biol 133: 1293-1305. PubMed ID: 8682865

Hopmann, R. and Miller, K. G. (2003). A balance of capping protein and profilin functions is required to regulate actin polymerization in Drosophila bristle. Mol. Biol. Cell 14: 118-128. 12529431

Janody, F. and Treisman, J. E. (2006). Actin capping protein alpha maintains vestigial-expressing cells within the Drosophila wing disc epithelium. Development 133: 3349-3357. PubMed ID: 16887822

Jezowska, B., Fernandez, B. G., Amandio, A. R., Duarte, P., Mendes, C., Bras-Pereira, C. and Janody, F. (2011). A dual function of Drosophila capping protein on DE-cadherin maintains epithelial integrity and prevents JNK-mediated apoptosis. Dev Biol 360: 143-159. PubMed ID: 21963538

Johnson, R. I., Seppa, M. J. and Cagan, R. L. (2008). The Drosophila CD2AP/CIN85 orthologue Cindr regulates junctions and cytoskeleton dynamics during tissue patterning. J. Cell Biol. 180(6): 1191-204. PubMed ID: 18362180

Oh, H., Reddy B. V. and Irvine K. D. (2009). Phosphorylation-independent repression of Yorkie in Fat-Hippo signaling. Dev. Biol. 335: 188-197. PubMed ID: 19733165

Pollard T. D. and Borisy G. G. (2003) Cellular motility driven by assembly and disassembly of actin filaments. Cell 112: 453-465. PubMed ID: 12600310

Rauskolb, C., Pan, G., Reddy, B. V., Oh, H. and Irvine, K. D. (2011). Zyxin links fat signaling to the hippo pathway. PLoS Biol 9: e1000624. PubMed ID: 21666802

Reddy B. V. and Irvine K. D. (2008). The Fat and Warts signaling pathways: new insights into their regulation, mechanism and conservation. Development 135: 2827-2838. PubMed ID: 18697904

Rogers, S. L., Wiedemann, U., Stuurman, N. and Vale, R. D. (2003). Molecular requirements for actin-based lamella formation in Drosophila S2 cells. J. Cell Biol. 162(6): 1079-88. PubMed ID: 12975351

Sansores-Garcia, L., Bossuyt, W., Wada, K., Yonemura, S., Tao, C., Sasaki, H. and Halder, G. (2011). Modulating F-actin organization induces organ growth by affecting the Hippo pathway. EMBO J 30: 2325-2335. PubMed ID: 21556047

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date revised: 25 April 2021

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