InteractiveFly: GeneBrief

washout: Biological Overview | References

Gene name - washout

Synonyms -

Cytological map position - 48E6-48E6

Function - signaling

Keywords - cytoskeleton, actin nucleation, oogenesis

Symbol - wash

FlyBase ID: FBgn0033692

Genetic map position - 2R: 8,068,145..8,069,878 [+

Classification - WH2 domain

Cellular location - cytoplasmic

NCBI links: EntrezGene
wash orthologs: Biolitmine
Recent literature
Nagel, B. M., Bechtold, M., Rodriguez, L. G. and Bogdan, S. (2016). Drosophila WASH is required for integrin-mediated cell adhesion, cell motility and lysosomal neutralization. J Cell Sci [Epub ahead of print]. PubMed ID: 27884932
The Wiskott-Aldrich Syndrome Protein and SCAR Homologue (WASH) is a conserved actin nucleation promoting factor controlling Arp2/3 complex activity in endosomal sorting and recycling. Previous studies have identified WASH as an essential regulator in Drosophila development. This study shows that homozygous wash mutant flies are viable and fertile. Drosophila WASH has conserved functions in integrin receptor recycling and lysosome neutralization. WASH generates actin patches on endosomes and lysosomes mediating both functions. Consistently, loss of WASH function results in cell spreading and cell migration defects of macrophages, and an increased lysosomal acidification that affects efficient phagocytic and autophagic clearance. WASH physically interacts with the vacuolar ATPase subunit Vha55 that is crucial to establish and maintain lysosome acidification. As a consequence, starved flies lacking WASH function show a dramatic increase in acidic autolysosomes causing a reduced lifespan. Thus, these data highlight a conserved role for WASH in the endocytic sorting and recycling of membrane proteins like integrins and the V-ATPase that increase the likelihood of survival under nutrient deprivation.
Verboon, J. M., Decker, J. R., Nakamura, M. and Parkhurst, S. M. (2018). Wash exhibits context dependent phenotypes and, along with the WASH Regulatory Complex, regulates Drosophila oogenesis. J Cell Sci. PubMed ID: 29549166
WASH, a Wiskott Aldrich Syndrome (WAS) family protein, has many cell and developmental roles related to its function as a branched actin nucleation factor. Similar to mammalian WASH, which is embryonic lethal, Drosophila Wash was found to be essential for oogenesis and larval development. Recently, however, Drosophila wash was reported to be homozygous viable. This study verified that the original wash null allele harbors an unrelated lethal background mutation, however, this unrelated lethal mutation does not contribute to any Wash oogenesis phenotypes. Significantly, it was found that the homozygous wash null allele retains partial lethality leading to non-Mendelian inheritance, that this allele's functions are subject to its specific genetic background, and that the homozygous stock rapidly accumulates modifications that allow it to become robust. Accordingly, Wash was found to play an important role in oogenesis and also that this role involves the WASH Regulatory Complex. Finally, this study showed that another WAS protein, SCAR/WAVE, plays a similar role in oogenesis and that it is upregulated as one of the modifications that allows the wash allele to survive in the homozygous state.

Wiskott-Aldrich Syndrome (WAS) family proteins are Arp2/3 activators that mediate the branched-actin network formation required for cytoskeletal remodeling, intracellular transport and cell locomotion. Wasp and Scar/WAVE, the two founding members of the family, are regulated by the GTPases Cdc42 and Rac, respectively. By contrast, linear actin nucleators, such as Spire and formins, are regulated by the GTPase Rho. A third WAS family member, called Washout (Wash), has Arp2/3-mediated actin nucleation activity. This study shows that Drosophila Wash interacts genetically with Arp2/3, and also functions downstream of Rho1 with Spire and the formin Cappuccino to control actin and microtubule dynamics during Drosophila oogenesis. Wash bundles and crosslinks F-actin and microtubules, is regulated by Rho1, Spire and Arp2/3, and is essential for actin cytoskeleton organization in the egg chamber. These results establish Wash and Rho as regulators of both linear- and branched-actin networks, and suggest an Arp2/3-mediated mechanism for how cells might coordinately regulate these structures (Liu, 2009).

The actin cytoskeleton consists of linear and branched filament networks required for processes ranging from cell division to migration (Chhabra, 2007; Faix, 2006; Goley, 2006). How these two networks function and are coordinated is of major interest, as their misregulation results in infertility, immunodeficiency, and tumor metastasis in humans. Linear actin filament networks, required for cytokinesis and filopodia formation, are regulated by nucleators and bundling proteins, which enhance filament formation rates and control filament organization, respectively. Examples include Spire and the formin Cappuccino (Capu), which exhibit both nucleation and bundling activities and are essential for oocyte development during Drosophila oogenesis. Both Spire and Capu are regulated by the GTPase Rho1 of the Rho family of small GTPases, which is upstream of other linear nucleators, such as Diaphanous, and is considered a key regulator of linear filament formation (Liu, 2009).

Branched or dendritic actin filament networks, which are required for phagocytosis and lamellipodia formation, are primarily regulated by the Arp2/3 complex and by nucleation-promoting factors that associate with Arp2/3 and actin monomers to nucleate daughter filaments off of existing mother filaments (Goley, 2006; Takenawa, 2007). Like Spire and Capu, Arp2/3 is essential for Drosophila oogenesis, specifically for maintaining proper nurse cell cyto-architecture and function (Hudson, 2002). One family of Arp2/3 activators, the Wiskott-Aldrich Syndrome (WAS) protein family, has been shown to function downstream of Rho GTPases to mediate the branched-actin network formation required for cytoskeletal remodeling, intracellular transport and cell locomotion. WASP and SCAR/WAVE, the two founding subclasses of the family, are activated by the GTPases Cdc42 and Rac, respectively (Stradal, 2004; Takenawa, 2007). Two new WAS subclasses, WASH and WHAMM, have recently been reported (Campellone, 2008; Linardopoulou, 2007) and have been shown to exhibit Arp2/3-mediated branched nucleation activity. Which GTPases might regulate them, however, is not known (Liu, 2009).

This study reports that Drosophila Wash functions downstream of Rho1 and interacts with Spire and Capu to regulate actin and microtubule organization during Drosophila oogenesis. Wash nucleates actin in an Arp2/3-dependent manner, and exhibits F-actin and microtubule bundling and crosslinking activity that is regulated by a pathway involving Rho1, Spire and Arp2/3. Wash genetically interacts with Rho1, Capu, Spire and Arp2/3, and is essential for actin cytoskeleton organization during oogenesis. These results establish Wash and Rho as regulators of both linear- and branched-actin networks, and suggest an Arp2/3-mediated mechanism of cytoskeletal control through which cells might coordinately regulate linear and branched architectures (Liu, 2009).

It has been suggested that Rho1 regulates the timing of ooplasmic streaming by regulating the MT/microfilament crosslinking that occurs at the oocyte cortex (Rosales-Nieves, 2006). In this model, crosslinking antagonizes the formation of the dynamic subcortical MT arrays that are required for ooplasm streaming, but does not require the actin-nucleation activity of these proteins. The current model depends on the presence of SpirC and the cortical localization of Rho1, Capu, the Spire isoforms, and now Wash during late-stage oocytes. Support for this model comes from a recent study demonstrating that chickadee, encoding fly Profilin, is required for the formation of cortical actin bundles in the oocyte, and that Capu and Spire anchor the minus ends of MTs to a scaffold made from these cortical actin bundles (Wang, 2008). These results suggest dual or multifaceted biochemical roles for these proteins in regulating developmental processes. Consistent with this concept, non-actin-nucleating roles for other formins (i.e. actin severing/depolymerization, MT stabilization, signaling, and transcriptional regulation) are beginning to be reported (Liu, 2009).

St Johnston and colleagues have recently proposed an alternative model in which Capu and Spire are required to organize an isotropic mesh of actin filaments in the oocyte cytoplasm that suppresses the motility of kinesin, a plus-end directed MT motor protein that is required for ooplasmic streaming (Dahlgaard, 2007). Their model was formulated with the assumptions that the SpirC isoform does not exist, that spirRP is a null allele, and that the cortical localization of Capu and Spire is lost in late-stage oocytes. This study found these assumptions not to be the case. mRNA and protein evidence is provided for the existence of the SpirC isoform. The existence of SpirC is also supported by ESTs from the Drosophila Genome Project. The spirRP allele affects only the SpirA and SpirD isoforms; it does not affect the SpirC isoform because this isoform has a unique 5' end. Ectopic SpirC expression would not be expected to rescue spirRP because it is already being expressed. The cortical localization of Capu and the Spire proteins during the late stages is masked by intense yolk auto-fluorescence in the green channel when using live imaging of GFP fusions, but can be observed by fixing, by antibody staining, or by the use of ChFP ('cherry' fusion protein). In addition, a subsequent study has shown that kinesin is not required for this cytoskeletal reorganization, suggesting that Capu and Spire might not act as indirect kinesin regulators, but as direct modulators of the MT cytoskeleton (Wang, 2008). One possibility is that Capu and Spire are bundling and crosslinking MTs to Profilin-dependent F-actin at the oocyte cortex, as has been demonstrated in vitro (Liu, 2009).

Since the discovery of Arp2/3 activators and other actin-nucleation promoting factors, much of the work examining the functions of these proteins has been focused on the properties of their nucleation activities. Recent studies, however, have begun reporting novel biochemical activities for actin nucleators, including MT stabilization activity by mammalian Diaphanous, filopodia inhibition by WAVE/Arp2/3, and F-actin and MT bundling and crosslinking by Spire and Capu (Rosales-Nieves, 2006). Consistent with this, not all disease-associated WASP mutations are predicted to affect its actin-nucleation activity (Notarangelo, 2008). The current results contribute to this growing list of actin nucleators with significant non-nucleation activities, since this study shows that Wash is both an Arp2/3 activator and a crosslinker/bundler of F-actin and microtubules. What is unique about Wash, however, is that its combination of biochemical activities suggests that it is an important intermediary molecule functioning at the intersection of linear and branched actin architectures, with Spire, Rho and Arp2/3 acting as the factors that direct these dual functions of Wash. Based on these findings, the following model is proposed for Wash function in the context of Drosophila oogenesis. In the nucleation pathway, upstream signals and factors, possibly Rho, induce Wash activation, which acts with Arp2/3 to promote branched filament formation and cytoskeletal integrity in nurse cells. In the crosslinking/bundling pathway, Wash bundles and crosslinks filaments of actin and MTs, under the control of Rho and SpirD, to maintain cortical bundle stability in the oocyte and to prevent premature ooplasmic streaming. Together with Capu and Spire (Rosales-Nieves, 2006), Wash maintains the correct timing of ooplasmic streaming by preventing the formation of the microtubule tracks required for motor proteins to drive cytoplasmic flow. The dual functions of Wash might also be regulated spatially by Arp2/3 and depend on the availability or concentration of Arp2/3. Since the nucleation activity of Wash is Arp2/3 dependent, Wash-mediated actin nucleation might require some threshold concentration of locally available Arp2/3; for example, at the ring canals. Spatiotemporal regulation is also possible through the changing levels of Arp2/3 during oogenesis. Arp2/3, for example, might transiently accumulate at the oocyte cortex during the onset of streaming to disrupt Wash bundling activity (Liu, 2009).

These findings contribute to previous studies examining the functions of Wasp and Scar in Drosophila, and together describe a spectrum of phenotypes that illustrate the multiple functions exerted by WAS family members in development. Scar has been shown to be required for axon development, egg chamber structure, adult eye morphology and myoblast fusion; Wasp has been demonstrated to be required for Notch-mediated cell-fate decisions, rhabdomere microvilli formation, bristle development and myoblast fusion; and Wash is required for pupal development (Linardopoulou, 2007) and oogenesis, as described in this study. Mutants in various subunits of Arp2/3 have also been described, offering additional insight into how Wash, Wasp and Scar shape the cytoskeleton during development (Hudson, 2002; Stevenson, 2002). Interestingly, the spectrum of Arp2/3 mutant phenotypes reported does not completely overlap with all of the phenotypes associated with these WAS family mutants. This might be because Arp2/3 has not been examined in all of the processes in which WAS members play a role, or it might be an indication that WAS members have additional, Arp2/3-independent functions, which is the case for Wash. The current observations support the idea that these and other actin nucleators, such as Capu and Spire, are required at different times or locations during development, and are thus tightly regulated spatiotemporally by Rho GTPases and other factors (Liu, 2009).

The data indicate that Wash acts as a downstream effector of Rho. Indeed, Rho is shown to regulates the bundling/crosslinking activity of Wash through the relief of SpirD inhibition. However, Rho does not enhance the ability of Wash to induce Arp2/3-mediated actin nucleation, raising the question of how or whether Rho might regulate the Arp2/3-associated functions of Wash. Interestingly, the results are consistent with studies examining the Cdc42 regulation of Wasp in Drosophila, which conclude that Cdc42 activation of Wasp is not required for Wasp function in myoblast fusion or bristle development. Although Wasp exhibits a strong and specific interaction with active Cdc42GTP in vitro, these studies provide strong evidence that, at least for the subset of developmental processes examined, Wasp is not regulated upstream by Cdc42GTP. As previously noted, Drosophila Wasp differs from mammalian homologs in that it is not auto-inhibited; Cdc42, therefore, might not be required for the activation of its actin nucleation-promoting functions. This might also be the case for Wash, as it too appears to be constitutively active, and might act as a downstream effector of Rho only where its bundling/crosslinking activities are concerned. The data, however, do not rule out the possibility that the nucleation activity of Wash is regulated by a complex in vivo. In fact, recent reports have shown that two proteins originally associated with Scar regulation, Abi and Kette, control Wasp function in Drosophila as well. It remains to be determined whether Abi and Kette also regulate Wash function, and whether Rho might play a role in mediating these interactions (Liu, 2009).

Wash requires Arp2/3 for actin nucleation, but, interestingly, this association appears to disrupt the ability of Wash to bundle and crosslink F-actin and microtubules, as a loss of F-actin/MT bundling favored branching actin filaments. This suggests that Arp2/3 might act as a molecular switch that shifts Wash function from bundling to nucleation and, in terms of cytoskeletal remodeling, supports the hypothesis that Arp2/3 regulates the balance between linear and branched actin architectures in the cell. This is predicated on the assumption that the Wash bundling/crosslinking and nucleation-inducing activities are mutually exclusive, and would represent a previously uncharacterized function of Arp2/3. However, scenarios cannot be ruled out in which nucleation and bundling might coexist. F-actin bundling might be preserved if the branched-actin structures created by Wash and Arp2/3 in vitro are bundled by Wash in parallel (form angled, branching bundles rather than the tortuous bundles observed under non-Arp2/3 conditions), or if filaments emanating from vertices are clamped together by Wash at the branching point to form angled bundles that branch from these vertices. An example of this latter case has been reported in a recent study examining the concerted actions of N-Wasp and Hsp90 to nucleate branched actin filaments (via N-Wasp activation of Arp2/3) and clamp the angled filaments to form a linear bundle (mediated by Hsp90). Wash therefore, in having both nucleation and bundling activities, might perform both functions simultaneously in the presence of Arp2/3. At the very least, Arp2/3 abolishes the ability of Wash to bundle MTs and crosslink them to actin, and so might contribute to regulating crosstalk between the actin and microtubule cytoskeletons. Further studies examining the molecular interactions of WAS family members and Arp2/3 will be invaluable for understanding the full range of cytoskeletal regulation in the cell (Liu, 2009).

In motile cells the actin cytoskeleton can be represented as a dynamic sum of two general geometries - strands or bundles of linear actin filaments, and broad dendritic networks of branched filaments. The mechanisms by which these two networks are remodeled and coordinated are areas of intense investigation and are important for understanding how processes such as lamellipodia and filopodia formation occur. It is intriguing to note that, in the latter case, the biochemical properties of Wash suggest that it might play a role in the convergent extension model of filopodia formation, whereby uncapped actin filaments nucleated from a dendritic branched-actin array are captured at the cell periphery and bundled to form long extensions (Mattila, 2008). Wash, as both an Arp2/3 activator and an F-actin bundling protein, is in an ideal position in which to carry out both the nucleation and the bundling functions, and might thus be an important regulator of filopodia formation alongside previously discovered molecules (Mattila, 2008). The presence of Spire and Arp2/3 at the dendritic bed and active Rho at the cell membrane could form two zones of differential activity to switch Wash function from nucleation to bundling and crosslinking. This form of spatial regulation is analogous to how Rho, Cdc42 and Rac define regions of differential activity during wound healing and cell adhesion. Further investigation into the role of Wash in filopodia and lamellipodia formation will be important, as these protrusions play essential roles in wound healing, substrate adhesion and neurite outgrowth (Liu, 2009).

In humans, the misregulation of WAS members results in disorders such as Wiskott-Aldrich Syndrome, and cancer metastasis. As a new member of the WAS family, human WASH appears to also be clinically relevant. WASH has been reported to be overexpressed in a breast cancer cell line and might, like the overexpression of N-WASP and Scar/WAVEs, contribute to metastasis (Leirdal, 2004). Moreover, the subtelomeric location of human WASH places it at high risk for deletion and rearrangement, as subtelomeres are hotspots of meiotic interchromosomal sequence transfers (Linardopoulou, 2005). The data presented in this study demonstrate that Wash is essential for development in Drosophila, and suggest that Wash might function in actin organization in other contexts. Further work will be required to understand how Wash and other WAS family members coordinate linear- and branched-actin networks during oogenesis and other cellular processes, and how the misregulation of these processes results in disease (Liu, 2009).

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).

Human subtelomeric WASH genes encode a new subclass of the WASP family

Subtelomeres are duplication-rich, structurally variable regions of the human genome situated just proximal of telomeres. This study reports that the most terminally located human subtelomeric genes encode a previously unrecognized third subclass of the Wiskott-Aldrich Syndrome Protein family, whose known members reorganize the actin cytoskeleton in response to extracellular stimuli. This new subclass, WASH, is evolutionarily conserved in species as diverged as Entamoeba. WASH is essential in Drosophila. WASH is widely expressed in human tissues, and human WASH protein colocalizes with actin in filopodia and lamellipodia. The VCA domain of human WASH promotes actin polymerization by the Arp2/3 complex in vitro. WASH duplicated to multiple chromosomal ends during primate evolution, with highest copy number reached in humans, whose WASH repertoires vary. Thus, human subtelomeres are not genetic junkyards, and WASH's location in these dynamic regions could have advantageous as well as pathologic consequences (Linardopoulou, 2007).

The single WASH ortholog in Drosophila, which has been named washout (wash; CG13176), is structurally and phylogenetically distinct from the two WASP family members already characterized in this species. Homozygous mutations in either Wasp or Scar result in zygotic lethality, although some of the Wasp mutants ('escapers') survive until adulthood and appear morphologically normal, but are lethargic and passive in their behavior. wash gene products, like those of Wasp and Scar, are provided maternally, and wash transcripts appear to be distributed uniformly throughout the early embryo, based on RNA in situ hybridizations performed by the Berkeley Drosophila Genome Project (Linardopoulou, 2007).

To investigate washout function, a P-element insertion line, P{EPgy2}CG13176EY15549 was obtained, with a P-element insertion at the beginning of the washout coding region. These flies are homozygous viable with no apparent phenotype. Numerous imprecise excision alleles of this insertion line were generated that are lethal when homozygous. In one of the alleles, washδ185, more than half of the coding region (up to the VCA module) is deleted and two stop codons at positions 11 and 12 are introduced; no flies homozygous for this allele survive to adulthood. Flies bearing precise excisions of the P-element insertion in the homozygous state were viable, indicating that the recessive lethality of washδ185 is due to disruption of washout (Linardopoulou, 2007).

To determine the stage at which lethality occurs in wash?185 homozygous flies, flies carrying the wash?185 allele were crossed to a balancer chromosome (a multiply inverted chromosome that suppresses recombination) carrying GFP, which allows for easy detection and separation of washδ185 homozygous embryos. Analyses of 200 washδ185 homozygous embryos revealed that the majority of them (98%) hatched, although none of them produced an adult fly. Further analysis showed that these washδ185 animals die at the transition from 3rd larval instar to prepupal stage. While flies heterozygous for the washδ185 allele have wild-type pupal morphology, washδ185 homozygotes fail to contract their body and evert their spiracles prior to secreting their pupal cuticle, resulting in an elongated appearance (Linardopoulou, 2007).


Search PubMed for articles about Drosophila Washout

Campellone, K. G., Webb, N. J., Znameroski, E. A. and Welch, M. D. (2008). WHAMM is an Arp2/3 complex activator that binds microtubules and functions in ER to Golgi transport. Cell 134: 148-161. PubMed ID: 18614018

Chhabra, E. S. and Higgs, H. N. (2007). The many faces of actin: matching assembly factors with cellular structures. Nat. Cell. Biol. 9: 1110-1121. PubMed ID: 17909522

Dahlgaard, K., Raposo, A. A., Niccoli, T. and St Johnston, D. (2007). Capu and Spire assemble a cytoplasmic actin mesh that maintains microtubule organization in the Drosophila oocyte. Dev. Cell 13(4): 539-53. PubMed ID: 17925229

Faix, J. and Grosse, R. (2006). Staying in shape with formins. Dev. Cell 10: 693-706. PubMed ID: 16740473

Goley, E. D. and Welch, M. D. (2006). The ARP2/3 complex: an actin nucleator comes of age. Nat. Rev. Mol. Cell. Biol. 7: 713-726. PubMed ID: 16990851

Hudson, A. M. and Cooley, L. (2002). A subset of dynamic actin rearrangements in Drosophila requires the Arp2/3 complex. J. Cell Biol. 156: 677-687. PubMed ID: 11854308

Leirdal, M., Shadidy, M., Rosok, O. and Sioud, M. (2004). Identification of genes differentially expressed in breast cancer cell line SKBR3: potential identification of new prognostic biomarkers. Int. J. Mol. Med. 14: 217-222. PubMed ID: 15254768

Linardopoulou, E. V., Williams, E. M., Fan, Y., Friedman, C., Young, J. M. and Trask, B. J. (2005). Human subtelomeres are hot spots of interchromosomal recombination and segmental duplication. Nature 437: 94-100. PubMed ID: 16136133

Linardopoulou, E. V., Parghi, S. S., Friedman, C., Osborn, G. E., Parkhurst, S. M. and Trask, B. J. (2007). Human subtelomeric WASH genes encode a new subclass of the WASP family. PLoS Genet. 3: e237. PubMed ID: 18159949

Liu, R., Abreu-Blanco, M. T., Barry, K. C., Linardopoulou, E. V., Osborn, G. E. and Parkhurst, S. M. (2008). Wash functions downstream of Rho and links linear and branched actin nucleation factors. Development 136(16): 2849-60. PubMed ID: 19633175

Mattila, P. K. and Lappalainen, P. (2008). Filopodia: molecular architecture and cellular functions. Nat. Rev. Mol. Cell. Biol. 9: 446-454. PubMed ID: 18464790

Notarangelo, L. D., Miao, C. H. and Ochs, H. D. (2008). Wiskott-Aldrich syndrome. Curr. Opin. Hematol. 15: 30-36. PubMed ID: 18043243

Rosales-Nieves, A. E., et al. (2006). Coordination of microtubule and microfilament dynamics by Drosophila Rho1, Spire and Cappuccino. Nat. Cell Biol. 8(4): 367-76. PubMed ID: 16518391

Speese, S. D., Ashley, J., Jokhi, V., Nunnari, J., Barria, R., Li, Y., Ataman, B., Koon, A., Chang, Y. T., Li, Q., Moore, M. J. and Budnik, V. (2012). Nuclear envelope budding enables large ribonucleoprotein particle export during synaptic Wnt signaling. Cell 149(4): 832-846. PubMed ID: 22579286

Stevenson, V., Hudson, A., Cooley, L. and Theurkauf, W. E. (2002). Arp2/3-dependent pseudocleavage [correction of psuedocleavage] furrow assembly in syncytial Drosophila embryos. Curr. Biol. 12: 705-711. PubMed ID: 12007413

Stradal, T. E., Rottner, K., Disanza, A., Confalonieri, S., Innocenti, M. and Scita, G. (2004). Regulation of actin dynamics by WASP and WAVE family proteins. Trends Cell. Biol. 14: 303-311. PubMed ID: 15183187

Takenawa, T. and Suetsugu, S. (2007). The WASP-WAVE protein network: connecting the membrane to the cytoskeleton. Nat. Rev. Mol. Cell. Biol. 8: 37-48. PubMed ID: 17183359

Verboon, J. M., Rincon-Arano, H., Werwie, T. R., Delrow, J. J., Scalzo, D., Nandakumar, V., Groudine, M. and Parkhurst, S. M. (2015a). Wash interacts with lamin and affects global nuclear organization. Curr Biol 25(6): 804-810. PubMed ID: 25754639

Verboon, J. M., Rahe, T. K., Rodriguez-Mesa, E. and Parkhurst, S. M. (2015b). Wash functions downstream of Rho1 GTPase in a subset of Drosophila immune cell developmental migrations. Mol Biol Cell 26(9): 1665-1674. PubMed ID: 25739458

Verboon, J. M., Nakamura, M., Davidson, K. A., Decker, J. R., Nandakumar, V. and Parkhurst, S. M. (2020). Drosophila Wash and the Wash regulatory complex function in nuclear envelope budding. J Cell Sci 133(13). PubMed ID: 32503943

Wang, Y. and Riechmann, V. (2008). Microtubule anchoring by cortical actin bundles prevents streaming of the oocyte cytoplasm. Mech. Dev. 125: 142-152. PubMed ID: 18053693

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date revised: 10 February 2010

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