Abelson kinase’s intrinsically disordered linker plays important roles in protein function and protein stability

The non-receptor tyrosine kinase Abelson (Abl) is a key player in oncogenesis, with kinase inhibitors serving as paradigms of targeted therapy. Abl also is a critical regulator of normal development, playing conserved roles in regulating cell behavior, brain development and morphogenesis. Drosophila offers a superb model for studying Abl’s normal function, because, unlike mammals, there is only a single fly Abl family member. Abl has multiple roles in embryonic morphogenesis, and we and others have begun to take Abl apart as a machine. This revealed many surprises. For instance, kinase activity, while important, is not crucial for all Abl activities, and the C-terminal F-actin binding domain plays a very modest role. This turned our attention to less well-known feature—the long intrinsically-disordered region (IDR) linking Abl’s kinase and F-actin binding domains. The past decade revealed unexpected, important roles for IDRs in diverse cell functions, by mediating multivalent interactions, enabling assembly of biomolecular condensates via phase separation. Previous work deleting conserved regions revealed an important role for a PXXP motif in the IDR, but did not identify any other essential regions. Here we extend this, deleting the entire IDR. This revealed essential roles for the IDR in embryonic and adult viability, and in cell shape changes and cytoskeletal regulation during embryonic morphogenesis. Strikingly, AblΔIDR acts as dominant negative, worsening the phenotype of the null mutant. AblΔIDR accumulates at >10-fold higher levels than wildtype Abl in both Drosophila embryos and cultured cells, suggesting important roles for the IDR in modulating protein stability.


Introduction
Biomedical research has dual goals: to uncover mechanisms underlying normal cellular function and to apply this understanding to develop better treatments in human disease. Perhaps no story better illustrates this than the discovery more than 60 years ago of the "Philadelphia chromosome", a translocation between chromosomes 9 and 22 present only in leukocytes from patients with chronic myelogenous leukemia. It provided the first molecular link between genetics and cancer, and ultimately led to the realization that Abelson kinase (Abl) is the initiating oncogene in many cases of chronic myelogenous and acute lymphoblastic leukemia (REN 2005). These translocations fuse the Bcr and Abl genes, removing a myristoylation sequence at Abl's N-terminus that inhibits kinase activation, rendering the kinase constitutively active. Drugs targeting Abl kinase activity like Gleevec (Imatinib) have emerged as paradigms of targeted therapy (SAWYERS et al. 2002;TALPAZ et al. 2002), and spurred development of similar inhibitors of other oncogenic kinases.
Of course, non-receptor tyrosine kinases like Src and Abl kinases did not evolve to cause cancer. Both play key roles in signal transduction, regulating embryonic development and tissue homeostasis. Abl family members regulate morphogenetic movements during embryogenesis in both mammals and Drosophila, and also play key roles in neural development, axon outgrowth, and synaptogenesis (reviewed in (MORESCO AND KOLESKE 2003;BRADLEY AND KOLESKE 2009;KHATRI et al. 2016;KANNAN AND GINIGER 2017). They act downstream of diverse receptors, including receptor tyrosine kinases or receptor serine/threonine kinases, as well as the cellmatrix and cell-cell adhesion receptors, integrins and cadherins. Downstream, Abl family members activate cytoskeletal effectors to directly regulate cell behavior, though transcriptional effectors are also important.
Abl's structure facilitates the link between cell signaling and cytoskeletal regulation. All Abl family members share a highly conserved set of N-terminal domains with Src (Fig. 1A). These include a Src homology 2 (SH2) domain that binds specific peptides carrying a phosphorylated tyrosine and an SH3 domain that binds specific proline-rich peptides, both allowing interactions with upstream receptors and downstream effectors.
These are immediately followed by the conserved tyrosine kinase domain (COLICELLI 2010). However, unlike Src, Abl family members have long C-terminal extensions, with a C-terminal F-actin-binding domain (FABD) separated from the N-terminal module by a long, poorly conserved linker that is predicted to be intrinsically disordered (the intrinsically disordered region or IDR). Different family members share peptide motifs within the IDR that bind or are predicted to bind actin, microtubules, Ena/VASP family members, and SH3 domain containing proteins. The only peptide motif in the IDR clearly conserved between mammals and Drosophila is a PXXP motif that in mammals binds both SH2/SH3 adapters like Crk and NCK and the actin regulator Abl interacting protein (Abi) (HOSSAIN et al. 2012;GREGOR et al. 2019).
Mammals have two Abl family members, Abl and Abl-related gene (Arg), with partially redundant functions in development and tissue homeostasis. Abl single mutant mice die neonatally with thymic and splenic atrophy, T and B cell lymphopenia, osteoporosis, and cardiac defects (SCHWARTZBERG et al. 1991;TYBULEWICZ et al. 1991;HARDIN et al. 1995;HARDIN et al. 1996;LI et al. 2000;QIU et al. 2010b). Conditional knockout confirmed roles in T cells (ZIPFEL et al. 2004;GU et al. 2007;HUANG et al. 2008). Arg single mutant mice are viable with grossly normal brains, but exhibit multiple behavioral defects (KOLESKE et al. 1998), likely linked to a reduced ability to maintain dendrites (LIN et al. 2013). Arg mutants also exhibit subtle defects in muscle development . In contrast, loss of both Abl and Arg leads to embryonic lethality at day 11, with a failure to complete neural tube closure. Conditional double knockout has revealed additional important redundant roles in cerebellar (QIU et al. 2010a), cerebral (MORESCO et al. 2005, and endothelial development and barrier function (CHISLOCK ).
Drosophila has a single Abl family member, simplifying analysis of its roles in development. In the 1980s Michael Hoffmann and his lab identified the first mutations in fly Abl, as part of a pioneering effort to define the normal roles of human oncogenes (HENKEMEYER et al. 1987). Others built on these early efforts. Like its mammalian homologs, Abl plays important roles in embryonic and postembryonic neural development, with Abl acting downstream of diverse axon guidance receptors, including DCC/Frazzled, Robo, Plexin, Eph, and Notch (reviewed in (KANNAN AND GINIGER 2017). Genetic and cell biological analyses also revealed Abl's downstream effectors, the most prominent of which is Enabled (Ena), which binds the growing end of actin filaments and promotes their elongation. Abl negatively regulates Ena , through a mechanism that remains unclear. Trio, a GTP exchange factor (GEF) for the small GTPase Rac, is also an Abl effector.
Subsequent analysis of embryos lacking both maternal and zygotic Abl revealed additional roles outside of the nervous system. Abl regulates diverse events ranging from the actin-dependent cellularization process, to apical constriction of mesoderm precursors, cell intercalation during germband elongation, and collective cell migration during germband retraction, dorsal closure, and head involution (GREVENGOED et al. 2001;GREVENGOED et al. 2003;FOX AND PEIFER 2007;TAMADA et al. 2012). In these events, regulation by and of the cadherin-based cell adhesion machinery plays a role, while Ena remains a critical downstream target (GREVENGOED et al. 2001;GREVENGOED et al. 2003;FOX AND PEIFER 2007).
Abl kinase activity has been a focus of much attention, particularly after the success of Abl kinase inhibitors in the treatment of leukemia. However, the simplistic picture of Abl as a kinase acting solely by phosphorylating downstream proteins rapidly proved inaccurate. Kinase-dead Abl rescues defects in both adult viability and retinal development (HENKEMEYER et al. 1990). Analysis of its role in embryonic development suggests kinase activity is important for roles in both axon patterning and in morphogenesis, but a kinase-dead mutant retains significant residual function (O'DONNELL AND BASHAW 2013;ROGERS et al. 2016). More limited analysis implicated the SH2 domain in axon guidance (O'DONNELL AND BASHAW 2013). The extended C-terminal region of Abl, including both the IDR and the C-terminal FABD, is essential for function, as abl 1 , which encodes a stable protein truncated soon after the kinase domain, behaves genetically as a null allele (HENKEMEYER et al. 1987). Similar results were seen in mice where a truncated protein resembled the null (SCHWARTZBERG et al. 1991;TYBULEWICZ et al. 1991). The simplest explanation would be that this reflected an essential function of the FABD. However, surprisingly Abl lacking the FABD fully rescues viability and fertility, though detailed analysis of axon patterning and synergistic effects with loss of kinase activity suggest the FABD does play a supporting role (O'DONNELL AND BASHAW 2013;ROGERS et al. 2016;CHEONG AND VANBERKUM 2017).
These data opened up potential roles for the IDR. The past decade revealed unexpected and important roles for IDRs in diverse cell functions (OLDFIELD AND DUNKER 2014), through their ability to mediate multivalent interactions, including those enabling assembly of "biomolecular condensates". These condensates organize proteins and RNAs into non-membrane bound cellular compartments that perform diverse functions, ranging from regulating transcription to RNA processing/RNP assembly to the DNA damage response to cellular signaling (BANANI et al. 2017;HOLEHOUSE AND PAPPU 2018). Biomolecular condensates assemble by multivalent interactions among their protein and RNA components, leading to "phase separation". While not all proteins containing IDRs have been shown to form biomolecular condensates, intriguingly proteins containing SH2 and SH3 domains were among the first proteins shown to assemble by this mechanism (LI et al. 2012), and Abl clearly can assemble into a large macromolecular complex (e.g. (GREGOR et al. 2019).
Abl's IDR is not highly conserved in primary sequence, even between the two mammalian paralogs (Fig.   1A). Only a single peptide is conserved among fly and mammalian family members-the PXXP SH3-domain binding motif in the N-terminal quarter of the IDR. Other peptide motifs, including a predicted Ena binding site, are conserved over shorter phylogenetic distances; e.g., among insect Abl proteins. There are also functional motifs present only in single family members, including the microtubule and second actin interaction sites in mammalian Arg (MILLER et al. 2004;COURTEMANCHE et al. 2015). Two groups assessed the function of the Drosophila Abl IDR, taking different approaches. Our lab individually deleted short conserved regions of 12-56 amino acids (conserved regions 1 to 4 (CR1-CR4)), in the context of a GFP-tagged full length Abl protein driven by its endogenous promotor (Fig. 1B). We measured rescue of embryonic and adult viability, morphogenetic movements in the embryo, and axon outgrowth in the embryonic central nervous system (ROGERS et al. 2016). Cheong and VanBerkum took a more comprehensive approach, deleting successively smaller fractions of the C-terminal region, including the FABD. They began by dividing it in half, then in quarters and then focused in on two smaller regions, with smaller deletions and point mutations. They expressed their mutant proteins in the background of zygotic abl mutants using the GAL4-UAS system and assessed rescue of axon pathfinding (CHEONG AND VANBERKUM 2017). Both approaches led to similar surprising conclusions. Only a single region of the IDR plays a major role in Abl function-the region containing the conserved PXXP motif. Surprisingly, however, this motif was extremely important, as its deletion caused as reduced Abl function more than loss of kinase activity or even loss of both kinase activity and the FABD.
Subsequent analyses support the idea that this motif acts by interactions with the adapter protein Crk (SPRACKLEN et al. 2019) and with the actin-regulatory WAVE-regulatory complex (CHEONG et al. 2020). The finegrained dissections of the IDR by Cheong and VanBerkum suggest other regions of the IDR may have more subtle roles in axon guidance.
Our initial analyses did not fully probe the function of the IDR, as perhaps the simplest test of its function-completely deleting the IDR while leaving the FABD intact-was missed. We thus generated a mutation cleanly removing the IDR in the context of a GFP-tagged Abl construct driven by its endogenous promotor. This revealed surprising dominant-negative activity of Abl∆IDR and a potential role for the IDR in regulation of Abl protein stability. These observations provide important insights into Abl function.

Creating a mutant to test the role of the IDR in Abl function
Abl is a multidomain protein which uses both its kinase activity and its protein interaction domains to create a signaling hub, integrating upstream signals and activating downstream effectors. Our lab previously created a series of abl mutants to assess the role of kinase activity and other domains and motifs in Drosophila (Fig. 1B;(ROGERS et al. 2016). The base construct was a P-element transgene containing a wild type abl gene driven by a 2kb fragment of the 5' upstream endogenous abl promoter (Tn Abl WT:GFP), which can fully rescue ablMZ mutant embryos (FOX AND PEIFER 2007). This construct has a C-terminal GFP tag that does not impair its rescuing ability, and which allows direct visualization of Abl localization in live embryos. These included mutants deleting short conserved regions in the IDR (AblDCR1-DCR4, Fig 1B), but we did not fully remove the IDR to assess its full set of roles. To do so, here we created a similar transgene that essentially deletes the entire IDR-below we refer to this as Abl∆IDR ( Fig. 1B; amino acids 679-1398 are deleted; see Methods for details). We added a 15 amino acid flexible linker (GGS)5) in place of the IDR to reduce the likelihood of disrupting folding of the adjacent kinase domain and FABD ( VAN ROSMALEN et al. 2017). We introduced this transgene into the Drosophila genome in two ways-by P element-based transformation (selecting an insertion on then second chromosome), and by site-specific integration on the left arm of the 2nd chromosome (at 22A3). We then used these transgenes to assess the roles of the IDR in Abl function.

Abl∆IDR does not rescue adult viability and has apparent dominant negative effects on embryonic morphogenesis
Because of its critical roles in embryonic morphogenesis and neuronal development, Abl is essential for both embryonic and adult viability (HENKEMEYER et al. 1987;GREVENGOED et al. 2001). The ability to rescue viability thus offered an initial test for our Abl∆IDR mutant protein. Abl is maternally contributed and this maternal contribution is sufficient for embryonic development (GREVENGOED et al. 2001). However, most abl null mutants die as pupae-the few that escape are functionally sterile and die soon after eclosing (HENKEMEYER et al. 1987). We thus tested the ability of Abl∆IDR to rescue adults that were heterozygous for the putative null allele abl 4 (FOX AND PEIFER 2007) and a Deficiency, Df(3L)st-7 Ki. that removes the abl gene (abl 4 /Df), as we had for our earlier mutants (ROGERS et al. 2016). A targeted transgene encoding Abl∆IDR provided partial but incomplete rescue of adult viability. While unrescued abl 4 /Df adults had only 20% the viability of those rescued by our wildtype abl transgene, Abl∆IDR; abl 4 /Df adults eclosed at 48% the rate of those rescued by the wildtype transgene ( Fig.1C ; Table 1). In contrast, Abl∆FABD fully rescued adult viability, and even a mutant lacking both kinase activity and the FABD provided substantial rescue (ROGERS et al. 2016); 82% of the wildtype transgene). However, Abl∆CR1, lacking the PXXP motif in the IDR, did not provide substantial rescue (31% viability relative to the wildtype transgene; Fig. 1C; Table 1; (ROGERS et al. 2016).
These data suggest that the IDR is important for Abl function.
Unlike the abl 4 /Df escapers, Abl∆IDR; abl 4 /Df females lived long enough to mate and produce fertilized eggs. We thus asked whether Abl∆IDR rescued the lethality of embryos lacking both maternal and zygotic Abl, by crossing these females to males who were heterozygous for abl 4 /+ and carried the transgene. Abl∆IDR did not rescue the viability of maternal/zygotic mutants (50% of the progeny), and, surprisingly, even 30% of embryos that inherited a paternal zygotic wildtype abl gene died before hatching and the rest (20%) died as first instar larvae (Fig. 1D, Table 1). In contrast, Abl∆FABD provided full rescue of embryonic viability ( Fig. 1D; Table 1; (ROGERS et al. 2016). To roughly assess the rescue of embryonic morphogenesis by Abl∆IDR, we examined the cuticles of the dead embryos. To our surprise, the cuticle phenotype was extremely severe, with all embryos exhibiting strong disruption of epidermal integrity, including those in which only fragments of cuticle were secreted ( Fig. 2A vs B-D). These morphogenetic phenotypes are more severe than those characteristically seen in abl maternal/zygotic mutant embryos (GREVENGOED et al. 2001;ROGERS et al. 2016).
However, the limitations of this approach are that since unrescued abl 4 /Df mutant females are sterile, we could not compare embryonic morphogenesis of their progeny to those rescued by Abl∆IDR.
To circumvent this, we used the FLP/FRT/DFS approach (CHOU et al. 1993) to generate females whose germlines are homozygous for abl 4 , either in the presence of one of our transgenes or in the absence of any transgene as a control. This approach allowed us to compare maternal/zygotic abl mutants (ablMZ), who are homozygous for the null allele, with similar mutants that have one of our transgenes contributed both maternally and zygotically. We used abl transgenes inserted at the same chromosomal location via phiC integrase. ablMZ mutants generated by the FLP/FRT/DFS approach are embryonic lethal (GREVENGOED et al. 2001), and there is only partial rescue of viability in the 50% of embryos that receive a wild-type abl gene paternally (9% overall embryonic viability ( Fig. 1E; Table 1). Strikingly, Abl∆IDR; ablMZ mutants had an even higher embryonic lethality (1% overall embryonic viability; probability that viability is lower than ablMZ p<0.0001; by Fisher's Exact test; Table 1). In contrast, our GFP-tagged wildtype transgene, provided strong rescue (39% viability (ROGERS et al. 2016); we attribute the lack of full rescue to other mutations that have accumulated on the abl 4 chromosome), as did the Abl∆FABD transgene (35% viability; (ROGERS et al. 2016).
We next examined embryonic cuticles, as they allow us to assess cell fate choice, major morphogenetic movements like germband retraction, head involution, and dorsal closure, along with epidermal integrity.
ablMZ mutants have multiple defects in these processes (GREVENGOED et al. 2001;ROGERS et al. 2016 with smost exhibiting strong defects in head involution and failure of full germband retraction. Many also fail in dorsal closure, and a small fraction (15%) have defects in epidermal integrity. Our transgene encoding wildtype Abl largely rescued these defects (Fig. 2F,G;(ROGERS et al. 2016). Our previous analysis revealed that neither kinase activity nor the FABD is essential for rescuing these cuticle defects, while Abl∆CR1, lacking the conserved PXXP motif in the IDR, largely rescued epidermal integrity but only partially rescued germband retraction and dorsal closure (Fig. 2F,G;(ROGERS et al. 2016). In contrast, however, Abl∆IDR; ablMZ mutants had even more severe cuticle defects than unrescued ablMZ mutants. For example, the fraction of embryos with the more severe epidermal defects more than doubled, from 15% to 33% (Fig. 2F,G; probability that the cuticle defects are worse than ablMZ p<0.0001; by Fisher's Exact test). This epidermal disruption phenotype was similar to that we observed in the progeny of Abl∆IDR; abl 4 /Df females ( Fig. 2B-D). Taken together, the increased embryonic lethality and higher proportion of severe cuticle defects in these experiments and the unexpectedly severe cuticle phenotype seen in our initial abl 4 /Df experiments, suggested to our surprise that expressing Abl∆IDR not only fails to rescue loss of Abl, but actually worsens some aspects of the abl null mutant phenotype in embryonic development.

Abl∆IDR does not effectively rescue defects in cellularization or mesoderm invagination
Abl has diverse roles in embryonic development, ranging from regulating actin dynamics during syncytial development and cellularization to regulating apical constriction of mesodermal cells to regulating cell shape change and collective cell migration during germband retraction and dorsal closure. Our cuticle data suggested that Abl∆IDR was substantially impaired in morphogenesis. To examine this more closely, we used immunofluorescence and confocal microscopy to examine cell shape changes and cytoskeletal regulation during embryonic development, as we had done to assess the roles of kinase activity, the FABD, and the conserved motifs in the IDR (ROGERS et al. 2016).
The first events of embryogenesis requiring Abl function are the characteristic dynamics of the actin cytoskeleton during the syncytial stages and cellularization. Maternal/zygotic abl mutants (ablMZ) have defects in both processes, and thus accumulate multinucleate cells at the end of cellularization (GREVENGOED et al. 2003); Fig. 3A vs B, red arrows). Abl∆IDR did not rescue these defects, and thus Abl∆IDR; ablMZ mutants accumulated multinucleate cells ( Fig. 3A vs C, red arrows). Abl is also required for the first event of gastrulation, in which cells along the ventral midline apically constrict in a coordinated way and invaginate as a tube (FOX AND PEIFER 2007). The invaginating cells then go on to become mesoderm, while the ectodermal cells close the gap and form a straight midline. In ablMZ mutants, apical constriction is poorly coordinated, leaving some mesodermal cells on the surface. Ectodermal cells eventually close the gap, but the resulting midline is not straight (Fig.3D vs. E, blue arrows). Once again, Abl∆IDR did not fully rescue these defects (Fig. 3F). This latter phenotype is particularly interesting as Abl∆CR1 did rescue mesoderm invagination (ROGERS et al. 2016).

Abl∆IDR does not rescue defects in germband retraction or dorsal closure
The morphogenetic events in which Abl's roles have been analyzed in greatest detail are two of the final morphogenetic movements of embryogenesis: germband retraction and dorsal closure (GREVENGOED et al. 2001;ROGERS et al. 2016). These events are easily visualized by staining embryos with antibodies to E-cadherin (Ecad) to outline cells. At the end of stage 11 of wildtype embryogenesis, the caudal end of the embryo is curled up on the dorsal side. During stage 12, the germband retracts, ultimately positioning the tail end of the embryo at the posterior end of the egg, and thus leaving structures like the spiracles at the posterior end ( Fig.   2A, arrow) and out of the dorsal view. At this stage, the ventral and lateral side of the embryo are enclosed in epidermis, but the dorsal side is covered by a "temporary" tissue, the amnioserosa (AS, Fig. 4A). During dorsal closure, the epidermis and the amnioserosa work in parallel to completely enclose the embryo in epidermis  4A,B, red arrows). Actin=based protrusions from leading edge cells also aid in cell matching/alignment between the two sheets. ablMZ mutants have defects in both germband retraction and dorsal closure (GREVENGOED et al. 2001;ROGERS et al. 2016). Germband retraction is not completed and the spiracles are thus positioned dorsally (Fig. 4C, green arrow). Dorsal closure proceeds very abnormally and often fails to go to completion. The leading edge is highly wavy rather than straight (Fig. 4C, blue arrows) and zippering at the two canthi is slowed (Fig. 4C, red arrows). Tissue tearing is often observed at the border between the leading edge and amnioserosa, leaving underlying tissue exposed (Fig 4C, asterisk).
We thus asked whether these defects are rescued by Abl∆IDR. Occasional Abl∆IDR; ablMZ embryos succeeded in proceeding through closure, but even these exhibited defects in germband retraction, with the spiracles positioned dorsally (Fig. 4D, green arrow). In most embryos closure was highly aberrant. The leading edge was wavy instead of straight (Fig. 4D,E,F vs. A,B,blue arrows). Zippering at the canthi was slowed (Fig.   4E,F red arrows) and often did not proceed uniformly, with zippering slower or absent at the anterior end ( Fig.   4G, red arrows). As we observed in unrescued ablMZ mutants, tearing occurred between the leading edge and the amnioserosa (Fig. 4F, asterisk). In a subset of Abl∆IDR; ablMZ embryos, the phenotype at stage 13/14 was even more severe. These embryos had reduced epidermal coverage ( Fig. 4H-J), suggesting earlier cell death. They also exhibited deep, un-retracted segmental grooves during dorsal closure (Fig. 4H,I, green arrows), another known phenotype of ablMZ mutants (ROGERS et al. 2016). Some Abl∆IDR; ablMZ embryos had numerous very large cells (Fig. 4J), which we suspect are a remnant of the multinucleate cells that arise during cellularization and gastrulation. This severe class of embryos likely represents the subset whose cuticles show substantial epithelial disruption (Fig. 2). Together, these data reveal that Abl∆IDR fails to rescue defects in germband retraction or dorsal closure. Intriguingly, our previous analysis revealed that kinase activity and the FABD are largely dispensable for these morphogenetic events, while the CR1 PXXP motif in the IDR plays a role (ROGERS et al. 2016).

Abl∆IDR does not rescue defects in leading edge cell shape or in actin regulation
We next explored the role of Abl's IDR at the cellular and subcellular level. During dorsal closure the leading edge cells assemble a contractile actin cable that exerts tension along the dorsal cell margin. This cable maintains a straight leading edge and together with amnioserosal apical constriction elongates epidermal cells along the dorsal-ventral axis (reviewed in (HAYES AND SOLON 2017;KIEHART et al. 2017). The cable is anchored cell-to-cell at leading edge adherens junctions. In wildtype embryos tension along the cable is balanced among the cells and thus they exhibit relatively uniform shapes (Fig. 5A, arrows), with slight deviation at the segmental grooves (Fig. 5A, arrowheads). Loss of Abl disrupts leading edge cell shapes, with some cells hyper-constricted and other splayed open, presumably due to failure of the leading edge actin cable in some cells (GREVENGOED et al. 2001;ROGERS et al. 2016). We thus examined if Abl∆IDR rescued these cell shape defects. It did not. Abl∆IDR; ablMZ embryos exhibited penetrant defects in leading edge cell shape, with splayed open and hyperconstricted cells (Fig. 5B,C magenta vs. yellow arrows). We also observed groups of cells that failed to elongate (Fig 5B,C,E red asterisks), as we had previously observed in ablMZ mutants (GREVENGOED et al. 2001;ROGERS et al. 2016). Finally, most embryos exhibited another ablMZ mutant phenotype (GREVENGOED et al. 2001;GREVENGOED et al. 2003): large, presumably multinucleate cells, which in some embryos were very frequent (Fig. 5D, yellow asterisks; the frequency of multinucleate cells appeared substantially higher than was seen in un-rescued ablMZ mutants). Cell shape defects and multinucleate cells were even observed in the occasional embryos which managed to close dorsally (Fig. 5E). From these data we conclude that Abl's IDR is essential for regulating leading edge cell shape.
One of the key roles of Abl family kinases is regulation of the cytoskeleton. Drosophila Abl regulates the actin cytoskeleton through effectors like the actin polymerase Enabled (Ena). Our previous analysis suggests an important role for Abl regulation of Ena and actin at the leading edge during dorsal closure (GREVENGOED et al. 2001;GATES et al. 2007;ROGERS et al. 2016). In wildtype embryos Ena localizes to the cell junctions of both amnioserosal and epidermal cells, but is strongly enriched in the tricellular junctions of leading edge cells, where the actin cable is anchored (Fig. 6A, red arrows; (GATES et al. 2007;MANNING et al. 2019). Ena is also somewhat enriched at tricellular junctions of more ventral epidermal cells (Fig. 6A, yellow arrows). In ablMZ mutants the uniform localization of Ena to leading edge tricellular junctions is lost (ROGERS et al. 2016). We thus asked whether Abl∆IDR can restore leading edge Ena localization. While Ena remained enriched at some leading edge tricellular junctions of Abl∆IDR; ablMZ mutants ( Fig 6B,C,D red arrows), its uniform enrichment was lost, even though enrichment at lateral epidermal tricellular junctions remained (Fig 6B,C,D yellow arrows). At many leading edge tricellular junctions Ena was weak or absent (Fig 6B,C,D cyan arrows), and at other places Ena spread across the leading edge (Fig 6B,C,D green arrows), all features we previously observed in ablMZ mutants and in embryos lacking the CR1 PXXP motif (ROGERS et al. 2016).
The altered cell shapes observed in ablMZ mutants reflect defects in the leading edge actin cable (ROGERS et al. 2016). In wildtype embryos the actin cable extends relatively uniformly across the leading edge (Fig. 6E, arrows), joined cell to cell at leading edge tricellular junctions. In contrast, in Abl∆IDR; ablMZ embryos, the leading edge actin cable was discontinuous, with regions of reduced intensity (Fig. 6F, cyan arrows) interspersed with regions of elevated actin intensity (Fig. 6F, green arrows), as we previously observed in ablMZ mutants (ROGERS et al. 2016). Actin levels were also elevated at many lateral epidermal tricellular junctions (Fig. 6F, yellow arrows) a featured shared by ablMZ mutants (ROGERS et al. 2016) and by embryos in which Ena levels were artificially elevated (NOWOTARSKI et al. 2014). These results indicate that Abl∆IDR does not rescue the defects in Ena localization or actin regulation seen after loss of Abl.

Abl∆IDR protein is more stable than WT Abl protein
Perhaps the most surprising aspect of our phenotypic analysis of Abl∆IDR were the indications that expression of this protein actually enhanced rather than rescued the phenotype of ablMZ mutants. We first  (Fig. 7B,C). Cortical enrichment of both wildtype Abl:GFP and Abl∆IDR:GFP was diminished during dorsal closure (Fig. 7D,E). Thus Abl∆IDR encodes an apparently stable protein that retains the ability to associate with the cortex.
We next visualized the Abl:GFP and Abl∆IDR:GFP proteins live, without fixation. While cortical enrichment was obvious for Abl:GFP (Fig. 7F), it was much less apparent for Abl∆IDR:GFP (Fig. 7G)-instead, the entire cell appeared to be filled with protein. These data suggested a second possibility: a difference in expression or accumulation levels. All of our transgenes were driven by the endogenous abl promotor, which drives expression of transgenes at normal levels (FOX AND PEIFER 2007) and in our second set of transgenes we targeted all to the same chromosomal location to reduce the possibility of position effects. Immunoblotting had previously revealed that our wildtype GFP-tagged Abl and each of our previously analyzed mutants accumulate at levels similar to endogenous wildtype Abl (ROGERS et al. 2016). We thus repeated this analysis with Abl∆IDR.
To our surprise, in embryos, AblDIDR protein accumulates to substantially higher levels than that of wildtype GFP-tagged Abl (Fig. 8A); quantitative immunoblots revealed that protein levels are elevated 11-fold ( Fig. 8B). This cannot be attributed to chromosomal position effects, as we observed similar elevation in protein levels with flies carrying two independently generated Abl∆IDR transgenes (flies carrying the Pelement -mediated transgenes generated for our initial experiments and the phiC targeted transgenes).
Because this result was so surprising, also we expressed our transgenic proteins in a well-characterized Drosophila cultured cell line, S2 cells, where they were driven by the heterologous metallothionein promotor.
Strikingly, AblDIDR protein also accumulated to a significantly higher level than wildtype Abl protein in transfected S2 cells (Fig. 8C). This observation ruled out the possibility that the higher levels of AblDIDR protein accumulation are solely due to differences in transcription, since in the embryos, transcription of both wildtype and Abl∆IDR transgenes are driven by the same 2kb upstream abl promoter region, while in S2 cells, transcription of transgenes encoding wild type Abl and AblDIDR was driven by the same metallothionein promoter and the plasmids encoding them had essentially identical transfection efficiencies. Consistent with what we observed in live embryos, the enrichment of Abl:GFP in the S2 cell lamellipodium was obscured when we examined Abl∆IDR (Fig. 8D), consistent with the possibility that its elevated levels saturated normal binding sites in the lamellipodium and filled the cell. These data suggest that AblDIDR protein is more stable and resistant to degradation than wildtype Abl, and that Abl's IDR contains an element important for regulating Abl protein levels.

Discussion
The important roles of Abl kinase in embryonic development, the nervous system, adult homeostasis and oncogenesis make understanding its molecular function essential for both basic scientists and clinicians. Abl is a complex multidomain protein and we and others have assessed the roles of kinase activity and its many protein interaction domains. Here we further explore the roles of its intrinsically disordered linker (IDR), revealing this region to be essential for protein function in Drosophila morphogenesis, and important in regulating protein stability.
As one of the first protein kinases implicated in cancer, attention initially focused on Abl's kinase activity.
This clearly is critical for function of the Bcr-Abl fusion protein found in chronic myeloid and acute lymphocytic leukemia, and drugs targeting kinase activity revolutionized treatment of these disorders (SAWYERS et al. 2002;TALPAZ et al. 2002). However, studies of Abl's normal roles in both Drosophila and in mammals suggest kinase activity, while important, is not essential, as proteins lacking kinase activity retain residual function in vivo (HENKEMEYER et al. 1990;MILLER et al. 2004;ROGERS et al. 2016 Abl's IDR is an interesting but poorly understood feature of Abl. IDRs are found in diverse proteins and have attracted increasing interest as regions mediating multivalent interactions, thus playing a role in some cases in phase transitions leading to the assembly of biomolecular condensates (BANANI et al. 2017; HOLEHOUSE AND PAPPU 2018). They contain regions of low-complexity sequence that are not well conserved, which mediate relatively non-specific interactions. IDRs also can contain short conserved motifs that mediate specific protein interactions, as is the case in Abl. Our previous analysis focused on four motifs that are well conserved among different insects, which we referred to as CR1 to CR4 (ROGERS et al. 2016). To our surprise, three of these, including a putative consensus binding site for the Abl effector Ena, are dispensable for rescuing viability and fertility (ROGERS et al. 2016). However, the PXXP motif embedded in CR1 proved important for function-Abl∆CR1 mutants exhibited reduced adult and embryonic viability and had defects in most but not all aspects of Abl function during embryonic morphogenesis. Cheong and VanBerkum similarly found important functions for this motif in supporting adult viability and embryonic axon guidance (CHEONG AND VANBERKUM 2017). However, the data from both groups reveal that Abl∆CR1 retains residual function.
Cheong and VanBerkum extended this analysis by deleting larger regions of the IDR, singly and in combination.
These data further support the idea that the PXXP motif is the only individually essential region of the IDR.
However, their gain-of-function assays and analysis of effects on protein localization suggest that the region containing the Ena-binding motif also contributes to axon localization and subtly to function.
Here we cleanly deleted the IDR while leaving the FABD intact, allowing us to directly determine whether other regions of the IDR have additional functions. Our new data strongly support this idea. In our assays of embryonic morphogenetic events in which Abl has a known role, Abl∆IDR failed to rescue mesoderm invagination and maintenance of epidermal integrity, whereas Abl∆CR1, lacking only the PXXP motif, retained full or substantial function (ROGERS et al. 2016). Consistent with our data, Cheong and VanBerkum found that deleting the first quarter of the IDR had stronger effects than simply mutating the PXXP motif (CHEONG AND VANBERKUM 2017;CHEONG et al. 2020). In fact, loss of the IDR reduced Abl function more substantially than any of our other previous alterations, including simultaneously eliminating kinase activity and the FABD (ROGERS et al. 2016), demonstrating its critical role in Abl function.
Surprisingly, in our assays of embryonic morphogenesis, expressing Abl∆IDR actually worsened the phenotype of ablMZ mutants, substantially elevating the frequency of embryos with severe disruption of epidermal integrity. We similarly observed drastic disruption of epidermal integrity when we used the Abl∆IDR transgene to rescue the progeny of abl 4 /Df females, once again a phenotype more severe than that of ablMZ mutants. The disruption of epidermal integrity we observe is likely a consequence of the early defects in syncytial development and cellularization, leading to the formation of multinucleate cells. Other mutants, including those that disrupt syncytial development and cellularization in different ways, as is seen in embryos mutant for the septin peanut, lead to a similarly disrupted cuticle phenotype (ADAM et al. 2000). Intriguingly, embryos maternally and zygotically mutant for the adapter protein Crk, which in mammals can bind the Abl What accounts for these seemingly "dominant negative" effects of Abl∆IDR? In our view, there are several possibilities, which are not mutually exclusive. First, it is possible that the allele we use as an abl null allele (FOX AND PEIFER 2007), abl 4 , actually encodes a very low levels of partially functional protein, via readthrough of the stop codon or a low level of downstream re-start. This could explain why the phenotype of the progeny of Abl∆IDR; abl 4 /Df females is even worse than the phenotype of Abl∆IDR; ablMZ mutants. In this scenario, Abl∆IDR could interfere with the function of this residual Abl protein by forming inactive complexes with it or with some of its effectors or regulators. Consistent with this, Cheong and VanBerkum saw strong dominant enhancement of mutants lacking the axon guidance modulators Frazzled and Slit when they overexpressed an Abl mutant lacking the first quarter of the IDR (CHEONG AND VANBERKUM 2017). It also is possible that the Deficiency we used, Df(3L)st-7 Ki, also deletes a gene that enhances the abl null phenotype, as there are many known mutants that exhibit this phenotype (e.g., (GERTLER et al. 1989;GERTLER et al. 1990;FORSTHOEFEL et al. 2005).
The Abl∆IDR protein has an additional property that may account for its dominant negative effects and which also casts light on the regulation of Abl activity: it accumulates at levels substantially higher than wildtype Abl. We observed this effect with two different transgenes driven by the endogenous abl promotor inserted at different chromosomal locations, and, importantly, also observed it when we expressed Abl∆IDR in cultured Drosophila cells driven by a heterologous promotor. These data imply that the IDR contains sequences that regulate Abl protein stability. None of our previous deletions of conserved motifs within the IDR (CR1-CR4) affected Abl levels (ROGERS et al. 2016), nor did the larger deletions of portions of the IDR made by Cheong and VanBerkum (CHEONG AND VANBERKUM 2017) suggesting this effect either involves a different region of the IDR or that it is a property of the IDR as a whole.
IDRs have clearly defined roles in regulating protein stability. Almost 80% of known degrons reside in disordered regions (GUHAROY et al. 2016), while computational predictions suggest a large fraction of ubiquitylation sites are in disordered regions (RADIVOJAC et al. 2010;PEJAVER et al. 2014). When we used the computational prediction software UbPred to identify potential ubiquitination sites within Abl (RADIVOJAC et al. 2010), 19 of 24 medium and high confidence predicted ubiquitination sites were located within Abl's IDR, and three more were in the unstructured N-terminal region ( Figure 9A,B). The presence of an IDR in a protein also accelerates proteasomal degradation, and they can act as initiation sites for proteolysis (PRAKASH et al. 2004).
Additionally, Ng et al. found that presence of IDRs may serve an important role in mediating ubiquitination in response to heat shock (Ng et al, 2013). Taken together, this evidence strongly suggests that Abl's IDR may play a role in ubiquitin-mediated protein turnover as a mechanism for Abl proteostasis. It will be interesting to determine whether this is a conserved property of the IDR across the Abl family, and by what mechanism this occurs. Mammalian Abl is regulated by the ubiquitin-proteasome system (ECHARRI AND PENDERGAST 2001) and Abl can be ubiquitinated by the E3 ligase Cbl (SOUBEYRAN et al. 2003). Mammalian Arg is also ubiquitinated in response to oxidative stress (CAO et al. 2005). Future work will determine if this is a conserved property of the IDR across the Abl family, and by what mechanism this occurs. work was supported by National Institutes of Health Grants R01 GM47957 and R35 GM118096 to M.P., and E.M.R was supported by a Leukemia and Lymphoma Society Career Development Program Fellowship Grant 5339-08.

Transgenic Fly Lines
To create the AblDIDR transgene, a pair of overlapping PCR products were generated with Phusion high fidelity DNA polymerase (NEB) using pUAS-Abl:GFP (FOX AND PEIFER 2007) as a template. pUAS-Abl:GFP contains 2kb of 5' upstream promoter from the endogenous abl gene as well as an in frame eGFP tag. The DIDR deletion was introduced by mutagenic DNA oligonucleotide primers in the overlapping section of the PCR products. In addition, a 15 amino acid flexible linker (GGS)5 ) was added at the location of the deletion -the hydrophilic glycine and serine residues are unlikely to form secondary structures, reducing the likelihood that the linker will interfere with the folding and function of the adjacent kinase and FABD domains . The two overlapping PCR products were joined by PCR stitching and cloned into the Xba/Not fragment of pUASg-Abl:GFP to make pUASg-AblDIDR:GFP. The primers used for mutagenesis were as follows : AblDIDR Forward:

CGA3'
Italicized regions contain the code for the 15 aa flexible linker and the bold regions are complementary to the abl sequence. The resulting coding sequence spanning the deletion is: …TSGVATGGGAGGSGGSGGSGGSGGSATPIAKLTEP… The pUASg-AblDIDR:GFP transgene was inserted via Pelement transposition, and we were able obtain multiple independent lines, including on the 2 nd chromosome.

Fly Stocks, viability and phenotypic analysis of abl mutants and statistical tests
All experiments were done at 25°C unless noted. y w served as wildtype in our experiments. For assessing rescue of adult viability, we generated zygotic abl mutants by crossing Df(3L)st-7 Ki/TM3 Sb females to transgene/transgene; abl 4 /TM3 Sb males, and selecting for Ki and against Sb ( abl 4 /Df(3L)st-7 Ki ). We set the fraction of progeny with this genotype seen when using the wildtype abl transgene (AblWT; 27%) as 100%, and other genotypes were normalized to this. Adult viabilities were compared by Fisher's Exact test (GraphPad). For this, the number of viable mutant adult flies (# of abl 4 /Df adults) was compared to the estimated number of non-viable flies. The number of non-viable flies was estimated by subtracting the number of viable mutant adult flies from the expected number if they were fully viable (# of abl 4 /TM3 plus Df/TM3 divided by 2). We used two methods to generate embryos maternally and zygotically abl mutant (ablMZ): 1) using the dominant female sterile method (Chou and Perrimon, 1996)  Fisher's Exact test (GraphPad). For each genotype, the number of cuticles falling into the two more severe classes (i.e. Dorsal closure failure, and Epidermal integrity defect) were grouped to a single defective category, and compared to the number of cuticles in the two less severe categories (wildtype and Strong defects in germband retraction). Similarly, cuticle scores of each mutant transgene in the abl 4 mutant background were compared to the wildtype transgene (AblWT) using the same approach.

Embryo live imaging
Embryos from flies that homozygous for either the transgene encoding Abl WT or AblDIDR were dechorionated in 50% bleach and mounted in halocarbon oil (series 700; Halocarbon Products, River Edge, NJ) between a gas-permeable membrane (Petriperm; Sartorius, Edgewood,NJ) and a glass coverslip and imaged in a Z-series of 1µM slices on a Zeiss LSM-5 Pascal confocal microscope.

Immunofluorescence
To examine embryos by immunofluorescence, flies were allowed to lay eggs on apple juice/agar plates with yeast paste for times calculated to obtain embryos at the right stages. Embryos were collected, dechorionated in 50% bleach, washed in 0.1% Triton-X, and fixed in 1:1 Heptane/3.7% Formaldehyde diluted in PBS for 20 minutes at room temperature. Embryos were then devitillenized by shaking in 1:1 heptane /methanol, when prepared for phalloidin staining, hand-devitillenized. Embryos were then blocked in Blocking Solution (PBS/0.1% Triton-X/1% Normal Goat Serum) for ≥ 30 min, incubated in primary antibody diluted in Blocking Solution overnight at 4°C and washed 3X in Blocking Solution. Embryos were then incubated in secondary antibody in Blocking Solution for 2 hours at room temperature and washed 3X in Blocking Solution.
Embryos were mounted on glass slides in Aquapolymount (Polysciences, Inc). Primary and secondary antibodies were: (anti-Dcad, 1:100; anti-Enabled, 1:500 (all from the Developmental Studies Hybridoma Bank and anti-mouse and anti-rat IgG Alexa Fluors 568 and 647, from Molecular Probes); some secondary antibodies were preabsorbed with fixed y w embryos. For F-actin staining TRITC labeled phalloidin (Sigma) was used at a dilution of 1:500 to 1:1000.
For S2 cells, resuspended cells were allowed to attach for 1 hour onto a ConcanavalinA coated glass coverslip. Cells were then fixed 10 minutes in 10% formaldehyde HL3 buffer (70 mM NaCl; 5 mM KCl; 1.5 mM CaCl2-2H2O; 20 mM; MgCl2-6H2O; 10 mM NaHCO3; 5 mM trehalose; 115 mM sucrose; 5 mM HEPES; pH 7.2) followed by four 10 minute washes in PBS with 0.1% Triton-X (PBST) and two brief washes with ddH2O. During the last PBST wash, TRITC, labeled phalloidin was added to a dilution of 1:1000. A drop of Aquapolymount was added to the coverslips, and the coverslips were mounted on pedestals of dried nail polish on a glass slide, and sealed with nail polish. Imaging of embryos and S2 cells was done on a Zeiss LSM-5 Pascal or Zeiss 710 scanning confocal microscopes. Images were processed using ZEN 2009 software. Photoshop CS6 (Adobe) was used to adjust input levels so that the signal spanned the entire output grayscale and to adjust brightness and contrast.

Immunoblotting
Embryonic extracts for immunoblotting were prepared by resuspending embryos in an equal volume of 2X SDS-PAGE Sample buffer(100 mM Tris-Cl (pH 6.8);4% SDS; 0.2% bromophenol blue; 20% glycerol; 200 mM Bmercaptoethanol) and homogenizing with a pestle in a microfuge tube. To make S2 cell extracts 1mL of resuspended S2 cells were spun down in a microfuge tube, the media was removed, and the pellet resuspended in an equal volume of 2X SDS PAGE Sample buffer. The samples were boiled for 5 min, spun to clear debris, and 10 ul of the resulting extract run on a 7.5% SDS-PAGE gel, and transferred to a nitrocellulose membrane. To detect the transgenic GFP-tagged Abl proteins we used anti-GFP (JL-8, 1:500 or 1:1000, Clontech). Anti-aTubulin (Sigma, 1:10000) or anti-Pnut (Developmental studies Hybridoma Bank,1:30) were used as loading controls. Detection was done using HRP-conjugated anti-mouse IgG secondary antibody (Pierce, 1:50000), and the ECL plus substrate kit (Pierce).

Quantification of Abl DIDR and Abl WT protein levels
Four immunoblots of embryo extracts from homozygous stocks of the targeted Abl WT and Abl DIDR transgenes were used to quantify relative levels of Abl WT and Abl DIDR proteins in the embryos. Scans of Western blot film exposures were opened and converted to grayscale images in Adobe Photoshop. The resulting image was opened in ImageJ as a JPEG and the pixels were inverted. Rectangular ROIs of the exact same dimensions, and just large enough to contain the thickest band were drawn around the Abl protein and loading control bands. An ROI was also drawn around an unexposed area of the film for background subtraction. The mean gray value (MGV) of the ROIs for Abl and loading control proteins, and background were determined. The background subtracted MGVs of the AblDIDR and Abl WT bands were adjusted for any loading differences by dividing them by the MGVs of their background subtracted loading controls. The background and loading control adjusted AblDIDR and Abl WT levels were expressed as a ratio of AblDIDR/Abl WT, normalized to the level of Abl WT which was assigned a value of 1. To determine statistical significance an unpaired t-test was used (GraphPad).

Expression of Abl proteins in S2 cells
To express Abl and Abl DIDR proteins in S2 cells, the Abl and Abl DIDR coding regions were cloned by Gateway Technology(Invitrogen) into pMT, a vector for metal inducible protein synthesis via the metallothionein promoter. To make pMT Abl::GFP and pMT AblDIDR::GFP, Phusion Polymerase was used to amplify the Abl and Abl DIDR coding regions using pUASt-attP-Abl:GFP and pUASt-attP-AblDIDR:GFP as a template with the following primers: AblGFP Gateway Forward: 5'CACCATGGGGGCTCAGCAGGGCAA 3' AblGFP Gateway Reverse:

5'CCTGTTAAGCGCATTGGAGATCTGA3'
pMT Abl or pMT AblDIDR::GFP DNAs were transfected into S2 cells grown in Sf-900 II SFM medium (Invitrogen) in the wells of 6 well plates (35mm) using Effectene transfection reagent(Qiagen) according to the manufacturer instructions. Six hours after the transfection, CuSO4 was added to 500 mM to induce expression of the transgenes. Cells were allowed to induce for 24 hrs and were used for both Western Blots and Immunufluorescence microscopy. Transfection efficiency was estimated by counting GFP positive cells on a dozen 143um X 143um fields on slides for immunofluorescence and dividing by the total number of cell (for blot in Fig 7c transfection efficiency: Abl WT= 65%(n=205) and AblDIDR=57%(n=109) Probababilty that viability is higher than than adults with no transgene Probababilty that rescue is worse than that by Abl WT Probababilty that rescue is better or worse than that by Abl WT Probababilty that lethality is worse than that of Abl WT Probababilty that lethality is better or worse than that of ablMZ Figure 1. Generating Abl∆IDR and testing its ability to rescue adult and embryonic viability. A. Diagram of human Abl and Arg and Drosophila Abl, showing conserved domains/motifs as well as motifs in the IDR that vary between family members. B. Illustration of the mutant Abl proteins we previously tested and our new Abl∆IDR mutant. It was designed to remove essentially the entire IDR, leaving only a few amino acids at each end to ensure we did not disrupt folding of the kinase domain or FABD. A 15 aa flexible linker was added in its place. C. Assessment of the ability of Abl∆IDR to rescue the viability of abl 4 /DfAbl adults, normalized to rescue by our wildtype Abl transgene, and compared to rescue by some of our previously tested mutants. Full data sets with statistical significance for C-E are in Table 1. D. Assessment of the ability of Abl∆IDR to rescue embryonic viability of the progeny of abl 4 /DfAbl females mated to abl 4 /+ males, compared to rescue by some of our previously tested mutants. Line indicates 100% embryonic viability. E. Assessment of the ability of Abl∆IDR to rescue embryonic viability of the progeny females with germlines homozygous for of abl 4 mated to abl 4 /+ males, compared to rescue by some of our previously tested mutants. Line indicates degree of rescue by our wildtype Abl transgene. Embryos, genotypes indicated, anterior left. A-C. Cellularization, Phalloidin stained to reveal f-actin. A.
Defects in actin regulation during syncytial development and cellularization led to the formation of multinucleate cells (red arrows). C. Abl∆IDR; ablMZ mutant. Abl∆IDR fails to rescue the defect in cellularization, and thus multiple multinucleate cells are observed. D-F. Stage 8 embryos, stained with antibodies to Ecad to visualize cell shapes. D. In wildtype mesoderm invagination is completed normally leaving a straight and even midline (blue arrows). E. ablMZ mutant. Defects in mesoderm invagination leave the ventral midline wavy and uneven (blue arrows). Also note the continued presence of multinucleate cells (red arrows). F. Abl∆IDR; ablMZ mutant. Abl∆IDR fails to fully rescue the defect in mesoderm invagination, leaving a wavy midline (blue arrows). Multinucleate cells remain (red arrows). Scale bar=15 µm.