Transcriptional Regulation

During Drosophila development, the Jun N-terminal kinase signal transduction pathway regulates morphogenetic tissue closure movements that involve cell shape changes and reorganization of the actin cytoskeleton. The genome-wide transcriptional response to activation of the JNK pathway has been analyzed in the Drosophila embryo by serial analysis of gene expression (SAGE) and loci encoding cell adhesion molecules and cytoskeletal regulators were identified as JNK responsive genes. The role in embryogenesis of one of the upregulated genes, chickadee (chic), encoding a Drosophila profilin, was analyzed genetically. chic-deficient embryos fail to execute the JNK-mediated cytoskeletal rearrangements during dorsal closure. This study demonstrates a transcriptional mechanism of cytoskeletal regulation and establishes SAGE as an advantageous approach for genomic experiments in the fruitfly (Jasper, 2001).

With SAGE, virtually every transcript in a sample RNA population can be identified and quantitated by generating specific 14 bp sequence tags from a defined position. Concatemers of such tags are then sequenced, and the frequency with which a given tag is detected represents a direct measure of the abundance of the corresponding mRNA (Jasper, 2001).

SAGE in Drosophila has become particularly powerful with the availability of the Drosophila genomic sequence. Due to the comparatively small size of the fly genome (1.2 x 108 bases of euchromatin), the 14 bases of a SAGE tag (2.7 x 108 possibilities) are typically sufficient to locate the respective transcript in the genome without having to rely on further information (Jasper, 2001).

The dorsal closure process takes place between embryonic stages 13 and 16, corresponding to 10-16 hr of development at 25°C. To capture the relevant changes of gene expression involved in setting up and executing dorsal closure, SAGE libraries were generated from 4-16 hr old Drosophila embryos in which the JNK pathway was either repressed by the ubiquitous expression of a dominant-negative form of Basket (BskDN) or ubiquitously activated due to expression of a constitutively active form of Hemipterous (Hepact). A library from wild-type embryos was prepared as a reference. The expression of the transgenes was dependent on Gal4 that was expressed ubiquitously under the control of the armadillo promoter starting at around 4.5 hr after egg laying. The effect of the BskDN and Hepact molecules on the transcription of target genes was therefore limited to the period of embryogenesis relevant for dorsal closure (Jasper, 2001).

Among the upregulated genes identified, several were known from previous studies to interact genetically or biochemically with components of the JNK pathway or to be required for embryonic dorsal closure. However, dpp and puc, until now the only known JNK-responsive genes, were not among them. Despite a significant upregulation of both dpp and puc in Hepact-expressing embryos as detected by RT-PCR, significant numbers of the corresponding tags were not obtained in the SAGE experiment. The absence of dpp and puc in this analysis is due to the low expression levels of these regulatory transcripts. To generate statistically relevant SAGE data for such rare messages, greater numbers of tags will have to be sequenced (Jasper, 2001).

Consistent with the proposed role of JNK signaling in reorganization of the actin cytoskeleton, this analysis identified several cytoskeletal regulators as JNK target genes. One example is the Drosophila homolog of the profilin gene, chickadee, which is strongly upregulated in Hepact-expressing embryos. Although mutants for chic have been described as defective in a number of actin-dependent processes, a role in dorsal closure has not yet been reported. Mutants lacking chic function were examined. In addition to pleiotropic defects observed in cuticle preparations, about 30% of these embryos secreted a very thin cuticle with obvious dorsal closure defects. The lateral epidermis failed to stretch normally during the closure process, confirming that the defects detected were not secondary effects caused by the weak cuticle (Jasper, 2001).

To further investigate the proposed role of chic downstream of JNK signaling in dorsal closure, whether chic and hep mutations interact genetically was examined. When female flies homozygous for the X-linked hypomorphic mutation hep1 are crossed to wild-type males, the male offspring die with mild dorsal closure defects: only 30% of these embryos are completely open, whereas in the remaining 70%, the segments a5-a8 close normally. In contrast, when hep1 homozygous females are crossed to chic221 heterozygous males, the dorsal open phenotype is enhanced and the number of completely open embryos in the offspring is increased to around 65%. Thus, the gene dose of chic becomes critical in embryos with compromised hep function. In an independent experiment, it was found that in chic heterozygous embryos the phenotypic effects of Hepact expression are suppressed. Together with the molecular data, these genetic interactions suggest that chic is required downstream of hep for normal dorsal closure (Jasper, 2001).

To understand the cellular function of Hep-induced chic expression in the embryo in more detail, the actin cytoskeleton in the relevant genotypes was examined. Hepact-expressing embryos display ectopic foci of actin polymerization among the lateral epithelial cells and show increased actin polymerization in leading edge cells, resulting in a stronger actin cable compared to wild-type embryos. In contrast, the leading edge of embryos lacking JNK activity shows a less prominent actin cable overall, which is occasionally disrupted. Significantly, the same phenotype can be observed in chic mutant embryos, consistent with Chic and Hep acting in the same pathway (Jasper, 2001).

Actin-based filopodia that extend dorsally from wild-type leading edge cells have been proposed to mediate the movement and proper alignment of the lateral epidermal sheets during dorsal closure. In hep-deficient embryos, these structures do not form. chic mutants share this phenotype and show an almost complete lack of these filopodia, indicating that this defect is a consequence of insufficient profilin expression in JNK pathway mutants (Jasper, 2001).

Transcriptional regulation of Profilin during wound closure in Drosophila larvae

Injury is an inevitable part of life, making wound healing essential for survival. In postembryonic skin, wound closure requires that epidermal cells recognize the presence of a gap and change their behavior to migrate across it. In Drosophila larvae, wound closure requires two signaling pathways [the Jun N-terminal kinase (JNK) pathway and the Pvr receptor tyrosine kinase signaling pathway] and regulation of the actin cytoskeleton. In this and other systems, it remains unclear how the signaling pathways that initiate wound closure connect to the actin regulators that help execute wound-induced cell migrations. This study shows that chickadee, which encodes the Drosophila Profilin, a protein important for actin filament recycling and cell migration during development, is required for the physiological process of larval epidermal wound closure. After injury, chickadee is transcriptionally upregulated in cells proximal to the wound. JNK, but not Pvr, mediates the increase in chic transcription through the Jun and Fos transcription factors. Finally, it was shown that chic-deficient larvae fail to form a robust actin cable along the wound edge and also fail to form normal filopodial and lamellipodial extensions into the wound gap. These results thus connect a factor that regulates actin monomer recycling to the JNK signaling pathway during wound closure. They also reveal a physiological function for an important developmental regulator of actin and begin to tease out the logic of how the wound repair response is organized (Brock, 2012).

The traditional model of the actin cytoskeleton in cell migration, based on in vitro cell culture and biochemical assays, provides a useful framework for the mechanics of how cell migration is regulated. However, there is need for in vivo studies in order to answer important questions that are not addressed by the current model: 1. Is there a role for Profilin-mediated recycling during wound-induced migration of differentiated cells in vivo? 2. Is there a role for transcriptional regulation of actin regulators during such migrations? This latter question emerges because the basic model generally assumes that migratory cells have an intact actin-regulatory apparatus that needs only to be activated to initiate and sustain migration. While this assumption may be correct for migrating cells in developmental contexts one could imagine that initially non-migratory differentiated cells may need more than their resting complement of actin regulators in order to effect long-distance migration (Brock, 2012).

Unwounded larval epidermal cells have an even distribution of actin and Profilin throughout the cytoplasm and are thought to be non-migratory. These fully differentiated epithelial cells secrete an apical cuticle and a basal lamina. They respond to the physiological signal of tissue damage by partially dedifferentiating and becoming migratory. This study shows that the leading edge cells form multiple actin-based structures including a discontinuous cable, filopodia, and lamellipodia. A working model is proposed where the basal levels of Profilin are sufficient to make actin-based structures, but wound-induced transcription of chic is required for the cells to efficiently migrate. The lack of actin-based structures at the wound edge in cells lacking Profilin would indicate that if Formin-mediated actin nucleation is involved in wound healing, it likely requires Profilin. An epidermal sheet lacking detectable Profilin fails to close wounds or form any actin-based structures at the wound edge whereas a sheet containing only a basal level of Profilin (i.e., one that is lacking proteins that transcriptionally regulate Profilin after wounding, such as JNK, Fos, or Jun) forms actin structures at the wound edge, but is ultimately unable to efficiently migrate and close the wound. This model is complicated by the fact that cells lacking JNK, Fos, or Jun also have defects in dedifferentiation, as these cells do not stop secreting cuticle following wounding. Thus, the possibility cannot completely excluded that the lack of wound closure is due to defects in dedifferentiation. However, it is entirely possible that upregulation of actin-binding regulators is an important component of the dedifferentiation process, as this involves returning to a state during which these cells were competent to migrate (Brock, 2012).

Current wound closure models have identified two signaling pathways that are important for healing. One is Pvr signaling, where the secreted VEGF-like ligand Pvf1 activates the Pvr receptor. Currently, only a few proteins are suspected of being downstream of Pvr signaling, but Profilin is not among them. Given that epidermal cells lacking Pvr are unable to mobilize actin to the wound edge, Pvr is likely upstream of actin regulatory proteins that initiate actin polymerization at the leading edge of migrating cells. The second pathway is JNK signaling, which is required for closure but not for actin polymerization at the wound edge. Naively, it was anticipated that wound-induced chic expression would be regulated by Pvr since epidermal expression of UAS-chicRNAi also blocks actin accumulation at the wound edge. Surprisingly, this is not the case. chiclacZ expression is instead regulated by JNK signaling, as it is in the developing embryo during DC . This data reveals that although the JNK signaling pathway is not required for actin nucleation at the wound edge it contributes to actin dynamics through regulating expression of chic and perhaps other genes important for migration (Brock, 2012).

How does JNK signaling activate chic transcription after wounding? Although the upstream signal for the JNK signaling pathway is still unknown, the kinase cascade is well-defined and is thought to culminate with the phosphorylation of the transcription factors, DJun and DFos. These two proteins are commonly thought to act as a dimer (AP-1) to mediate transcriptional activation of target genes. In the early DC studies chickadee expression was shown to depend on the JNK signaling pathway. This study did not address the roles of DJun and DFos in particular, although these transcription factors are required for DC. In wound healing contexts, however, it appears that DFos can act without DJun to activate a ddc-wound reporter and a msn-lacZ wound reporter. This study found that both DJun and DFos are required to activate chic. Additionally, two consensus binding sequences for the AP-1 transcription factor (TGANTCA) are located upstream of the chic start codon (depending on the message isoform the sites are located in the 5’UTR, the first intron, or the promoter region), indicating that it is at least possible that the upregulation of chic transcription is directly accomplished by Jun and Fos. The consensus sequence is also located upstream of the human Pfn1, indicating that there is potential for this regulation to be conserved. This suggests that in the migrating cells at the wound edge, DFos can act either as a homodimer, with unidentified binding partners, or with DJun to regulate the necessary transcriptional targets (Brock, 2012).

In Drosophila embryonic models of wound closure both the contractile actin cable and filopodial processes are important for wound closure, but their relative contributions are still unclear. There has been debate over whether the cable mediates closure through contraction, through serving as a platform for extension of processes into the wound gap, or through a combinaton of these functions. From the data shown in this study it seems that actin-based contraction is not a major contributor to larval wound closure. First, the actin concentrations that appear at larval wound edges are discontinuous. Second they do not appear to be locally contractile given that the cells behind prominent concentrations do not obviously taper toward the wound. This is similar to what has been observed in the embryonic Xenopus epithelium where actin cables form but differently shaped wounds do not round up as would be expected from cable contraction. Thus it would appear that in larvae the actin concentrated at the wound edge primarily facilitates process extension into the wound gap (Brock, 2012).

This study has establish a connection between a known wound-induced signaling pathway, JNK signaling, and Profilin-mediated regulation of the actin cytoskeleton. It is speculated that transcriptional induction of actin-regulators may be a general feature of cell migration in differentiated cells as suggested by a recent study of cells undergoing EMT. By connecting upstream signaling pathways to downstream actin dynamics, this work begins to unravel the logic of how the cellular movements required for wound closure are orchestrated (Brock, 2012).

Imp promotes axonal remodeling by regulating profilin mRNA during brain development

Neuronal remodeling is essential for the refinement of neuronal circuits in response to developmental cues. Although this process involves pruning or retraction of axonal projections followed by axonal regrowth and branching, how these steps are controlled is poorly understood. Drosophila mushroom body (MB) γ neurons provide a paradigm for the study of neuronal remodeling, as their larval axonal branches are pruned during metamorphosis and re-extend to form adult-specific branches. This study identified the RNA binding protein Imp as a key regulator of axonal remodeling. Imp is the sole fly member of a conserved family of proteins that bind target mRNAs to promote their subcellular targeting. Whereas Imp is dispensable for the initial growth of MB γ neuron axons, it is required for the regrowth and ramification of axonal branches that have undergone pruning. Furthermore, Imp is actively transported to axons undergoing developmental remodeling. Finally, it was demonstrated that profilin mRNA is a direct and functional target of Imp that localizes to axons and controls axonal regrowth. This study reveals that mRNA localization machineries are actively recruited to axons upon remodeling and suggests a role of mRNA transport in developmentally programmed rewiring of neuronal circuits during brain maturation (Medroni, 2014).

In cultured vertebrate neurons, ZBP1 mediates the transport of β-actin mRNA to axons, a process required for the chemiotropic response of growth cones to guidance cues. Whether these observations reflect a general requirement for ZBP1 and axonal mRNA transport during brain development has remained unclear. This study found that Imp, the Drosophila ZBP1 ortholog, accumulates in the cell bodies of a large number of neural cells in adult brain. Strikingly, Imp was additionally observed in the axonal compartment of a subpopulation of mushroom body (MB) neurons. MBs are composed of three main neuronal types (αβ, α'β', and γ) with specific axonal projection patterns and developmental programs. α'β' and αβ neurons are generated during late larval stage and early metamorphosis and are maintained until adulthood. γ neurons are born during late embryogenesis and early larval stages and undergo extensive remodeling during metamorphosis. Imp was found to be enriched in adult γ neuron axons where it colocalized with FasciclinII, but it was not detected in the axons of nonremodeling MB neurons (αβ and α'β' neurons). To test whether Imp is expressed in αβ and α'β' neurons, brains expressing GFP in γ and αβ-core neurons were labelled with antibodies against Imp and Trio, a protein specifically expressed in adult α'β' and γ neurons. Imp was not detected in αβ-core neurons but accumulated in the cell bodies of both α'β' and γ neurons. Thus, both the expression and subcellular distribution of Imp are tightly regulated in Drosophila MB neurons (Medroni, 2014).

To investigate whether Imp axonal translocation is developmentally regulated, the distribution of Imp within γ neurons was examined at different stages. In third-instar larvae, Imp accumulated exclusively in the cell bodies and was not observed in axons. During metamorphosis (pupariation), MB γ neurons first prune the distal part of their axons and then re-extend a medial branch to establish adult-specific projections. Six hours after puparium formation (APF), Imp was weakly detected in γ neuron axons. Such an axonal accumulation of Imp was visible at the time larval γ neurons have completed the pruning of their axonal processes (18 hr APF). During the subsequent intensive growth phase, Imp was enriched at the tip of axons, where it accumulated in particles. Thus, the translocation of Imp to axons is developmentally controlled, and correlates with axonal remodeling (Medroni, 2014).

To test whether Imp is required for γ axon developmental remodeling, the morphology was analyzed of adult homozygous mutant neurons generated using the MARCM (mosaic analysis with a repressible cell marker) system. Clones in which the entire progeny of a neuroblast was mutant exhibited a reduced number of cells and an altered morphology. Although wild-type adult γ axons typically span the entire medial lobe, a mixture of elongated and nonelongated axons was observed upon imp inactivation. To better visualize the morphology of mutant neurons, single labeled neurons were analyzed. Wild-type adult γ neurons extend one main axonal process that reaches the extremity of the MB medial lobe. Several secondary branches typically form along this main axonal process. In contrast, about 50% of imp γ axons failed to reach the end of the medial lobe. These defects did not result from axon retraction, as the proportion of defective axons did not increase with age. Interestingly, mutant axons of normal length but lost directionality were observed, suggesting that imp may be required for the response of γ axons to guidance cues during metamorphosis. imp mutant neurons also exhibited an overall decrease in the complexity of axonal arborization patterns characterized by a reduced number of terminal branches. Both phenotypes were significantly suppressed upon expression of a wild-type copy of Imp in γ neurons, revealing that imp acts cell autonomously to control axonal regrowth and branching (Medroni, 2014).

To determine whether Imp function in axonal growth correlates with its accumulation in axons, the requirement for Imp was investigated in two neuronal cell types where it is exclusively detected in cell bodies: larval γ neurons and α'β' neurons. Both single larval γ neurons and single adult α'β' neurons mutant for imp projected their axons normally. Furthermore, larval γ neuron neuroblast clones exhibited a normal morphology, confirming that imp is not necessary for initial axon growth. These results show that Imp is specifically required for the growth and branching of remodeling γ axons and suggest that its translocation to axons is critical for this function (Medroni, 2014).

To address whether Imp is transported actively to the axons of regrowing γ neurons, a live-imaging protocol was developed using cultured pupal brains expressing functional GFP-Imp fusions specifically in γ neurons). The culture conditions supported efficient axonal growth, as MB neurons from cultured brains grew similarly to their counterparts developing inside the pupa. Fast confocal imaging of axon bundles revealed that GFP-Imp fusions accumulated in particles undergoing bidirectional movement. In contrast, no particles could be detected upon expression of GFP alone. Motile GFP-Imp particles were distributed into three classes: particles with a strong net anterograde (56%) or retrograde (36%) movement and particles with little net bias (8%). Individually tracked particle trajectories were broken into segments to calculate velocities. Segmental velocities distributed over a wide range, with mean anterograde and retrograde segmental velocities of 0.98 ± 0.05 microm/s and 0.73 ± 0.03 microm/s, respectively. Furthermore, curves matching the graph of a quadratic function were obtained upon plotting of the mean square displacement (MSD) values over time, indicating that GFP-Imp particles undergo directed transport rather than diffusion. To assess the role of microtubules (MTs) in this process, brains were treated with colchicine. This treatment abolished MT dynamics, as revealed by the loss of EB1-GFP comets characteristic of growing MT plus ends. Strikingly, motile GFP-Imp particles were no longer observed under these conditions. These results demonstrate that Imp is a component of particles undergoing active MT-dependent transport during midpupariation, consistent with a role of Imp in the transport of selected mRNAs to regrowing γ axons (Medroni, 2014).

Previous in vitro studies have revealed that the axons of immature neurons are enriched in mRNAs encoding regulators of the actin cytoskeleton that play critical roles in axonal growth and guidance. To identify Imp mRNA targets, an immunoprecipitation RT-PCR-based screen was performed for mRNAs encoding actin regulators. Imp was found to selectively associate with chickadee (chic) mRNA, which encodes the G-actin binding protein Profilin. As revealed by affinity pull-down assays, endogenous Imp associated with the chic 3' untranslated region (UTR), but not with the chic coding sequence. To test whether Imp can interact with chic mRNA directly, the binding of recombinant MBP-Imp to the chic 3' UTR was analyzed in electrophoretic mobility shift assays. Retarded complexes formed in the presence of the chic 3' UTR, but not in the presence of a nonrelated RNA (y14). Furthermore, no significant interaction was observed when other MBP-tagged proteins were used. Notably, two discrete complexes were detected in the presence of low amounts of Imp, whereas higher-order complexes were formed with increasing amounts of Imp. Formation of these complexes was outcompeted by the addition of nonlabeled RNAs corresponding to the chic 3' UTR, but not to the chic coding sequence. Altogether, these results show that Imp associates with chic mRNA in vivo and that it can bind directly and specifically to the chic 3' UTR (Medroni, 2014).

To test whether chic mRNA localizes to the neurites of regrowing γ neurons, in situ hybridization was performed on pupal and adult brains. The poor signal-to-noise ratio obtained with this method at these stages, combined with the relatively low levels of axonally localized mRNAs, did not allow chic transcripts or reporters to be unambiguously detected in axons. Thus chic reporter constructs expressed under the control of the γ-specific 201Y-Gal4 driver was used and fluorescent in situ hybridizations was used on dissociated neurons extracted from 24 hr APF pupae and cultured for 3-4 days. chic reporter mRNAs could be observed in the neurites of γ neurons at a significantly higher frequency than control gfp mRNAs. Furthermore, chic mRNA and Imp colocalized in developing neurites, consistent with their association within mRNA transport complexes (Medroni, 2014).

To test whether the region of chic bound by Imp is required for chic mRNA localization to developing neurites, the distribution of reporters containing both the chic coding sequence and 3' UTR was compared with that of reporters lacking the chic 3' UTR. Transcripts with the chic 3' UTR localized more efficiently than transcripts lacking it, suggesting that Imp binding to the 3' UTR promotes chic axonal targeting. To exclude an effect of Imp on chic mRNA stability, the levels of chic transcripts were analyzed in cultured S2R+ cells. No significant differences in chic mRNA and Chic protein levels could be observed upon imp inactivation in these conditions (Medroni, 2014).

To functionally test the importance of chic regulation in vivo, the phenotypes associated with chic downregulation were examined. Consistent with described roles of Profilin in regulating F-actin polymerization and axonal pathfinding, it was observed that chic mutant γ neurons fail to properly extend their axons. More importantly, overexpression of chic significantly rescued the axonal growth defects observed in imp mutant neurons. Similar results were obtained with two independent UAS-chic transgenes, but not with overexpression of another regulator of F-actin polymerization (enabled). These results suggest that imp controls axonal remodeling by regulating chic expression in vivo and reveal that forced accumulation of Chic protein in axons can partially compensate for the loss of imp function (Medroni, 2014).

In conclusion, the finding that Drosophila Imp is required for γ axon regrowth but is dispensable for initial axonal growth suggests a novel and specific function of axonal mRNA targeting in developmental remodeling of the brain. Furthermore, these results highlight mechanistic similarities between developmental axonal regrowth and postinjury axonal regeneration, a process known to depend on axonal mRNA transport. Finally, this study uncovers that the translocation of Imp to γ axons is tightly linked to their developmental remodeling program. This reveals that mRNA transport machineries are subject to precise spatiotemporal regulation and may be specifically recruited in the context of developmental rewiring of the brain. It will now be interesting to identify the signals controlling the localization and the activity of mRNA transport machineries during this process (Medroni, 2014).

Protein Interactions

Drosophila Enabled (Ena) was first identified as a genetic suppressor of mutations in the Abelson tyrosine kinase and subsequently was shown to be a member of the Ena/vasodilator-stimulated phosphoprotein family of proteins. All members of this family have a conserved domain organization, bind the focal adhesion protein zyxin, and localize to focal adhesions and stress fibers. Members of this family are thought to be involved in the regulation of cytoskeleton dynamics. The Ena protein sequence has multiple poly-(L-proline) residues with similarity to both profilin and src homology 3 binding sites. Ena can bind directly to Chickadee, the Drosophila homolog of profilin. Furthermore, Ena and profilin are colocalized in spreading cultured cells. The proline-rich region of Ena is responsible for this interaction as well as for mediating binding to the src homology 3 domain of the Abelson tyrosine kinase. These data support the hypothesis that Ena provides a regulated link between signal transduction and cytoskeleton assembly in the developing Drosophila embryo (Ahern-Djamali, 1999).

To identify target proteins for the C-terminal 243 amino acids of Ena, a yeast two-hybrid screen was performed. The C-terminal 243 amino acids of Ena, which include the consensus binding site for profilin and a proline-rich consensus site for binding the Abl SH3 domain, were fused to the DNA binding domain of the yeast transcription factor GAL4 and used to screen a Drosophila third-instar larval library whose inserts were fused to the activation domain of GAL4. The separately expressed domains are unable to activate transcription of the reporter genes HIS3 and LacZ unless a protein-protein interaction takes place. Of 20.5 million clones screened, 9 interacted with Ena as assessed by expression of both the HIS and LacZ reporter genes. One of these clones carried a cDNA encoding full-length Chickadee. The interaction is specific, because a construct with the Ena N-terminal domain fused to the DNA binding domain of GAL4 does not interact with the same isolated Chickadee clone. Of the seven remaining clones, two were partial Ena cDNAs and the other five are unique sequences that are yet to be described (Ahern-Djamali, 1999).

The region of the Ena protein used as bait in the yeast two-hybrid screen contains several matches to a putative profilin binding site. To test whether these sequences are important for mediating the interaction with Chickadee, DNA encoding Ena amino acids 440-490 that contain these putative binding sites, and DNA encoding Ena amino acids 490-684 were fused to the DNA binding domain of the yeast transcription factor GAL4. Yeast were cotransformed with each of these constructs, and the chickadee cDNA was fused to the activation domain of GAL4 and tested for activation of transcription of the reporter genes HIS3 and LacZ. An interaction is detected when chickadee is cotransformed with Ena amino acids 440-490 and not Ena amino acids 490-684, suggesting that this interaction is mediated by proline-rich sequences in Ena. Ena and Chickadee have also been shown to interact in vitro (Ahern-Djamali, 1999).

Profilin has been shown to be localized to cortical microfilament webs and leading lamellae of spreading or locomoting cells. In Drosophila, profilin is expressed ubiquitously throughout development, as for example, the high levels of profilin in the ventral nerve cord of stage 16 embryos. The Ena protein is localized to actin stress fibers and focal adhesions in cultured cells and is localized to the axonal tracts of the developing Drosophila embryonic central nervous system, although the small size of these cells makes higher-resolution localization difficult. Because Ena and Chickadee interact in vitro and are expressed in the nervous system of Drosophila embryos, it was speculated that these two proteins might interact in vivo in regions of dynamic actin remodeling. The subcellular distribution of transfected Drosophila Ena and endogenous profilin were compared in cultures of spreading Ptk2 cells. Ena and profilin colocalize to the periphery of the spreading cells. The colocalization, together with the biochemical interactions, suggests that Ena and profilin associate in vivo (Ahern-Djamali, 1999).

The proline-rich region of Ena contains multiple consensus binding sites for the SH3 domain in addition to the profilin binding sequences. It has been shown with a filter binding assay that Ena binds the Abl and Src SH3 domains in vitro. The SH3 binding specificity of Ena was examined further by using a solution binding assay. Ena was expressed in Drosophila S2 cells, and the transfected cell lysates were incubated with a series of GST-SH3 fusions. Ena binds specifically to the Drosophila and murine Abl-SH3 domains and the murine src SH3 domain. Ena also bind to the C-terminal but not to the N-terminal SH3 domain of Drk. The two Ena peptides most closely matching the Abl consensus binding motif partially and specifically block Ena binding to the Abl SH3 domain, although they do not block Src SH3 domain binding. Peptides derived from Ena proline-rich sequences most closely matching the optimal sequences for Src or Drk SH3 binding do not compete for Ena binding with any of the SH3 domains tested (Ahern-Djamali, 1999).

To determine whether the proline motifs identified in the peptide binding experiment as Abl SH3 binding sites are sufficient to mediate Abl SH3 binding, site-directed mutagenesis was employed to change eight prolines to alanine, thereby eliminating many of the PXXP motifs present in the sites. Serial two-fold dilutions of transfected cell lysates containing either the mutant Ena protein (Ena8 P to A) or wild-type Ena were tested for solution binding to the Abl SH3 domain. At higher concentrations of protein, it is difficult to detect an effect of the proline-to-alanine mutations on binding. However, at lower concentrations of the Ena proteins, binding of the mutant protein is markedly reduced when compared with the wild-type Ena protein. To examine the in vivo effect of the proline-to-alanine mutations on Ena function, transgenes expressing wild-type Ena and the Ena8 P to A mutant proteins were tested for their ability to rescue ena mutant lethality. The ena8 P to A transgene rescues the embryonic lethality associated with loss-of-function mutations in ena as well as the wild-type ena transgene. The ena8 P to A-rescued flies are phenotypically normal and have viability and fertility comparable to wild-type ena-rescued flies. Thus, the proline-to-alanine mutations present in Ena8 P to A are not sufficient to disrupt an essential function of the Ena protein (Ahern-Djamali, 1999).

Interestingly, there is some overlap in the binding sites for the Abl SH3 domain and some of the putative binding sites for P to A profilin. Because mutation of the prolines in these overlapping regions reduces binding to the Abl SH3 domain, it was hypothesized that these mutations might also disrupt binding to Chickadee. The mutant ena8 P to A cDNA was subcloned in the pGex expression vector, and the resulting mutant Ena fusion protein, GST-Ena8 P to A, was compared with wild-type GST-Ena in solution binding assays for its ability to pull down Chickadee from serial dilutions of lysates prepared from adult Drosophila. The GST-Ena8 P to A fusion protein and wild-type GST-Ena pull down approximately equivalent amounts of Chickadee, suggesting that different amino acids may be important for Ena binding to profilin and the Abl-SH3 domain, despite the overlap observed in some of their putative binding sites. It is worth noting that no SH3 domain-bearing proteins were isolated in the yeast two-hybrid screen that identified profilin as a binding partner for Ena. Perhaps the proline-rich sequences present in the Ena bait are not the most important for binding to SH3 domains. Alternatively, the conditions in the yeast two-hybrid screen may not favor detection of an interaction between an SH3 domain and proline-rich sequences. Another possibility is that Ena's interaction with the Abl SH3 domain may be less physiologically relevant than its interaction with Chickadee. Indeed, mutations that disrupt binding to the Abl SH3 domain in vitro have no effect in vivo when they are expressed from a heterologous promoter. It will be important to identify critical amino acids for the interaction between chickadee and Ena and to examine whether these mutations have any in vivo effects (Ahern-Djamali, 1999).

Chickadee/profilin interacts with Cappuccino in a two-hybrid screen for proteins that bind to Cappuccino. This, together with the similarity of mutant phenotypes, suggests that profilin and Cappuccino may interact during development (Manseau, 1996).

Drosophila capulet (capt), a homolog of the adenylyl cyclase-associated protein that binds and regulates actin in yeast, associates with Abl in Drosophila cells, suggesting a functional relationship in vivo. A robust and specific genetic interaction is found between between capt and Abl at the midline choice point where the growth cone repellent Slit functions to restrict axon crossing. Genetic interactions between capt and slit support a model where Capt and Abl collaborate as part of the repellent response. Further support for this model is provided by genetic interactions that both capt and Abl display with multiple members of the Roundabout receptor family. These studies identify Capulet as part of an emerging pathway linking guidance signals to regulation of cytoskeletal dynamics and suggest that the Abl pathway mediates signals downstream of multiple Roundabout receptors (Wills, 2002).

Genetic experiments suggest that Abl interacts with a number of actin regulatory proteins to control cytoskeletal assembly. Given the functional redundancy observed between CAP and Profilin in yeast, it was thought that Capt and Profilin might participate in some form of protein complex regulated by the Abl kinase. S2 cells were transfected with full-length Drosophila Abl (dAbl), Drosophila Src64 (dSrc), or the truncated mammalian v-Abl and then Capt immunoprecipitations were assayed with anti-Profilin (Chic) and anti-actin antibodies. No significant binding of Capt and Profilin were seen in cells transfected with dSrc or v-Abl or in untransfected controls where endogenous dAbl is expressed at very low levels. However, an association of Capt with Profilin and with actin was observed when dAbl was elevated, suggesting a model where Abl, Capt, and Profilin function together in a cytoskeletal protein complex (Wills, 2002).

Active macromolecular transport between the nucleus and cytoplasm proceeds through nuclear pore complexes and is mostly mediated by transport receptors of the importin beta-superfamily. Exportin 6 (Exp6) has been identified as a novel family member from higher eukaryotes; it mediates nuclear export of profilin-actin complexes. Exp6 appears to contact primarily actin, but the interaction is greatly enhanced by the presence of profilin. Profilin thus functions not only as the nucleotide exchange factor for actin, but can also be regarded as a cofactor of actin export and hence as a suppressor of actin polymerization in the nucleus. Even though human and Drosophila Exp6 share only approximately 20% identical amino acid residues, their function in profilin-actin export is conserved. A knock-down of Drosophila Exp6 by RNA interference abolishes nuclear exclusion of actin and results in the appearance of nuclear actin paracrystals. No indications was found for a major and direct role for CRM1 in actin export from mammalian or insect nuclei (Stuven, 2003).

chickadee: Biological Overview | Evolutionary Homologs | Developmental Biology | Developmental Biology | References

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