BIOLOGICAL OVERVIEW

When first identified (reviewed by Thomas, 1997), members of the Src family of protein tyrosine kinases (Src PTKs) were determined to be transforming proteins, encoded by oncogenic retroviruses. Drosophila Src oncogene at 64B has been shown to play a role in ring canal morphogenesis during oogenesis (Dobson, 1998). Before describing the biology of Drosophila Src oncogene at 64B, here termed Src64, a digression into the complex structural and functional world of Src biology is necessary; a brief overview of this information is provided to put Src developmental effects in Drosophila into a broader context.

At present, nine distinct Src PTKs have been identified in vertebrates and two in Drosophila. The Src PTKs share a common domain structure consisting of the following features from N- to C-terminal: an amino-terminal myristylation site, a region unique to Src PTKs, SH3 and SH2 domains, the protein tyrosine kinase catalytic domain and a carboxy-terminal negative regulatory region. The three domains that follow a region unique to all Src PTKs represent modular structures found in many classes of cellular proteins: the catalytic domain possesses tyrosine-specific protein kinase activity; the SH3 and SH2 domains are protein-binding domains present in lipid kinases, protein and lipid phosphatases, cytoskeletal proteins, adaptor molecules, transcription factors, and other proteins (reviewed by Thomas, 1997).

With regard to the SH3 domains of Src PTKs in particular, they are composed of 50 amino acids. The SH3 domain is important for intra- as well as intermolecular interactions that regulate Src catalytic activity, Src localization, and recruitment of substrates. SH3 domains bind short contiguous amino acid sequences rich in proline residues. All SH3 domain ligands contain a core consensus sequence of P-X-X-P; however, amino acids surrounding the prolines confer additional affinity and specificity for individual SH3 domains. SH3 ligands can bind in either one of two orientations: NH2 COOH (Class I) or COOH NH2 (Class II). The SH3 binding pocket has two hydrophobic grooves that contact the core X-P-X-X-P sequence. A second region contacts the residues N-terminal (class I) or C-terminal (Class II) to the proline core. Proteins that have been shown to interact with Src PTK SH3 domains, either in vitro or in vivo, include p68sam, p85 phosphatidylinositol-3' kinase (PI 3-K), and paxillin (reviewed by Thomas, 1997).

The SH2 domain also controls the repertoire of proteins interacting with Src PTKs. Binding interactions mediated by the SH2 domain function in regulating the catalytic activity of Src PTKs, as well as the localization of Src or its binding proteins. All SH2 domains bind to short contiguous amino acid sequences containing phosphotyrosine, and the specificity of individual SH2 domains lies in the 3-5 residues following the phosphotyrosine (+1, +2, +3, etc). Amino acids preceding phosphotyrosine may also be important for regulating binding affinity. Structural studies on Src family SH2 domains have shown that the ligand-binding surface of SH2 domains is composed of two pockets. One pocket contacts the phosphotyrosine; the other pocket contacts the +3 amino acid residue following the phosphotyrosine. Src family kinases show a preference for leucine at this position. Proteins that have been shown to interact with the Src SH2 domain in vivo include the focal adhesion protein FAK (focal adhesion kinase), p130cas (see CAS/CSE1 segregation protein), p85 PI 3-K, and p68sam (reviewed by Thomas, 1997).

Several of the vertebrate Src PTKs are expressed in specific hematopoietic lineages where their participation in receptor-mediated signaling is required for proper development and cellular function. The more broadly expressed Src PTKs (Src, Fyn and Yes) are activated in response to growth factors (such as PDGF, EGF, FGF and CSF-1) that signal through the activation of receptor tyrosine kinases (RTKs). Consistent with a role in RTK signaling, the inhibition of Src PTKs blocks mitogenesis in response to these growth factors. In addition to the involvement of Src PTKs in receptor-mediated signaling, the inhibition of Src PTKs blocks the G2-M transition of the cell cycle in fibroblasts (reviewed by Thomas, 1997).

There are multiple ways to activate Src family kinases. These include displacement of the intramolecular interactions of the SH2 or SH3 domains by high-affinity ligands or modification of certain residues, dephosphorylation of pY527 (phosphotyrosine 527 of Src) by a tyrosine phosphatase, or phosphorylation of Y416. The SH2 domain interacts with pTyr 527 of Src and adjacent residues in the negative regulatory tail. The primary sites of tyrosine phosphorylation in vivo are Y527 in c-Src, and the corresponding tyrosine in other Src PTKs. This residue is phosphorylated by the cytoplasmic tyrosine kinase Csk. Loss of Y527 phosphorylation leads to activation of Src catalytic activity. It is thought that Csk-mediated tyrosine phosphorylation of the C-terminal tail promotes an intramolecular interaction between the SH2 domain and the phosphorylated tail, keeping the kinase in a closed, inactive conformation. Biochemical and structural studies of Src suggest that the autophosphorylation site within the catalytic domain is also important for regulation of kinase activity. Phosphorylation of analogous residues within the catalytic domain of kinases induces a conformational change that allows the kinase to assume an active conformation. This site of phosphorylation corresponds to Y416 in c-Src, the endogenous cellular Src of mammals, which is not phosphorylated in inactive wild type Src, but is constitutively phosphorylated in activated oncogenic Src mutants. Mutation of Y416 diminishes the transforming potential of both v-src (the Src oncogene of viruses) and some oncogenic variants of c-Src, suggesting that phosphorylation of this residue may be important in vivo (reviewed by Thomas, 1997).

Numerous studies suggest that the Src PTKs also function to regulate the actin cytoskeleton. Transformation of fibroblasts with activated Src PTKs causes disruptions in the actin cytoskeleton. These changes are associated with increased tyrosine phosphorylation of many cytoskeletal associated proteins. These include proteins involved in cell substrate adhesion (tensin, vinculin, talin, paxillin, FAK, beta1 integrin, p130 cas, AFAP110); cell-cell adhesion (plakoglobin, beta-catenin, p120 cas), and other proteins thought to regulate the actin cytoskeleton (p190 rhoGAP and cortactin). Src PTK participation in cytoskeletal regulation is also supported by studies of src-deficient mice. These mice suffer from osteopetrosis, a bone remodeling disorder caused by a failure of osteoclast function. Examination of the src - osteoclasts shows that they are deficient in the formation of ruffled borders and have defects in the underlying actin cytoskeleton. Studies of fibroblasts derived from mice lacking Csk, a negative regulator of Src PTKs, have provided additional evidence for Src PTK involvement in actin cytoskeleton regulation. These cells have disrupted actin cytoskeletons and increased phosphorylation of p120, FAK, paxillin, tensin and cortactin. Furthermore, the removal of src activity suppresses the cytoskeletal defects of csk - cells and returns the phosphorylation of tensin and cortactin to normal levels (Dodson, 1998 and references).

Concluding this digression into Src biological structure and function is a description of Tec29 (formerly, and more properly termed Btk family kinase at 29A), a second Drosophila non-receptor tyrosine kinase, originally cloned because of sequence homology to the kinase domain of v-src. As will be described in the protein interaction section, Tec29 interacts with Src64 in ring canal development in Drosophila oocytes (Roulier, 1998 and Guarnieri, 1998). In vertebrates (Roulier, 1998 and references), Tec family kinases interact with a variety of membrane associated and cytoplasmic proteins. For example, Tec family members can act in a cytokine-stimulated signaling pathway: Tec and Btk act downstrem of janus kinase (JAK) to link interleukin 6 signaling to PI-3 kinase. Tec family members may also link signals to the cytoskeleton, since they bind via their Src homology 3 (SH3) domains to WASP and Vav, both of which are associated with the actin cytoskeleton. Tec kinases also interact with Src family kinases. In vitro binding data indicate direct interactions between proline-rich regions of Tec family kinases and the SH3 domains of Src family members. Thus mammalian Src family kinases are directly upstream of Tec family kinases in signaling pathways. This heirarchy appears to be conserved in Drosophila (Roulier, 1998 and Guarnieri, 1998).

Drosophila Tec29 shows a dynamic expression pattern throughout development ( Gregory, 1987; Vincent, 1989; Katzen, 1990 and reviewed in Roulier, 1998). Tec29 mRNA is contributed maternally to the embryo and is first localized uniformly with refinement during gastrulation, followed by expression in segmental stripes. Subsequently, it is expressed strongly in the developing salivary primordia and invaginating salivary glands, as well as in other epithelial tissues that undergo defined cell movements, including regions of the head. During early embryogenesis, Tec29 is expressed as a 66 kDa protein isoform, p66, which is membrane-localized, while the expression in the central nervous system (CNS) beginning during germband retraction is of a smaller 55 kDA isoform, p55 (Vincent, 1989 and Wadsworth, 1990). Expression continues during larval stages in the CNS as well as in the lymph glands, the site of hemocyte production. All third instar imaginal discs express Tec29 at moderate levels, except the eye disc, in which expression is lower. Pupal expression is strongest in the lamellocytes, the differentiated hemocytes responsible for the removal of larval tissues during metamorphosis. The immune-associate expression of Tec29 in Drosophila is particularly interesting in light of its roles in immune system development in the vertebrate Tec family members. Although the cloning and expression pattern of Tec29 have been reported previously, until recently no mutations have been available for the functional analysis of this gene.

Drosophila Src64 plays a role in ring canal morphogenesis during oogenesis. The biology of Drosophila ring canals (reviewed by Dodson, 1998) will be presented briefly, before specific discussion of Src64. Drosophila oocytes develop from one of 16 germline cells (which in turn have developed from a single germline stem cell). These 16 cells are interconnected by a network of 15 cytoplasmic bridges, termed ring canals. Of the 16 cells, the one cell destined to become the oocyte is connected to four ring canals. Therefore, the developing oocyte is connected directly (via 4 ring canals) to four of the 15 germline cells, and indirectly (via the ring canal network) to the remaining germline cells. These 15 remaining cells will differentiate as nurse cells. Early in oogenesis, the developing oocyte becomes transcriptionally inactive. Thus, most of the maternal products required for early embryogenesis are synthesized in the nurse cells and transported to the oocyte through the network of ring canals. Throughout most of oogenesis, the cytoplasmic transport from the nurse cells to the oocyte is gradual. At late stages (beginning at stage 11) of oogenesis, the nurse cells contract and rapidly transfer the remainder of their cytoplasmic contents to the oocyte. Following the completion of cytoplasmic transfer, the nurse cells degenerate.

The actin cytoskeletal rearrangements that occur during ring canal morphogenesis have been extensively studied. Shortly after the arrest of the mitotic cleavage furrow, one or more of the unidentified phosphotyrosine-containing proteins localizes to the outer rim of the presumptive ring canal (Robinson, 1994). After the final mitotic divisions give rise to the 16 germ-cell cluster, an inner rim forms at the ring canals. Initially, several proteins become localized to the inner rim of the ring canal. These include a ring-canal-specific product (HTS-RC) of the hu-li tai shao (hts) gene; F-actin, and additional phosphotyrosine-containing protein(s) (Robinson, 1994). The accumulation of F-actin is dependent on hts function since, in hts mutant egg chambers, the inner rim does not form at the majority of cytoplasmic bridges and only phosphotyrosine can be detected at most ring canals (Yue, 1992; Robinson, 1994).

Subsequent to the addition of F-actin, HTS-RC and phosphotyrosine protein(s), the Kelch protein (Kelch) also becomes localized to the inner rim of the ring canal (Xue, 1993). The function of Kelch is to maintain the compaction of the ring canal rim. In late stage kelch mutant egg chambers, F-actin diffuses into the inner lumen of the ring canals and partially blocks the transfer of nurse cell cytoplasm to the oocyte. In addition to these known components of the ring canal, the product of the cheerio gene is also required for proper ring canal formation. cheerio ring canals are small and lack F-actin, HTS-RC and Kelch. Furthermore, fusions between the nurse cell and the oocyte are frequently observed in cheerio egg chambers indicating that the integrity of the plasma membrane has been compromised (Robinson, 1997). To date, the cheerio gene has not been cloned, so it is not known whether its product is a ring canal component. Once the ring canals are established, they do not remain static. The rims of newly formed ring canals have diameters of 0.5-1 mm. By stage 11, at the onset of rapid cytoplasmic transfer from the nurse cells to the oocyte, the ring canals have attained their maximum size, with a diameter of roughly 10 mm. EM studies have shown that the early phase of growth (prior to stage 5) is accompanied by the addition of new actin filaments to the ring canal. After stage 5, there is an increase in total F-actin at the ring canal, but it is unclear as to whether this increase results from the addition of new filaments or the lengthening of existing filaments. However, during this developmental period, the filaments become organized into large bundles (Tilney, 1996).

Drosophila Src64 function has been shown to be required during oogenesis for ring canal morphogenesis. Females homozygous for any of three Src64 alleles have reduced fertility. Eggs laid by Src64 females hatch at reduced frequency, when compared to wild type. In addition, Src64 females lay fewer eggs than wild type, which may suggest that the defective eggs are resorbed as has been previously observed for other female-sterile mutations. In contrast, mutant Src64 males are fully fertile. The loss of female fertility is associated with a defect in cytoplasmic transfer from the nurse cells to the developing oocyte. Unlike wild-type egg chambers, 55% of late stage Src64 egg chambers have nurse cell cytoplasm remaining at the anterior end of the oocyte. As a result, eggs from Src64 females range from 50%-100% of the length of eggs oviposited by wild-type females. Both the reduction of female fertility and the defect in cytoplasmic transfer can be reverted by excision of the downstream P-element present in the Src64 PI allele. This indicates that these phenotypes are due to disruption of the Src64 gene (Dodson, 1998).

Incomplete cytoplasmic transfer is often indicative of defects in the actin cytoskeleton of the nurse cells. Two unique cytoskeletal features are particularly important for efficient transfer. The first is the formation prior to the rapid transfer phase (stage 10B) of actin cables, which hold the nurse cell nuclei in place. In the absence of these actin cables, the nurse cell nuclei become lodged in the ring canals and block transfer. Staining of Src64 egg chambers with fluorescein-conjugated phalloidin to visualize filamentous actin at stage 10B shows that the actin cables are present. The second important cytoskeletal structure of the nurse cells are the ring canals. In order to assess the role of SRC64 in ring canal morphogenesis, early to mid-stage mutant egg chambers were stained with fluorescein phalloidin and with antibodies directed against phosphotyrosine, HTS-RC or Kelch. These experiments show that the normal complement of 15 ring canals is present and that F-actin, HTS-RC and Kelch all localize properly at the ring canals. The intensity of the staining for these components does not differ appreciably from that observed for wild-type ring canals, but the ring canal morphology appears abnormal. In contrast, there is a significantly reduced level of anti-phosphotyrosine staining in Src64 egg chambers. This reduction is particularly dramatic at the ring canals, where only faint staining can generally be observed. However, mutant ring canals that maintain elevated anti-phosphotyrosine are observed occasionally. Reduced anti-phosphotyrosine staining is also observed in the cortical regions of the mutant nurse cells. These results indicate that Src64 function is required for the majority of phosphotyrosine accumulation at ring canals, but that the formation of the ring canals and the localization of known ring canal components does not depend on this accumulation (Dodson, 1998).

During the analysis of ring canal morphogenesis, it was observed that Src64 ring canals are smaller than their wild- type counterparts. This phenotype is particularly obvious in the later stages of oogenesis. This effect was quantitated by measuring the outer ring canal diameters during both mid and late stages (stages 5 and 10A) of oogenesis. In stage 5 wild-type egg chambers, the ring canals vary in size between 2.0 and 4.5 mm, with an average size of 3.1 mm. In contrast, Src64 PI ring canals vary in size from 1.0-3.5 mm with an average size 2.6 mm.The difference in ring canal size is more apparent at stage 10A. At this stage, wild-type ring canals vary between 6 and 14 mm with an average diameter of 9.5 mm. This represents a 3.1-fold increase in the average outer diameter of wild-type ring canals between stage 5 and stage 10A. In Src64 PI egg chambers, there is only a 2.3-fold increase in ring canal diameter between stages 5 and 10A. Src64 PI ring canals range from 3-10 mm with an average of 5.9 mm. These measurements indicate that ring canal growth is defective during both early and late phases of ring canal morphogenesis. In order to assess whether these smaller ring canals have other morphological abnormalities, F-actin-, HTS-RC- and Kelch-stained ring canals were examined at high magnification. The small ring canals appear normal except for the presence of a slightly concave inner rim that is reminiscent of the inner rims of earlier stage wild-type ring canals (Dodson, 1998).

Many (45%) stage 10A Src64 PI egg chambers contain fewer than 15 ring canals. This reduction in ring canal number is only observed in stage 9-10 egg chambers, indicating that some ring canals must degenerate during these later stages of oogenesis. One possible consequence of ring canal degeneration would be fusion between cells. Evidence for such fusions was sought by staining mutant egg chambers with fluorescein phalloidin to visualize the filamentous cortical actin at the nurse cell boundaries, and propidium iodide to visualize the nurse cell nuclei. At stages 9-10 approximately 70% of Src64 PI mutant egg chambers have fusions between nurse cells (Dodson, 1998).

The altered morphogenesis and stability of Src64 ring canals and their reduced phosphotyrosine content suggested that Src64 might be a ring canal component. To test this possibility, wild-type egg chambers were stained with anti-Src64 antibodies. Specific staining is detected in the cortex of the nurse cells and appears enriched at the ring canals in wild type egg chambers. Co-staining for F-actin and Src64 shows that Src64 localization overlaps F-actin at the ring canal. These results are consistent with Src64 being localized to the nurse cell and oocyte plasma membranes, as well as to the inner rim of the ring canals (Dodson, 1998).

Src64 regulates the localization of Tec29, a Tec-family kinase required for ring canal growth. A genetic screen was carried out to search for downstream components of the Src64 signaling pathway. Mutations affecting Tec29 dominantly enhance the Src64 ring canal phenotype. Loss of Tec29 function in the female germ line results in a phenotype strikingly similar to that caused by loss of Src64 function. In each case, the ring canals are reduced in size and phosphotyrosine content. Although Src64 and Tec29 mutations have similar effects on ring canal growth, there are differences in their phenotypes that suggest that the two proteins may have nonoverlapping functions. Neither nurse cell fusion nor ring canal detachment is observed in Tec29 mutant egg chambers. This phenotypic difference suggests that Src64 may activate biochemical pathways in which Tec29 does not participate (Guarnieri, 1998). In addition, while Src64 mutations have no effect on viability, animals lacking Tec29 function do not survive to adulthood, indicating a different requirement for Tec29 and Src64 in zygotic development. The phenotype associated with Tec29 lethality is a defect in the process of head involution in which several distinct morphogenetic movements during mid to late embryogenesis normally result in the positioning of the head primordia inside the embryo. The head skeletal defects in Tec29 mutants can be explained by the idea that the correct juxtaposition of tissues in the head during and after head involution is necessary for proper head skeleton sclerotization (Roulier, 1998). The embryonic phenotype correlates with the expression of Tec29 in regions of the head, including the labial and maxillary segments and the cleft between the acron and labrum (Katzen, 1990 and Erica M. Roulier, unpublished experiments, 1998 cited in Roulier, 1998).

Previous studies have suggested that Src64 may function at other times during Drosophila development. If so, what other roles might it play? SRC64 mRNA is broadly expressed throughout development with elevated levels detectable in the central nervous system (CNS) and developing visceral mesoderm of embryos and early pupae. High levels of Src64 are also detected in the photoreceptors of third instar eye imaginal discs (Simon, 1985). Since no Src64 alleles were available prior to Dodson's 1998 report, previous attempts to examine the role of Src64 have relied on ectopic expression of a kinase inactive Src64 protein. In some cases, kinase inactive forms of Src PTKs have been shown to mimic the effects of disrupting the corresponding gene. In the case of Src64, ubiquitous overexpression of the kinase inactive protein during embryogenesis leads to defects in CNS development. During eye development, expression under sevenless transcriptional control of the kinase inactive Src64 leads to the loss of photoreceptors (Kussick et al., 1993). These results suggested that Src64 might have an important role in CNS and photoreceptor development. However, no CNS or photoreceptor defects have been detected in Src64 animals. One possible explanation for this discrepancy is that the amount of Src64 required during CNS and photoreceptor development is much lower than that required during oogenesis. Thus, the very low level of Src64 present in the mutant animals might be sufficient for these processes. Alternatively, expression of the kinase inactive Src64 may interfere with biochemical pathways in which Src64 either does not normally participate or in which Src64 function can be replaced by Src41, the second Drosophila Src PTKs (Takahashi et al., 1996). During embryonic and larval development, the expression of Src41 largely overlaps Src64, including high levels of expression in the CNS and visceral mesoderm (Takahashi, 1996). Thus Src41 may be able to compensate for a lack of Src64 in these tissues. Interestingly, the lack of maternally contributed Src41 in preblastoderm embryos suggests that the female germline is a tissue where Src41 expression may not overlap that of Src64. This difference in expression patterns may explain why the Src64 mutant phenotype is limited to defects in oogenesis. Ultimately, understanding the full range of Src64 and SrcPTK function during Drosophila development will require the identification of mutations in Src41 and any as yet unidentified Drosophila SrcPTKs (Dodson, 1998).

Toll receptors remodel epithelia by directing planar-polarized Src and PI3K activity

Toll-like receptors are essential for animal development and survival, with conserved roles in innate immunity, tissue patterning, and cell behavior. The mechanisms by which Toll receptors signal to the nucleus are well characterized, but how Toll receptors generate rapid, localized signals at the cell membrane to produce acute changes in cell polarity and behavior is not known. This study shows that Drosophila Toll receptors direct epithelial convergent extension by inducing planar-polarized patterns of Src and PI3-kinase (PI3K) activity. Toll receptors target Src activity to specific sites at the membrane, and Src recruits PI3K to the Toll-2 complex through tyrosine phosphorylation of the Toll-2 cytoplasmic domain. Reducing Src or PI3K activity disrupts planar-polarized myosin assembly, cell intercalation, and convergent extension, whereas constitutive Src activity promotes ectopic PI3K and myosin cortical localization. These results demonstrate that Toll receptors direct cell polarity and behavior by locally mobilizing Src and PI3K activity (Tamada, 2021).

Cell-surface receptors convert extracellular signals into changes in cell behavior that are essential for the formation, remodeling, and repair of multicellular tissues. Toll-like receptors (TLRs) are a conserved family of receptors with roles in dorsal-ventral patterning and cell-pathogen recognition in the innate immune system. More recently, members of the Toll-receptor family have also been shown to regulate cell behavior during the development of epithelial tissues and the nervous system. Toll receptors organize cell movement, direct nervous system wiring, repair wounds, and eliminate less fit cells through cell competition, defining new functions for this receptor family. In the immune system, Toll-like receptor (TLR) signaling activates the expression of immune-response genes by promoting the nuclear translocation of transcriptional regulators in the NF-κB, AP-1, and IRF families. However, how Toll receptors elicit fast-acting and localized responses in cells to generate spatially regulated changes in cell behavior is not understood (Tamada, 2021).

Tyrosine phosphorylation is a rapid and reversible mechanism that transduces extracellular information into molecular changes within cells. Cell-surface receptors such as growth-factor receptors initiate signaling cascades through their intrinsic tyrosine-kinase activity, which is activated by ligand binding and receptor dimerization. In addition, receptors that lack kinase activity can also participate in tyrosine-kinase signaling by recruiting nonreceptor tyrosine kinases. Src-family nonreceptor tyrosine kinases are essential for signaling by growth-factor receptors, T and B cell receptors, integrins, and cadherins, and have diverse roles in cell-cell adhesion, and cytoskeletal organization. A critical event in receptor-initiated tyrosine kinase signaling is often phosphorylation of the receptor itself, which generates binding sites that recruit downstream effector proteins. Although canonical TLR signaling involves a serine/threonine kinase cascade triggered by the recruitment of TLR-associated adaptor proteins, tyrosine kinases in the Src family have also been shown to facilitate the response to TLR signaling in cultured cells. However, the effects of Src-family kinases on Toll-receptor signaling in vivo, and whether tyrosine-kinase pathways mediate the rapid and localized effects of Toll receptors on cell behavior, are not known (Tamada, 2021).

Convergent extension is a conserved morphogenetic process that shapes developing organs and elongates the body axis of multicellular organisms. In Drosophila, three Toll-family receptors expressed in distinct striped patterns provide critical spatial cues that orient cell rearrangements and elongate the embryo along the head-to-tail axis. Toll receptors guide cell movements by directing the planar-polarized localization of proteins involved in actomyosin contractility and cell adhesion. However, the signaling pathways that generate spatially regulated changes in the organization and activity of the force-generating machinery downstream of Toll receptors are unknown. This study shows that a Src-mediated tyrosine-kinase pathway is essential for planar polarity and Toll-receptor signaling during convergent extension in Drosophila. Toll receptors generate a planar-polarized pattern of Src kinase activity, and Src in turn phosphorylates the Toll-2 C-terminal domain, promoting the association of Toll-2 with PI3-kinase (PI3K). Drosophila Src-family kinases, the regulatory and catalytic subunits of the PI3K complex, and tyrosine phosphorylation of the Toll-2 cytoplasmic domain are all necessary for planar polarity, cell intercalation, and convergent extension. These results identify a localized signaling mechanism by which Toll receptors induce rapid changes in cell polarity and behavior during development (Tamada, 2021).

Toll-family receptors are best known for their roles in signaling to the nucleus to activate the expression of immune-response genes. This study shows that Toll receptors also direct cell polarity and behavior during convergent extension by promoting the localized activities of Src and PI3K at the cell membrane. Toll receptors recruit active Src kinases to planar-polarized membrane domains, and localized Src activity generates spatially regulated actomyosin contractility by promoting the phosphorylation of two clusters of tyrosines in the Toll-2 cytoplasmic domain, creating docking sites for PI3K-reg. Src and PI3K are critical effectors of Toll-receptor signaling, as the loss of Src or PI3K recapitulates the effects of Toll-receptor mutants on planar polarity, cell intercalation, and convergent extension. Moreover, disruption of a single cluster of tyrosines in the Toll-2 C-terminal domain, which is phosphorylated in a Src-dependent fashion, inhibits Toll-2/PI3K complex formation and abolishes Toll-2 function during convergent extension. Taken together, these results identify Src and PI3K-reg as critical effectors of Toll-receptor signaling during convergent extension and reveal a noncanonical mechanism by which Toll receptors induce spatially regulated cell behavior by mobilizing two potent signaling proteins to specific subcellular domains (Tamada, 2021).

These results demonstrate that a critical symmetry-breaking event during convergent extension in Drosophila is the formation of localized Toll-2/PI3K complexes that generate tissue-wide patterns of actomyosin contractility. The upstream signals that activate Toll receptors in specific cellular domains are not known but could involve a localized extracellular ligand that increases Toll-2 binding to Src or enhances Src activity. Alternatively, mechanisms that promote Toll-receptor clustering could amplify the activity of associated Src kinases by enhancing their proximity to multiple substrates. Toll-2 and Toll-6 both interact with PI3K-reg and are tyrosine phosphorylated by Src kinases in vitro, suggesting that a Src/PI3K-signaling mechanism could link both Toll-2 and Toll-6 to PI3K. By contrast, Toll-8 is not phosphorylated by Src kinases, although it does associate weakly with PI3K-reg. Toll-8 could signal through a distinct mechanism, or it could communicate with Src and PI3K indirectly through heterodimerization with receptors such as Toll-6, which is expressed in many of the same cells. The defects in embryos lacking Src or PI3K appear to be more severe than the defects in Toll-2,6,8 mutants, suggesting that Src and PI3K may represent a common mechanism that mediates signaling downstream of multiple receptors during convergent extension (Tamada, 2021).

Tyrosine phosphorylation is a widely used signaling mechanism in multicellular organisms, with a particular enrichment of tyrosine phosphorylation at sites of contact between cells and with the extracellular matrix. Identifying the substrates required for the diverse roles of tyrosine-kinase signaling in vivo remains a formidable challenge. This study demonstrates that Src-dependent phosphorylation of a single cluster of tyrosines in the Toll-2 C-terminal domain is sufficient to eliminate Toll-2 function during convergent extension. In addition to Toll-2, Src kinases may also promote the phosphorylation of other proteins involved in Toll-receptor signaling. For example, Src-family kinases can phosphorylate Rho-kinase and recruit Rho-kinase and myosin II to focal adhesions and adherens junctions in fibroblasts and epithelial cells, and can phosphorylate and activate the Abl tyrosine kinase, which regulates planar polarized junctional dynamics during convergent extension. In addition, Src kinases can stabilize cell adhesion, enhance E-cadherin dynamics, influence cytoskeletal organization, and modulate cell polarity. The recruitment of active Src kinases to Toll-receptor complexes in Drosophila demonstrates an essential role for tyrosine-kinase pathways in signaling by Toll receptors and suggests a mechanism by which Toll receptors could trigger a wide range of cellular events that contribute to epithelial remodeling (Tamada, 2021).

PI3K is a conserved lipid kinase with widespread roles in cell growth, proliferation, survival, and polarity. This study demonstrates that PI3K directs planar polarity in the Drosophila embryo by promoting the localized assembly of contractile myosin networks in epithelial cells. These results are consistent with the altered cell topology and deregulated myosin activity observed in epithelial cells lacking the PI3K-antagonizing enzyme PTEN. The mechanisms by which PI3K influences tissue structure are not well understood. PI3K promotes the activity of the Rac GTPase in a number of cell types, but PI3K can also influence epithelial organization by activating PDK1, and PI3K can regulate the localization and activity of RhoA and Rho-kinase in fibroblasts and endothelial cells. PI3K could influence planar polarity by regulating Rho GTPase signaling, which has been linked to Toll-2 and is essential for convergent extension. The effectors that link Toll-2/Src/PI3K signaling to changes in actomyosin organization remain to be identified. These could include classical PI3K effectors that signal to the actin cytoskeleton or other proteins such as RhoGEFs that interact with the phospholipid product of PI3K, phosphatidylinositol (3,4,5)-trisphosphate (PIP3) (Tamada, 2021).

TLRs initiate signaling by recruiting adaptor proteins that assemble large multiprotein signaling complexes. The Toll-receptor signaling complexes involved in morphogenesis have not been defined. This study demonstrated that the C-terminal-most tyrosine cluster of Toll-2 is targeted by Src kinases and recruits Src and PI3K to the Toll-2 complex. This tyrosine cluster bears homology to immunoreceptor tyrosine-based activation motifs (ITAMs and hemITAMs). These motifs are present in several classes of immunoreceptors and their associated adaptor proteins and, when phosphorylated, create binding sites for downstream effector proteins. Toll-2 therefore displays features of multiple receptors in the immune system: an extracellular domain and intracellular TIR domain characteristic of TLRs and a C-terminal extension that couples to effectors through Src-family kinases and PI3K, reminiscent of other immunoreceptors such as C-type lectin-like receptors and the T cell receptor/CD3 complex. Although mammalian TLRs do not display this bipartite structure, tyrosine-rich adaptor proteins that associate with mammalian TLRs, such as BCAP and SCIMP, could create docking sites for the assembly of downstream signaling components. Toll-family receptors have conserved roles in regulating spatially regulated cell behaviors in flies and mammals, including axon growth, synapse formation, epithelial remodeling, and wound healing. An intriguing possibility is that Src and PI3K could represent a core module of a spatially regulated branch of Toll-receptor signaling that promotes the rapid and directional mobilization of cells during tissue development and immune defense (Tamada, 2021).

This study demonstrates a functional connection between Toll receptors and localized Src and PI3K activity during convergent extension in Drosophila. However, several limitations remain. First, phosphospecific antibodies to the C2 tyrosine cluster of Toll-2 could not be generated, and the other phosphospecific Toll-2 antibodies that were generated in this study worked only for western blots, but not for immunostaining. Therefore, it was not possible to analyze the spatiotemporal pattern of Toll-2 tyrosine phosphorylation in the embryo. Second, embryos were analyzed that had only a partial loss of Src42, PI3K-reg, and PI3K-cat function, due to essential roles of these proteins at earlier stages of development. Therefore, these proteins may have additional functions in this tissue that were not detected in this study. Third, focus was placed on protein localization and cell behavior in the adherens junction domain, and the results do not exclude the possibility that defects in basolateral regions of these cells or in other parts of the embryo could contribute to the phenotypes observed (Tamada, 2021).

Cullin-5 mutants reveal collective sensing of the nucleocytoplasmic ratio in Drosophila embryogenesis

In most metazoans, early embryonic development is characterized by rapid division cycles that pause before gastrulation at the midblastula transition (MBT). These cleavage divisions are accompanied by cytoskeletal rearrangements that ensure proper nuclear positioning. However, the molecular mechanisms controlling nuclear positioning are not fully elucidated. In Drosophila, early embryogenesis unfolds in a multinucleated syncytium. Nuclei rapidly move across the anterior-posterior (AP) axis at cell cycles 4-6 in a process driven by actomyosin contractility and cytoplasmic flows. In shackleton (shkl) mutants, this axial spreading is impaired. This study shows that shkl mutants carry mutations in the cullin-5 (cul-5) gene. Live imaging experiments show that Cul-5 is downstream of the cell cycle but is required for cortical actomyosin contractility. The nuclear spreading phenotype of cul-5 mutants can be rescued by reducing Src activity, suggesting that a major target of cul-5 is Src kinase. cul-5 mutants display gradients of nuclear density across the AP axis that were exploited to study cell-cycle control as a function of the N/C ratio. The N/C ratio is sensed collectively in neighborhoods of about 100 μm, and such collective sensing is required for a precise MBT, in which all the nuclei in the embryo pause their division cycle. Moreover, it was found that the response to the N/C ratio is slightly graded along the AP axis. These two features can be linked to Cdk1 dynamics. Collectively, this study revealed a new pathway controlling nuclear positioning and provides a dissection of how nuclear cycles respond to the N/C ratio (Hayden, 2022).

The tight control of the cell cycle and nuclear (cell) positioning and number is a ubiquitous feature of metazoan development and is crucial to the proper development of early embryos. This work has taken advantage of shkl mutants that have defects in nuclear spreading to identify a novel pathway involved in the control of cortical contractility and gain insights into how nuclei respond to changes in the N/C ratio. Through DNA sequencing and complementation tests, this study has identified shkl mutants as mutations of the ubiquitin ligase Cul-5. In the early embryo, Cul-5 does not regulate the cell-cycle oscillator but is required for Rho and myosin activities. Cul-5 restricts the levels of active Src kinase, which is a known regulator of the actomyosin cytoskeleton. Indeed, it was found that the cullin-5 phenotype could be largely rescued through a genetic reduction in Src activity and recapitulated through Src overexpression, indicating that a main function of Cul-5 is to downregulate Src activity. These results implicate the Cul-5/Src axis as a crucial pathway involved in the control of cortical contractility in early Drosophila embryos (Hayden, 2022).

In the early embryo, nuclei regulate their own positioning through PP1 activity that spreads from the nuclei to the cortex. This localized PP1 activity drives activation of Rho and myosin II accumulation in turn. The current results argue that Cul-5 and Src act in a pathway downstream or parallel to the cell cycle to regulate Rho activity. The molecular mechanisms by which Cul-5 and Src control Rho remain to be elucidated, as is the possible connection between the cell-cycle oscillator and Cul-5/Src activities. Since Src has been shown to regulate Rho GTPases in several contexts, these mechanisms are natural candidates for the regulation of cortical actomyosin regulation via the Cul-5/Src pathway (Hayden, 2022).

Control of the MBT by the N/C ratio is important in several species, including Drosophila and Xenopus but likely excluding zebrafish. This density of DNA (as well as nuclear size) can directly or indirectly impact multiple aspects of the MBT, namely zygotic gene expression and cell-cycle control. Previous experiments with embryos irradiated to generate different nuclear densities across the AP axis argued that nuclear cycles and zygotic activation of a large set of genes respond to the local N/C ratio. This study has exploited the changes in nuclear positioning in shkl embryos to generate a continuous range of nuclear densities. This property has lead insights into how the decision of nuclei to pause their cell cycles at the MBT is affected by the N/C ratio. The threshold for nuclear division was found to be about 70% of the density at nuclear cycle 14, which confirms previous results. This value-about halfway between the density at cycles 13 and 14-likely contributes to the robustness of the MBT. However, it is not sufficient for the robustness of the MBT. To ensure reliable lengthening of cycle 14 in all nuclei, the sensing of the N/C ratio must be averaged over hundreds of nuclei. Consistently, the results suggest that nuclei sense the local N/C ratio in neighborhoods of ~100 &mi;m. This length essentially coincides with the correlation length of the Cdk1 activity field, which is established via reaction-diffusion mechanisms. Additionally, it was found that a model based on uniform sensing of the N/C ratio fails to predict the behavior of a large fraction of nuclei. However, a model assuming a slightly higher N/C ratio threshold in the posterior is highly predictable and mainly misses the behavior of nuclei at the interface between the region of extra division and that of normal division. Thus, it is proposed that the N/C ratio is the major regulator of the cell cycle at the MBT and that no mechanism other than a slight spatial modulation of the N/C threshold is needed to account for nuclear behaviors. This spatial modulation likely reflects the fact that the rate of Cdk1 activation is also slightly graded across the AP axis. The Cdk1 activation gradient is dependent on the DNA replication checkpoint, which argues that the gradient might be controlled by an asymmetric distribution of factors controlling DNA replication and/or Chk1 activity. Alternatively, the DNA replication checkpoint and Cdk1 activity might be influenced by factors controlling AP patterning and expressed in gradients across the embryos. In the future, it will be interesting to understand the mechanisms and possible functional significance of this gradient (Hayden, 2022).

The precise coordination of biochemical and mechanical signals is a ubiquitous feature of embryonic development. In early Drosophila embryogenesis, it is necessary for the uniform positioning of nuclei and timing of the MBT. This work has identified a new pathway wherein Cul-5 regulates cortical contractility by restricting Src activity. The results investigating embryos with patchy divisions indicate that nuclei sense the N/C ratio in neighborhoods of ~100 μm and pause the cell cycle when the local density exceeds a threshold around 70% of the normal density at the MBT. Moreover, the threshold required to arrest the cell cycle is slightly graded across the AP axis and is coupled to the spatiotemporal dynamics of Cdk1. Quantitatively measuring biochemical and physical dynamics during specific morphogenic events will undoubtedly continue to reveal new insights into the mechanisms and regulations of these pathways (Hayden, 2022).

PROTEIN STRUCTURE

Amino Acids - 552

Structural Domains

In the region of the polypeptide responsible for kinase and transforming activity, the Drosophila amino acid sequence is 54% homologous with that from v-src; beginning with the tryosine-416 codon, it is 62% homologous with Drosophila Abl (Hoffman, 1983)

date revised: 18 July 2024

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