G protein ai subunit 65A: Biological Overview | References
Gene name - G protein αi subunit 65A
Cytological map position - 65D5-65D5
Function - signaling
Keywords - Asymmetric cell division
Symbol - G-iα65A
FlyBase ID: FBgn0001104
Genetic map position - 3L: 6,965,844..6,973,153 [-]
Classification - G protein alpha subunit
Cellular location - cytoplasmic
Drosophila neuroblasts divide asymmetrically by aligning their mitotic spindle with cortical cell polarity to generate distinct sibling cell types. Neuroblasts asymmetrically localize Gαi, Pins, and Mud proteins; Pins/Gαi direct cortical polarity, whereas Mud is required for spindle orientation. It is currently unknown how Gαi-Pins-Mud binding is regulated to link cortical polarity with spindle orientation. This study shows that Pins forms a "closed" state via intramolecular GoLoco-tetratricopeptide repeat (TPR) interactions, which regulate Mud binding. Biochemical, genetic, and live imaging experiments show that Gαi binds to the first of three Pins GoLoco motifs to recruit Pins to the apical cortex without "opening" Pins or recruiting Mud. However, Gαi and Mud bind cooperatively to the Pins GoLocos 2/3 and tetratricopeptide repeat domains, respectively, thereby restricting Pins-Mud interaction to the apical cortex and fixing spindle orientation. It is concluded that Pins has multiple activity states that generate cortical polarity and link it with mitotic spindle orientation (Nipper, 2007).
In complex, multicellular organisms, differentiated cell types are needed to perform diverse functions. One common mechanism for cellular differentiation is asymmetric cell division, in which the mitotic spindle is aligned with the cell polarity axis to generate molecularly distinct sibling cells. Asymmetric divisions have been proposed to regulate stem cell pool size during development, adult tissue homeostasis, and the uncontrolled proliferation observed in cancer. Thus, understanding how the mitotic spindle is coupled to the cell polarity axis is relevant to stem cell and cancer biology. This question was investigated in Drosophila neuroblasts, a model system for studying asymmetric cell division (Nipper, 2007).
Drosophila neuroblasts are stem cell-like progenitors that divide asymmetrically to produce a larger self-renewing neuroblast and a smaller ganglion mother cell (GMC) that differentiates into neurons or glia. Mitotic neuroblasts segregate factors that promote neuroblast self-renewal to their apical cortex and differentiation factors to their basal cortex. Precise alignment of the mitotic spindle with the neuroblast apical/basal polarity is required for asymmetric cell division and proper brain development: spindle misalignment leads to symmetric cell divisions that expand the neuroblast population and brain size (Nipper, 2007).
A key regulator of neuroblast cell polarity and spindle orientation is Partner of Inscuteable (Pins; LGN or mPins in mammals, GPR-1/2 in Caenorhabditis elegans). In metaphase neuroblasts, Pins is colocalized at the apical cortex with the heterotrimeric G protein subunit Gαi and the spindle-associated, coiled-coil Mushroom body defect protein (Mud; NuMA in mammals, Lin-5 in C. elegans). Pins and Gαi are interdependent for localization and for establishing cortical polarity (Yu, 2003; Schaefer, 2001). Pins also binds directly to Mud and recruits it to the apical cortex; Mud is specifically required to align the mitotic spindle with Gαi/Pins but has no apparent role in establishing cortical polarity (Nipper, 2007).
The mechanism underlying Pins regulation of cortical polarity and spindle-cortex coupling is unclear, and it is unknown how Gαi-Pins-Mud complex assembly is regulated. Pins has the potential to bind multiple Gαi·GDP molecules via three short GoLoco motifs, as do mammalian Pins homologs (Du, 2004), but the role of these multiple binding sites is unknown. Moreover, via its tetratricopeptide repeats (TPRs), Pins can bind Mud, but the stoichiometry and regulation of this interaction has not been explored. Furthermore, like its mammalian homolog LGN, the regions of Pins containing the TPRs and GoLocos interact, raising the possibility of cooperative "opening" of Pins by Gαi and Mud ligands. This study tested the role of Pins intra- and inter-molecular interactions in coupling cortical polarity with spindle orientation. Biochemistry, genetics, and in vivo live imaging were used to test the role of Pins intramolecular interactions and whether Gαi and Mud bind Pins independently, cooperatively, or antagonistically. It is concluded that Pins has multiple functional states -- a form recruited by a single Gαi to the apical cortex that is unable to bind Mud but sufficient to induce cortical polarity, and a form saturated with Gαi that recruits Mud and links cortical polarity to the mitotic spindle. The multiple Pins states are due to cooperative binding of Mud and Gαi to Pins and result in a tight link between apical cortical polarity and mitotic spindle orientation (Nipper, 2007).
The NH2-terminal half of Pins contains seven TPRs, and the COOH-terminal half contains three GoLoco motifs, which is termed here the GoLoco region, or GLR. Each of the three GoLocos has the potential to bind GDP-bound Gαi (Willard, 2004), whereas the TPRs bind the Mud protein. Before testing whether the Pins intramolecular interaction regulates Pins-Gαi-Mud complex assembly, of the relevant individual domain interactions were tested: TPR-Mud, GLR-Gαi, and TPR-GLR. (1) The Pins TPRs bind Mud with a 1:1 stoichiometry as judged by the elution profile of the TPR-Mud complex on a calibrated gel-filtration column, indicating that Pins contains a single Mud binding site. (2) Each of the three Pins GoLoco domains binds Gαi·GDP (hereafter Gαi) equally well in a qualitative pull-down assay as well as in a more quantitative assay measuring Gαi binding by using the fluorescence anisotropy of tetramethylrhodamine attached to the COOH terminus of the Pins GLR. A binding isotherm describing three equivalent, independent sites with submicromolar Gαi affinities (Kd = 530 ± 80 nM) fits the data well and yields a linear Scatchard relationship. It is concluded that each GoLoco in the Pins GLR binds Gαi with a similar affinity and without cooperativity in the absence of the TPRs, similar to a three-GoLoco region of the protein AGS3 (Adhikari, 2003). Finally, the interaction between the TPRs and GLR has an affinity of Kd = ~2 µM in trans, which may be enhanced in intact Pins because of the increase in effective concentration (Nipper, 2007).
To test whether the Pins intramolecular interaction regulates Pins-Gαi-Mud complex assembly, it was first determined whether Gαi or Mud binding disrupts TPR-GLR. Using a qualitative assay in which the TPRs and GLR are expressed as separate fragments, it was found that increasing concentrations of Gαi completely disrupt the trans TPR-GLR complex. The region of Mud that binds to Pins (Pins binding domain or PBD; contained within Mud residues 1825-1997) also disrupts the TPR-GLR complex, although not as efficiently as Gαi. Thus, Pins contains an intramolecular interaction that competes against both Gαi and Mud binding (Nipper, 2007).
Because Gαi and Mud are both coupled to the Pins intramolecular interaction, whether the two proteins bind cooperatively to Pins was tested by determining whether Gαi could enhance the affinity of Pins for Mud. 1 µM Pins binds weakly to a GST fusion of the Mud PBD. However, addition of Gαi induces a large increase in Pins binding and the formation of a Mud-Pins-Gαi ternary complex. It is concluded that Gαi increases the affinity of Pins for Mud (i.e., Gαi and Mud bind cooperatively to Pins) (Nipper, 2007).
Because Pins contains three GoLoco motifs and the Pins intramolecular interaction competes against Gαi binding, whether these Gαi binding sites are repressed equally in intact Pins was tested. Gel-filtration chromatography of full-length Pins and Gαi were used to determine how Gαi-GoLoco binding is affected by the intramolecular interaction. Pins elutes as a single peak with an elution volume consistent with the molecular weight for a monomer. Addition of low Gαi concentrations leads to formation of a 1:1 Gαi:Pins complex peak. Higher Gαi concentrations lead to the formation of a 3:1 Gαi:Pins complex with a very broad peak, suggestive of a lower affinity interaction. It is concluded that full-length Pins contains a single high-affinity Gαi-binding GoLoco and two low-affinity GoLocos (Nipper, 2007).
Because the three GoLocos are intrinsically equivalent, independent Gαi binding sites, the distinct Gαi binding behavior in full-length Pins suggests that Pins contains one GoLoco domain that is unregulated or only partially regulated by the intramolecular interaction and two GoLoco domains that are cooperatively repressed. To further explore this model, one or more GoLocos was inactivated by mutating a single critical arginine residue (Willard, 2004) to phenylalanine in the context of full-length Pins. These mutations do not inhibit the ability of the TPRs and GoLocos to interact. Inactivation of GoLoco1 (Pins δGL1; R486F) specifically abolishes the high-affinity 1:1 complex, whereas inactivation of either GoLoco 2 or 3 has no effect on the high-affinity complex. Therefore GoLoco1 is classified as a high-affinity GoLoco in the context of full-length Pins. Disruption of GoLocos 2 and 3 (Pins δGL2/3; R570F, R631F) leads to the formation of a 1:1 complex at low concentrations of Gαi, further confirming that GoLoco1 is not repressed by the TPRs. It is concluded that the three GoLoco motifs are differentially regulated by the Pins intramolecular interaction: Gαi shows unregulated high-affinity binding to GoLoco1 and low-affinity, cooperative binding to GoLocos 2 and 3 (Nipper, 2007).
It was next asked how Gαi binding to the different Pins GoLoco domains affects cooperative Gαi-Pins-Mud complex assembly. When GoLoco1 is inactivated (Pins δGL1), Gαi can still enhance Mud binding, in a manner similar to the WT Pins. The activation is more efficient, however, presumably because of the lack of Gαi "buffering" by GoLoco1. In contrast, in the Pins δGL2/3 mutant, Gαi does not enhance Mud binding even though it binds GoLoco1 with high affinity. Thus, Pins differentially regulates the ability of Gαi to promote Pins-Mud binding: Gαi binding to GoLoco1 has no effect on Pins-Mud binding, whereas Gαi binding to GoLocos 2 and 3 strongly enhances Pins-Mud association (Nipper, 2007).
These results suggest that Gαi binding to GoLocos 2 and 3 "opens" Pins to allow Mud binding to the TPRs. To directly monitor the Pins conformational transition between "closed" and "open" states, a Pins fluorescence resonance energy transfer (FRET) sensor was constructed with YFP and CFP at the NH2 and COOH termini, respectively. This type of sensor has been used successfully to monitor the conformational transition of a mammalian Pins homolog, LGN (Du, 2004). Surprisingly, addition of Gαi or Mud alone did not cause a significant change in the YFP-Pins-CFP FRET signal, even at high concentrations, suggesting that Gαi or Mud alone is insufficient to "open" Pins. The addition of both ligands together, however, leads to a large change in the FRET signal (nearly complete loss of energy transfer), indicating that Mud and Gαi are both required to induce the "open" Pins conformation. To test the model that Gαi binding to GoLoco1 cannot open Pins, a Pins δGL2/3 FRET sensor was analyzed. Mud and Gαi fail to induce the conformational change seen with the WT FRET sensor, consistent with Gαi binding at GoLoco1 not being coupled to the intramolecular interaction (Nipper, 2007).
Because Mud or Gαi alone are not able to "open" Pins, a simple model in which Mud and Gαi directly compete in a mutually exclusive fashion (e.g., sterically) with the intramolecular interaction can exclude be excluded. Although disruption of the Pins TPR-GLR interaction was observed in trans, this is likely to result from effective concentration effects in which the interaction is weaker when the two domains are not in the same polypeptide. It is concluded that Mud and Gαi allosterically modulate the TPRs and GoLocos, respectively, in a manner that leaves the intramolecular interaction intact but in a weakened state, poised to open upon binding of the second ligand. Thus, Pins can exist in a "closed" state (no Gαi or Mud bound), a "potentiated" closed state (with Gαi or Mud bound), and an "open" state (with both Gαi and Mud bound) (Nipper, 2007).
Based on the network of interactions present in Pins, Gαi binding to GoLoco1 should recruit Pins to the neuroblast apical cortex but not lead to Mud recruitment. To test this model, either HA:Pins WT or HA:Pins δGL2/3 was expressed in pins mutant neuroblasts and both Pins and Mud localization were examined. In third-instar larval central brain neuroblasts, both WT and δGL2/3 Pins localized to the apical cortex at metaphase. However, Mud was correctly recruited to the apical cortex in neuroblasts expressing WT Pins, and Mud recruitment in δGL2/3 neuroblasts was significantly reduced. Thus, Gαi binding to GoLoco1 is sufficient for Pins localization but not for efficient Mud targeting (Nipper, 2007).
To understand how cortically localized and Mud-recruiting Pins states are populated as Gαi accumulates at the apical cortex, Pins-Gαi binding was simulated based on the parameters described earlier. At low Gαi concentration, Pins with Gαi bound to GoLoco1 predominates because of its higher affinity relative to the other two GoLocos (which are repressed by the TPRs). Although this Pins form does not bind to Mud with high affinity, it was hypothesized that it is sufficient to induce aspects of cortical polarity (e.g., Insc polarization). At higher Gαi concentrations, GoLoco1 becomes saturated and binding can occur at GoLocos 2 and 3, allowing for Mud recruitment to the apical cortex. Thus, it is predictd that as Gαi accumulates at the apical cortex, it first recruits Pins in a form that is competent for cortical polarization but not spindle positioning. As Gαi levels further increase, however, GoLocos 2 and 3 become populated, weakening the intramolecular interaction and freeing the TPRs to recruit Mud to the apical cortex (Nipper, 2007).
The model that the population of Pins activation states is very sensitive to Gαi concentration was tested by examining Pins localization, Mud localization, and spindle orientation in larval neuroblasts with different levels of Gαi protein. The model strongly predicts that normal Gαi and Mud levels should "open" Pins to form a ternary complex at the apical cortex that is functional for spindle alignment, low Gαi levels would bind Pins GoLoco1 and recruit Pins to the apical cortex without allowing Mud binding or spindle orientation, and no Gαi protein would result in a failure to recruit Pins or Mud to the cortex. To test this model, larval neuroblasts were examined with normal, low, or no Gαi protein (WT zygotic mutants and maternal zygotic mutants, respectively). As expected, neuroblasts with WT levels of Gαi invariably colocalize Gαi, Pins, and Mud to an apical cortical crescent that is tightly coupled with the mitotic spindle, consistent with the activity of both Gαi and Mud "opening" Pins to form a ternary complex that is functional for spindle orientation. In contrast, neuroblasts with reduced Gαi levels formed robust Pins and Insc crescents but typically failed to localize Mud to the apical cortex and showed defects in spindle orientation. Neuroblasts lacking all Gαi protein fail to recruit Pins to the cortex and have spindle orientation defects (Yu, 2003; Schaefer, 2001). These results strongly support the model: low Gαi levels can recruit "closed" Pins to the cortex without recruiting Mud or promoting spindle orientation, whereas higher Gαi levels function together with Mud to "open" Pins and promote spindle orientation (Nipper, 2007).
To further test the model, time-lapse video microscopy was used to examine the dynamics of spindle behavior using a GFP-tagged microtubule-associated protein. In WT neuroblasts, the apical centrosome/spindle pole is anchored at the center of the Gαi/Pins/Mud crescent from prometaphase through telophase, although slight spindle rocking can be observed. In neuroblasts with reduced Gαi levels, where Gαi/Pins but not Mud are present at the apical cortex, it was found that the centrosome/spindle pole is not stably attached to the apical cortex and often shows excessive rotation. These data provide further support for the model that low levels of Gαi are sufficient to recruit Pins to the cortex via GoLoco1 binding but are insufficient to allow Pins to bind Mud and capture the apical spindle pole (Nipper, 2007).
Through interactions with Gαi and Mud, Pins regulates two fundamental aspects of asymmetric cell division: cortical polarity and alignment of the spindle with the resulting polarity axis. This study has investigated the mechanism by which Gαi regulates Pins interactions with the spindle orientation protein Mud. It was found that, although the three Pins GoLocos are intrinsically equivalent, independent Gαi binding sites, an intramolecular interaction with the Pins TPRs leads to differential Gαi binding. Gαi binding to GoLoco1 is not coupled to the Pins intramolecular interaction and therefore does not influence Mud binding but is sufficient to localize Pins to the cortex for Mud-independent functions (e.g., recruitment of Insc to the apical cortex). Gαi binding to GoLocos 2 and 3 destabilizes the Pins intramolecular interaction leading to cooperative Mud binding, and together the ligands induce an "open" Pins conformational state. This leads to a model in which Gαi induces multiple Pins activation states: one that localizes cortically but is not competent for Mud binding, and one that binds Mud linking localized Gαi to the mitotic spindle (Nipper, 2007).
Intramolecular interactions are common features of signaling proteins that typically act through "autoinhibition" of an enzymatic or ligand binding activity. Such interactions allow for coupling of regulatory molecule binding to an increase or decrease in downstream function, a critical aspect of information flow in signaling pathways. Pins is involved in the regulation of multiple downstream functions, and the results support the notion that the multiple Gαi binding sites present in Pins allow for the signal to branch into two pathways, one controlling cortical polarity and the other spindle positioning. A notable exception to the multiple GoLocos present in Pins-like proteins is the C. elegans Pins homologue GPR-1/2, which contains a single GoLoco domain. The lack of multiple GoLocos in GPR-1/2 may be consistent with their more limited role in C. elegans asymmetric cell division, where they regulate spindle positioning but not cortical polarity (Nipper, 2007).
In the model presented in this study, the Pins intramolecular interaction serves to regulate Mud binding. This may occur for several reasons. (1) Localization of Mud activity to the apical cortex appears to be important for aligning the spindle with the axis of cortical polarity. In this context, the Pins intramolecular interaction may be important for restricting Mud activity to the apical cortex. Mutant pins or Gαi neuroblasts may have low ectopic Mud activity at the basal or lateral cortex that leads to the observed misdirected spindle rotation seen in live neuroblast imaging. This observation is consistent with the previous observations that too little Mud (in mud mutant neuroblasts) results in spindle position defects without any rotation. (2) Mud activity may be affected by its interaction with Pins. For example, LGN binds to a region of NuMA near its microtubule binding site such that LGN binding to NuMA competes with microtubule binding (Nipper, 2007).
A unique feature of the Pins intramolecular interaction is that autoinhibition is incomplete. Binding of GoLocos 2 and 3 to Gαi is repressed by the TPRs, but binding to GoLoco1 is not. This has two important consequences. (1) Whereas the three GoLocos are intrinsically equivalent and independent Gαi binding sites, TPR repression of GoLocos 2 and 3 significantly lowers the affinity of these GoLocos relative to GoLoco1. This leads to preferential population of GoLoco1, which may be important for temporal regulation of asymmetric cell division by ensuring that cortical polarity is established before the spindle is positioned. (2) The TPRs appear to repress GoLocos 2 and 3 cooperatively (Gαi binding to 2 or 3 increases the affinity at the other site). Cooperativity is a common property of signaling pathways that is used generate complex input-output profiles. Pins exhibits both homotropic (Gαi) and heterotropic (Gαi and Mud) binding cooperativity. In both cases, cooperativity is not an inherent property of the binding sites but is generated through the competition that results from the intramolecular interaction between the TPRs and GoLocos. Such "cooperative repression" of inherently equivalent binding sites through intramolecular interactions may be a general mechanism for generating cooperativity in signaling proteins (Nipper, 2007).
Cellular signaling pathways exhibit complex response profiles with features such as thresholds and steep activation (i.e., ultrasensitivity). In a reconstituted mitotic spindle orientation pathway, activation of Drosophila Pins (LGN in mammals) by Gαi is ultrasensitive (apparent Hill coefficient of 3.1), such that Pins recruitment of the microtubule binding protein Mud (NuMA) occurs over a very narrow Gαi concentration range. Ultrasensitivity is required for Pins function in neuroblasts as a nonultrasensitive Pins mutant fails to robustly couple spindle position to cell polarity. Pins contains three Gαi binding GoLoco domains (GLs); Gαi binding to GL3 activates Pins, whereas GLs 1 and 2 shape the response profile. Although cooperative binding is one mechanism for generating ultrasensitivity, it was found GLs 1 and 2 act as 'decoys' that compete against activation at GL3. Many signaling proteins contain multiple protein interaction domains, and the decoy mechanism may be a common method for generating ultrasensitivity in regulatory pathways (Smith, 2011).
Complex input/output relationships generated by cell signaling networks allow for a multitude of cellular decision-making behaviors, such as bistability or hysteresis, which are necessary to implement diverse physiological processes. Ultrasensitivity is a building block for these types of behaviors, yet its molecular origins are poorly understood. While cooperativity is a well-described mechanism to generate ultrasensitivity, this study has uncovered a cellular regulatory system that uses another mechanism for obtaining sigmoidal responses with high apparent Hill coefficients (Smith, 2011).
It was found that activation of the mitotic spindle orientation protein Pins by Gαi is highly ultrasensitive, and this ultrasensitivity arises from a decoy mechanism as binding sites GLs 1 and 2 compete with the activating GL3 for the Gαi input. Cooperativity is commonly thought to be the source for ultrasensitivity in protein-protein interaction networks and protein-DNA interactions. However, the current observations of Pins activation are inconsistent with a cooperative mechanism for three reasons. First, activation of δGL1,2 occurs at a lower Gαi concentration than WT. Second, the sigmoidal response can be largely recapitulated through Gαi binding to GLs 1 and 2 in trans. Lastly, thresholding behavior is entirely dependent on the concentration of Pins present. These findings are supported by mathematical modeling and suggest that ultrasensitive responses can be generated without cooperativity from binary protein-protein interactions through a simple competition mechanism, similar to the competition that occurs in kinase signaling cascade (Smith, 2011).
Although competition and cooperativity are both potential origins of ultrasensitive responses, there are inherent differences between curves created by each of these mechanisms. Cooperativity- based ultrasensitivity can dramatically reduce the amount of input necessary to reach maximal output. For example, initial binding events of O2 are of low affinity and, without cooperativity, would require a large change in O2 concentration for saturation. The competition mechanism described in this study and in kinase cascades generates ultrasensitive responses from a threshold, as activation would occur in a graded fashion at low input concentrations without competition. Therefore, while yielding sigmoidal responses with high apparent Hill coefficients this mechanism may be more important for thresholding than the observed apparent steepness. Modeling studies have shown that multisite phosphorylation builds a good threshold, not necessarily a more switch-like response (although the Hill coefficient is often used as a measure of steepness, this single parameter is also influenced by the threshold). However, multisite phosphorylation is required for the bistable signaling nature of Xenopus oocyte maturation and cell cycle progression (Smith, 2011).
Expressing the nonultrasensitive δGL1,2 Pins failed to fully rescue the spindle positioning defect of the pinsP62 null allele relative to WT Pins, suggesting that ultrasensitive regulation of Pins is important for proper molecular function. The reduced spindle-orienting activity of the graded Pins mutant is caused by decreased pathway output because less apical Mud recruitment and spindle pole dynamics was seen relative to NBs expressing WT Pins. The δGL1,2 Pins spindle phenotype is similar to loss of Lis1 function, an adaptor protein that physically links the Gαi-Pins-Mud complex at the apical cortex to the Dynein motor protein, generating pulling forces on the spindle. Although ultrasensitivity is important for the robust spindle positioning observed in WT NBs, loss of ultrasensitivity had only a minor effect on spindle orientation, as all spindle angles measured in δGL1,2 Pins NBs were within 30° of the apical Pins crescent. This is likely because of redundant spindle-orienting cues in vivo as the mitotic spindle is not completely random in pinsP62 null NBs (Smith, 2011).
Why might ultrasensitive regulation of Pins be required for robust spindle-orienting function? In WT NBs, thresholding limits Pins output to the apical cortex where the Gαi input concentration is high. Thus, Pins is not activated at cortical sites where input concentration is low. In δGL1,2 Pins NBs, thresholding is absent such that Pins output can potentially occur both at the apical cortex and distal cortical regions. Loss of steepness results in only a slight difference in total Pins output between WT and δGL1,2 Pins in vitro (100% versus 85%), but transient activation of δGL1,2 Pins at cortical sites with low Gαi concentration could magnify this difference in vivo by titrating away Mud from the apical cortex. Thus, ultrasensitivity may be an important feature of the Gαi-Pins-Mud spindle orientation pathway, as it allows for generating maximal pathway output through spatial restriction of Pins activity. In this way, competition-based ultrasensitivity allows for increased pathway output by setting concentration thresholds to restrict signaling protein activity and may be a common theme in other regulatory pathways (Smith, 2011).
The modular architecture of signaling proteins is thought to be a means of coupling different inputs with new output functions, allowing for rapid evolution of new signaling functions. This feature is also important for creating signaling proteins that integrate multiple inputs to trigger a specific output. Protein modularity also can create new input/output relationships such as ultrasensitive responses through cooperative interactions between input domains. This analysis of Pins supports this idea but adds that modularity can shape pathway responses without cooperativity, simply by including multiple input domains. In this system it was shown that decoys can build either ultrasensitivity or thresholding depending on the affinities of the decoys relative to the activating site for the input. A high-affinity decoy sets a strong threshold, but lowering the decoy affinity can change thresholding into a more sigmoidal shaped curve, simply by blending the inflection point between thresholding and activation. This type of ultrasensitivity may be a fairly common component of cell signaling pathways, because autoinhibition and domain repeats are common features of cell signaling proteins. Thus, incorporating more domain repeats through genetic recombination events can modulate the response profile. The relative affinities of these sites could then be 'tuned' through point mutations to build thresholding behavior and/or apparent steepness into the signaling pathway (Smith, 2011).
Coupling of spindle orientation to cellular polarity is a prerequisite for epithelial asymmetric cell divisions. The current view posits that the adaptor Inscuteable (Insc) bridges between Par3 and the spindle tethering machinery assembled on NuMA-LGNGαiGDP, thus triggering apico-basal spindle orientation. The crystal structure of the Drosophila ortholog of LGN (known as Pins) in complex with Insc reveals a modular interface contributed by evolutionary conserved residues. The structure also identifies a positively charged patch of LGN binding to an invariant EPE-motif present on both Insc and NuMA (Mushroom body defect or Mud). In vitro competition assays indicate that Insc competes with NuMA for LGN binding, displaying a higher affinity, and that it is capable of opening the LGN conformational switch. The finding that Insc and NuMA are mutually exclusive interactors of LGN challenges the established model of force generators assembly, which this study revises on the basis of the newly discovered biochemical properties of the intervening components (Culurgioni, 2011).
This study reports the characterization of the PinsTPR dInscPEPT complex and provides a molecular explanation for the mutual exclusive interaction of Insc and NuMA to LGN. While this manuscript was in preparation, Zhu and coworkers arrived to similar conclusions analyzing the structure of the LGN-NuMA complex (Zhu, 2011).
A 38-residue fragment of Drosophila Insc encompasses the PinsTPR binding region. This fragment of Insc shares a high sequence similarity to functional homologues recently discovered in mammals, fully supporting the notion that the basic mechanism responsible for the recruitment of force generators at polarity sites is evolutionary conserved. With the exception of a short N-terminal α-helix, the InscPEPT is intrinsically unstructured, and lines on the scaffold provided by the superhelical TPR arrangement of Pins with an extended conformation. The interaction surface is organized around a core module involving the EPE motif of InscPEPT and the central TPR5-6 of Pins, whose specificity is primarily dictated by charge complementarity. The binding is further stabilized by polar and hydrophobic interactions contributed by the αA helix of InscPEPT. Not surprisingly, the large interaction surface characterizing the topology of the PinsTPR;InscPEPT heterotypic dimer accounts for an elevate;d binding affinity (of about 5 nM for the fly proteins and 13 nM for the human counterparts). The structure of mouse LGN191–350, corresponding to what is named TPR5-8, with Insc19–40 suggests that vertebrate proteins assemble with organizational principles similar to the fly ones. However, the short mouse constructs only depict the interaction of LGNTPR with the αA helix of InscPEPT, up to the first Glu of the EPE motif. Intriguingly, the mouse LGN;Insc interaction seems to be characterized by lower affinity compared to human and fly ones (with KD of 63 nM for LGNTPR5–8;Insc19–40, and of 47 nM for LGNTPR1–8;Insc20–57) (Zhu, 2011).
The evidence that NuMA forms a complex with the same LGNTPR domain associating to Insc raised the question of whether it binds in a similar manner. Indeed, comparison of the primary sequence of InscPEPT with the known LGN-binding portion of NuMA revealed the presence of an EPE triplet that turned out to be essential for the LGN recognition, with a similar molecular signature of the EPE motif of the InscPEPT. Notably, the NuMA ortholog in fly (Mud) codes for two consecutive EDE-EGE motifs in the Pins-binding region, whose interplay remains to be clarified. The structure of LGN in complex with NuMAPEPT fully supports the notion that the EPE-interaction module represents a common region required for docking unstructured ligands on the LGNTPR scaffold. In the case of NuMA, the interface is further contributed by a helical fragment forming a bundle with helices αA2 and αA3 of LGNTPR. The consequence of the partial overlap between the Insc and NuMA binding sites is that their concomitant loading on LGN is excluded (Zhu, 2011).
A key step during the assembly of the force generators is the opening of the LGN conformational switch that keeps the molecule in an inactive state. Binding of NuMA to LGNTPR induces the release of the intramolecular interactions holding the molecule in a closed form. In agreement with the similarity in the binding modes, it was demonstrated that also Insc disengages the LGN GoLoco motifs from the TPR domain. Together these findings imply that the GoLoco motifs contact the TPR repeats in the same region occupied by Insc and NuMA. Primary sequence inspection revealed that the GoLocos of both Pins and LGN do not contain EPE triplets, suggesting that either the head-to-tail interaction involves alternative TPR patches sterically occluded by the presence of Insc and NuMA, or that less conserved negatively charged triplets are accommodated on the same TPR5-6 of LGN (Zhu, 2011).
The well established model for force generators recruitment at polarity sites rests on the assumption that Insc and NuMA can be part of the same apically localized multisubunit complexes containing Par proteins. This model stems from colocalization experiments showing that in asymmetric mitoses Par3, Insc, LGN, and NuMA cluster together in apical crescents, complemented by coimmunoprecipitation assays in which LGN;Gαi were found in association with Par3;Insc and NuMA. The finding that Insc and NuMA are mutually exclusive partners of LGN is both unexpected and puzzling. In particular, the higher affinity characterizing the Insc binding to LGN shifts the balance of the unmodified proteins towards the Insc;LGN complex formation, which is instrumental in recruiting LGN with Par proteins at the onset of mitosis but cannot account for microtubule-pulling forces. What is the possible mechanism for transferring LGN from Insc to NuMA? The architecture of the InscPEPT;PinsTPR structure whereby an extended ligand is accommodated on a large domain allows a high degree of regulation of the interaction strength. Posttranslational modifications on either side of the dimer might locally alter the contacts without affecting the rest of the interface, as it has been demonstrated for the similarly organized complex between the cytoplasmic domain of E-cadherin and β-catenin. Such modulating modifications can in principle occur on Insc, NuMA, or on LGN. To date, no experimental information is available regarding putative Insc or NuMA modifications. More controversial is the literature relative to LGN phosphorylations. In mitotic Drosophila neuroblasts, Pins has been found phosphorylated by Aurora-A on Ser436 at about half of the linker connecting the TPR domain with the GoLoco motifs. Using an “induced polarity” assay in S2 cells, phospho-Ser436Pins was shown to trigger a redundant NuMA-independent spindle orientation pathway engaging the membrane associated Dlg protein. It is to date unclear if such pathway is conserved in vertebrates. Notably, during oriented symmetric cell divisions of MDCK cells, phosphorylation on a similarly positioned Ser401 of LGN functions in excluding force generators from the apical cortex in order to prevent apico-basal spindle orientation. In this context, phospho-Ser401LGN would selectively prevent binding of LGN to apical Gαi. Based on structural and biochemical results, it is difficult to provide a molecular explanation as to whether these LGN phosphorylations could also impact on the Insc and NuMA binding. Recent observations support the notion that the pool of NuMA;LGN;Gαi colocalizing with Par3;Insc in embryonic mouse skin progenitors is tightly regulated to set the balance between symmetric and asymmetric divisions, though no mechanism for this has been put forward. In summary, more has to be learned to understand what brings LGN from Insc to NuMA (Zhu, 2011).
An additional question relates to the mechanism maintaining effective NuMA;LGN;GαiGDP species at the correct cortical sites in the absence of Insc. Based on the knowledge acquired in this study, a step-wise model is proposed that can be schematized as follows (see Both NuMA and Insc open the LGN conformational switch): (1) in the early phases of mitosis LGN is brought to the apical membrane in conjunction with Par proteins by the high-affinity interaction with the preformed Par3;Insc complex. Binding of LGN to Insc triggers the conformational switch transition enabling the relocation of GαiGDP moieties previously distributed all around the plasma-membrane with Gβγ; (2) upon mitotic progression, when LGN is already at the correct sites, a yet unidentified molecular event alters the relative affinities of Insc and NuMA for LGN to shift the balance between the Insc-bound and the NuMA-bound LGN pools. It is hypothesized that the four Gαi subunits present on LGN at this stage are sufficient to transiently hold cortical NuMA;LGN;GαiGDP in proximity of Par proteins to allow directional microtubule pulling. It is speculated that NuMA;LGN;GαiGDP is a short-lived complex and disassemble, possibly assisted by a specialized GEF for Gαi such as as Ric-8A, releasing apo-LGN in the cytoplasm to start a new cycle. Such a dynamical interaction network would allow for a continuous regulation of the force exerted on astral microtubules throughout mitosis. Future attempts to validate the model in vivo will greatly benefit from the biochemical tools presented in this study (Zhu, 2011).
During asymmetric cell division, alignment of the mitotic spindle with the cell polarity axis ensures that the cleavage furrow separates fate determinants into distinct daughter cells. The protein Inscuteable (Insc) is thought to link cell polarity and spindle positioning in diverse systems by binding the polarity protein Bazooka (Baz; aka Par-3) and the spindle orienting protein Partner of Inscuteable (Pins; mPins or LGN in mammals). This study investigated the mechanism of spindle orientation by the Insc-Pins complex. Previously, two Pins spindle orientation pathways were defined: a complex with Mushroom body defect (Mud; NuMA in mammals) is required for full activity, whereas binding to Discs large (Dlg) is sufficient for partial activity. The current study examined the role of Inscuteable in mediating downstream Pins-mediated spindle orientation pathways. It was found that the Insc-Pins complex requires Galphai for partial activity and that the complex specifically recruits Dlg but not Mud. In vitro competition experiments revealed that Insc and Mud compete for binding to the Pins TPR motifs, while Dlg can form a ternary complex with Insc-Pins. These results suggest that Insc does not passively couple polarity and spindle orientation but preferentially inhibits the Mud pathway, while allowing the Dlg pathway to remain active. Insc-regulated complex assembly may ensure that the spindle is attached to the cortex (via Dlg) before activation of spindle pulling forces by Dynein/Dynactin (via Mud) (Mauser, 2012).
Spindle positioning is important in many physiological contexts. At a fundamental level, spindle orientation determines the placement of the resulting daughter cells in the developing tissue, which is important for correct morphogenesis and tissue organization. In other contexts, such as asymmetric cell division, spindle position ensures proper segregation of fate determinants and subsequent differentiation of daughter cells. This study examined the function of a protein thought to provide a 'passive' mark on the cortex for subsequent recruitment of the spindle orientation machinery. During neuroblast asymmetric cell division, Insc has been thought to mark the cortex based on the location of the Par polarity complex (Mauser, 2012).
Ectopic expression of Insc in cells that normally do not express the protein has revealed that it is sufficient to induce cell divisions oriented perpendicular to the tissue layer, reminiscent of neuroblast divisions. Expression of the mammalian ortholog of Inscuteable, mInsc, in epidermal progenitors has shown that this phenotype is not completely penetrant over time. Expression of mInsc leads to a transient re-orientation of mitotic spindles, in which mInsc and NuMA initially co-localize at the apical cortex. After prolonged expression, however, the epidermal progenitors return to dividing along the tissue polarity axis, a scheme in which mInsc and NuMA no longer co-localize. These results indicate that Insc and Mud can be decoupled from one another (Mauser, 2012).
This study examined the effect of Insc-Pins complex formation both in an induced polarity spindle orientation assay and in in vitro binding assays. The results indicate that Insc plays a more active role in spindle positioning than previously appreciated. Rather than passively coupling polarity and spindle positioning systems, Insc acts to regulate the activity of downstream Pins pathways. The Dlg pathway is unaffected by Inscuteable expression while the Mud pathway is inhibited by Insc binding (Mauser, 2012).
Recent work on the mammalian versions of these proteins explains the structural mechanism for competition between the Insc-Pins and Pins-Mud complexes. The binding sites on Pins for these two proteins overlap making binding mutually exclusive because of steric considerations. The observation of Insc dissociation of the Pins-Mud complex in Drosophila (this work) and mammalian proteins (LGN-NuMA) suggests that Insc regulation of Mud-binding is a highly conserved behavior (Mauser, 2012).
This competition between Mud and Insc for Pins binding is consistent with previous work done with a chimeric version of Inscuteable/Pins (Yu, 2000). This protein, in which the Pins TPR domain was replaced with the Inscuteable Ankyrin-repeat domain, bypasses the Insc-Pins recruitment step of apical complex formation. In these cells, the chimeric Insc-Pins protein was able to rescue apical/basal polarity and spindle orientation in metaphase pins mutant neuroblasts. As this protein lacks the Mud-binding TPR domain, Mud binding to Pins is not absolutely necessary for spindle alignment. Importantly, the PinsLINKER domain is still intact in the Insc-Pins fusion, implying that Dlg, not Mud, function is sufficient for partial activity, as observed in the S2 system (Mauser, 2012).
The Mud and Dlg pathways may play distinct roles in spindle positioning. The Dlg pathway, through the activity of the plus-end directed motor Khc73, may function to attach the cortex to the spindle through contacts with astral microtubules. In contrast, the Mud pathway, through the minus-end directed Dynein/Dynactin generates force to draw the centrosome towards the center of the cortical crescent. Fusion of the Pins TPR motifs, which recruit Mud, to Echinoid does not lead to spindle alignment, indicating that the Mud pathway is not sufficient for spindle alignment. The PinsLINKER domain does have partial activity on its own, however, and when placed in cis with the TPRs leads to full alignment. In this framework, the function of Insc may be temporal control, ensuring that microtubule attachment by the Dlg pathway occurs before the force generation pathway is activated (Mauser, 2012).
In the temporal model of Insc function, what might cause the transition from the Insc-Pins-Dlg complex, which mediates astral microtubule attachment, to the Mud-Pins-Dlg complex, which generates spindle pulling forces? By early prophase, Inscuteable recruits Pins and Gαi to the apical cortex. During this phase of the cell cycle, Mud is localized to the nucleus in high concentration. Apically-localized Pins binds Dlg, creating an apical target for astral microtubules. During early phases of mitosis, Inscuteable would serve to inhibit binding of low concentrations of cytoplasmic Mud to the Pins TPRs to prevent spurious activation of microtubule shortening pathways. After nuclear envelope breakdown, Mud enters the cytoplasm in greater concentrations and could then act to compete with Insc for binding to Pins, allowing Pins output to be directed into microtubule-shortening pathways (see Proposed model for Inscuteable regulation of spindle orientation). Future work will be directed towards testing additional aspects of this model (Mauser, 2012).
During asymmetric cell division, the mitotic spindle must be properly oriented to ensure the asymmetric segregation of cell fate determinants into only one of the two daughter cells. In Drosophila neuroblasts, spindle orientation requires heterotrimeric G proteins and the Gα binding partner Pins, but how the Pins-Gαi complex interacts with the mitotic spindle is unclear. This study shows that Pins binds directly to the microtubule binding protein Mud, the Drosophila homolog of Nuclear Mitotic Apparatus (NuMA) protein. Like NuMA, Mud can bind to microtubules and enhance microtubule polymerization. mud mutants form functional spindles and the neuroblasts are correctly polarized. Consistent with this, Brat and Numb form crescents in mud mutant neuroblasts, but the spindle is not aligned with them. Mitotic spindles in neuroblasts fail to align with the polarity axis. Therefore, the spindle orientation defect is a direct consequence of Mud loss of function. mud mutation can lead to symmetric segregation of the cell fate determinants Brat and Prospero, resulting in the misspecification of daughter cell fates and tumor-like overproliferation in the Drosophila nervous system. The data suggest a model in which asymmetrically localized Pins-Gαi complexes regulate spindle orientation by directly binding to Mud (Bowman, 2006; Izumi, 2006; Siller, 2006).
The role of heterotrimeric G proteins in asymmetric cell division is well studied in Drosophila. In embryonic neuroblasts, G proteins make three major contributions: (1) maintenance of the apical localization of Inscuteable and the Par complex, (2) regulation of spindle orientation at metaphase, and (3) generation of spindle asymmetry at anaphase. It is thought that both free Gβγ and Pins-Gαi, as well as Par complex members Baz and aPKC, have a role to play in the control of spindle asymmetry. Whether G proteins can directly regulate spindle orientation is less clear because of the complexity of G protein phenotypes. Misregulation of G proteins can cause Insc and Par complex delocalization as well as spindle orientation defects. As a result, it is difficult to determine whether it is actually G proteins that are responsible for spindle misorientation, or whether the orientation defect is a secondary consequence of a general loss of polarity. mud mutants, however, show spindle misorientation without Insc or Par delocalization. Since Mud binds to Pins and localizes asymmetrically in neuroblasts, this suggests that Pins-Gαi regulates spindle orientation through its interaction with Mud (Bowman, 2006).
In vertebrates, the Pins-Gαi complex is proposed to control the attachment of astral microtubules to the cortex through its interaction with NuMA. This model of spindle positioning is supported by an experiment in which overexpressed Pins causes spindle rocking movements that can be inhibited by coexpressing a short fragment of NuMA or disrupting astral microtubules with low concentrations of nocodazole. In Drosophila, astral microtubules are also important for spindle positioning. Mutations in centrosomin and asterless prevent the formation of centrosomes and astral microtubules, and neuroblasts in these mutant backgrounds often fail to coordinate the mitotic spindle with the crescent of cell fate determinants at metaphase. Abolishing astral microtubules pharmacologically produces similar results. It is proposed that Mud forms a complex with Pins and Gαi that regulates the attachment of astral microtubules to the cortex, and that this regulation is necessary for the mitotic spindle to assume the correct orientation in asymmetric cell division. In mud mutants, faulty microtubule-cortical attachment results in a failure to coordinate the mitotic spindle with the axis of polarity. Accordingly, the spindle assumes orientations that do not align with the crescents of Insc and Miranda, and regulators of cell size as well as cell fate determinants can be inherited symmetrically (Bowman, 2006).
The identification of Mud and LIN-5 as NuMA homologs indicates that three different model organisms use NuMA-like proteins to regulate spindle movements. During the first division of the C. elegans zygote, the mitotic spindle is set up along the A/P axis in the center of the cell. In anaphase, the spindle rocks vigorously as the posterior centrosome is displaced toward the posterior cortex. Following this division, mitosis begins in the daughter cells, which initially align their centrosomes transverse to the A/P axis. However, the spindle in the posterior cell eventually rotates 90° and orients along the A/P axis. These spindle rocking and displacement movements require the NuMA-like protein LIN-5. Because LIN-5 is found in a complex with the Pins-like GoLoco motif proteins GPR-1 and GPR-2, and because the phenotype of GPR-1/-2 loss of function is nearly identical to that of LIN-5, it is thought that LIN-5 and GPR-1/-2 act together to generate the forces required for spindle rocking and spindle orientation in mitosis (Bowman, 2006).
In rodents, NuMA, mammalian Inscuteable (mInsc), and G proteins regulate spindle orientation in the asymmetric division of self-renewing stem cells. Epidermal stem cells localize mInsc, NuMA, and Pins to an apical crescent and align the spindle parallel to the apical-basal axis. If apical localization of Pins and NuMA is disrupted, spindle orientation becomes randomized. In the developing neocortex, neural progenitors divide with their spindles orthogonal to the apical-basal axis for symmetric divisions and parallel to this axis for asymmetric divisions. Reliable coordination of the spindle with the apical-basal axis during asymmetric division requires mInsc, free Gβγ, and the Pins-like protein AGS3. If the function of any of these proteins is compromised, asymmetric divisions fail because of misoriented spindles. Furthermore, NuMA and Pins can create spindle-rocking movements during mitotis. This work shows that the NuMA-like protein Mud forms a complex with Pins and Gαi and is required for spindle orientation in asymmetrically dividing Drosophila neuroblasts. Taken together, these studies strongly suggest that asymmetric cell divisions in C. elegans, Drosophila, and vertebrates all use NuMA-Pins-Gαi complexes to regulate spindle orientation (Bowman, 2006).
In mud mutants, failure of asymmetric division leads to an expansion of the neuroblast pool. This places mud with lgl and brat in a class of genes in which zygotic loss of function produces ectopic neuroblasts. Because of the interaction of Pins with Mud, pins mutants could also be expected to have defective spindle orientation and symmetric divisions that produce two neuroblasts. Surprisingly, pins mutant neuroblasts do not overproliferate. In fact, they exhibit a mild underproliferation phenotype (Bowman, 2006).
How can the difference in the proliferative behavior of mud and pins mutant neuroblasts be explained? (1) The possibility that in addition to regulating spindle orientation, Mud directly inhibits proliferation by an unknown mechanism cannot be excluded. Since the overproliferation in mud mutants is mild compared to that in lgl or brat mutants, this seems unlikely. (2) Pins could be acting redundantly with Loco to regulate spindle orientation, so a potential pins mutant overproliferation is masked by the presence of Loco. Since Mud-C does not bind to Loco under the same conditions with which it binds to Pins, the notion that Loco substitutes for Pins by interacting with Mud is questionable. Alternatively, the proliferative differences could be explained by the localization of aPKC. A recent study in larval neuroblasts suggests that inheritance of cortical aPKC can confer the ability to self-renew. Since work in embryos has shown that Pins is required to maintain the apical localization of the Par complex, it follows that in pins mutant brains, aPKC localizes weakly to the cortex and cytoplasm of metaphase neuroblasts. By contrast, aPKC forms a cortical crescent in mud mutants. In this model, pins mutant daughter cells inheriting cytoplasmic aPKC are more likely to exit the cell cycle, while, in mud mutants, the daughter cells inheriting cortical aPKC continue to proliferate as neuroblasts. The data neither prove nor disprove this hypothesis (Bowman, 2006).
Asymmetric division of Drosophila neuroblasts (NBs) and the C. elegans zygote uses polarity cues provided by the Par proteins, as well as heterotrimeric G-protein-signalling that is activated by a receptor-independent mechanism mediated by GoLoco/GPR motif proteins. Another key component of this non-canonical G-protein activation mechanism is a non-receptor guanine nucleotide-exchange factor (GEF) for Galpha, RIC-8, which has recently been characterized in C. elegans and in mammals. The Drosophila Ric-8 homologue is required for asymmetric division of both NBs and pI cells. Ric-8 is necessary for membrane targeting of Galphai, Pins and Gbeta13F, presumably by regulating multiple Galpha subunit(s). Ric-8 forms an in vivo complex with Galphai and interacts preferentially with GDP-Galphai, which is consistent with Ric-8 acting as a GEF for Galphai. Ric-8 complexes with Pins through their mutual interactions with Galpha. Comparisons of the phenotypes of Galphai, Ric-8, Gbeta13F single and Ric-8;Gbeta13F double loss-of-function mutants indicate that, in NBs, Ric-8 positively regulates Galphai activity. In addition, Gbetagamma acts to restrict Galphai (and GoLoco proteins) to the apical cortex, where Galphai (and Pins) can mediate asymmetric spindle geometry (Wang, 2005).
In neuroblasts (NBs), two apically localized protein cassettes -- 1. Bazooka, Par3-DmPar6-DaPKC0 and 2. Galpha-Partner of Inscuteable [Pins, a GDP dissociation inhibitor (GDI) of Galpha], that are linked by Inscuteable (Insc) -- mediate all aspects of NB asymmetric division. These two conserved protein cassettes are spatially separated in pI cells of the sensory organ precursor (SOP) lineage: Pins-Galpha localizes to the anterior, whereas Baz-Par-6-DaPKC localizes to the posterior cortex. In both Drosophila and C. elegans asymmetry models, the activation of heterotrimeric G-protein signalling apparently occurs via a receptor-independent mechanism that is mediated by proteins containing GoLoco/GPR (G-protein regulatory) motifs with GDI activity (for example, Drosophila Pins and nematode GPR1/2), which can compete with Gbetagamma for GDP-Galpha. With respect to the spindle geometry of Drosophila NBs, Gbeta13FGgamma1 seems to have a more crucial role than Galpha and Pins in this process. By contrast, Galpha subunits, GOA-1 and GPA-16, and the GoLoco proteins GPR1/2 are essential in C. elegans, for the generation of a net posterior force that is necessary for asymmetric spindle positioning. Gbetagamma, in contrast, does not play a positive role in this process. More recently, RIC-8, a novel non-receptor guanine nucleotide-exchange factor (GEF) for Galpha, has been shown to be required for asymmetric spindle positioning in the C. elegans zygote. This study characterizes the role of the Drosophila Ric-8 homologue in neural progenitor asymmetric division (Wang, 2005).
Database searches of rat Ric-8A identified a putative Drosophila homologue, Ric-8 (CG15797, at cytological position 8D10 of the X chromosome), which shares ~31% amino-acid identity with rat Ric-8A. Ric-8 RNA is ubiquitously expressed with an abundant maternal component. In glutathione S-transferase (GST) pull-down assays, GST-Ric-8 interacts directly with Galpha in vitro. In co-immunoprecipitation experiments using embryonic extracts, Ric-8, similarly to Pins and Gbeta13F, interacts strongly with Galpha when GDP has been added in excess, but interacts poorly with Galpha in the presence of excess GTP-gammaS. This indicates that Ric-8 preferentially interacts with GDP-Galpha. These interactions are consistent with Ric-8 acting as a GEF for Galpha, similarly to its mammalian and nematode homologues (Wang, 2005).
To ascertain that the in vitro binding of Ric-8 with Galpha reflects an in vivo association, co-immunoprecipitation experiments were performed using embryonic extracts. Ric-8 was detected in immunocomplexes when precipitation was performed with anti-Galpha but not with the pre-immune control, indicating that Ric-8 complexes with Galpha in vivo. To further substantiate this interaction using a different approach, protein extracts from wild-type embryos were incubated with agarose beads coupled to bacterially expressed MBP-Galpha or MBP protein. Ric-8 was detected in the bound complex with MBP-Galpha but not MBP (Wang, 2005).
In Drosophila NBs, Galpha is present in at least two mutually exclusive complexes: a heterotrimeric complex with Gbeta13F, or with a GoLoco-containing protein, Pins, which acts as a GDI for, and can directly interact with Galphai. Conventional G-protein-coupled receptors (GPCRs) promote nucleotide exchange on the Galphai-Gbetagamma heterotrimeric complex, whereas the mammalian non-receptor GEF RIC-8A cannot act on the heterotrimer. To explore the molecular context in which Ric-8 might act on Galpha, whether Ric-8 can complex with Pins or Gbeta13F was examined in Drosophila using co-immunoprecipitation experiments with embryonic extracts. When precipitations were performed using anti-Ric-8, Pins was specifically detected in the immunocomplex; in precipitations using anti-Pins, Ric-8 was also specifically detected. No direct interaction was observed with Ric-8 and Pins in the in vitro binding assays, indicating that Ric-8 complexes with Pins through their mutual interactions with Galpha. To confirm these findings using a different approach, wild-type embryonic extracts were incubated with agarose beads coupled to bacterially expressed MBP-Ric-8 fusion protein. Pins but not Gbeta13F was found in the bound complex with MBP-Ric-8. Thus, Ric-8 preferentially binds to the GDP-Galpha-Pins complex, a similar finding to that seen in C. elegans embryos. This is in contrast to conventional GPCRs, which act on the heterotrimeric complex (Wang, 2005).
To determine the effects of ric-8 loss of function, several mutant alleles were isolated by imprecise excision of a P-element, EY05996. ric-8P587 removes the entire coding region (-953 bp to +1853 bp; ric-8 transcriptional start is +1), whereas ric-8P340 contains a larger deletion with unsequenced breakpoints. Both maternal and zygotic components were removed in the ric-8P340 and ric-8P587 germline clones (GLCs). These mutant embryos showed indistinguishable phenotypes, indicating that both are null alleles. Experiments were performed using embryos that were derived from ric-8P587 GLCs (Wang, 2005).
Galpha shows punctated, cytosolic distribution in dividing and non-dividing NBs of ric-8 GLCs, in contrast to the apical cortical crescents seen in wild-type NBs. Pins also seemed to be cytosolic, which is consistent with findings that Galpha is required for the recruitment of Pins to the cortex. The issue of whether Gbeta13F is also required for membrane targeting of Galpha was examined using a newly generated anti-Galpha antibody, as it was unclear whether the reported inability to detect Galpha in Gbeta13F mutant NBs by immunofluorescence was due to low sensitivity of the previously available antibody. The specificity of this new antibody was demonstrated by the absence of immunoreactivity in Galphai mutant embryos or nota in both immunofluorescence and Western experiment. It was found that Galpha was uniformly localized on the cortex of Gbeta13F GLC NBs, with clearly reduced intensity compared with the wild type. Pins was also uniformly cortical in Gbeta13F GLC NBs, which indicates that the residual Galpha on the cortex is sufficient to recruit Pins. The localization of Galpha and Pins in blastoderm embryos that were derived from ric-8 and Gbeta13F GLCs lends further support to these findings. Strikingly, Galpha and Pins localized as punctated, cytosolic 'spots' in ric-8 GLC embryos, whereas in both wild-type and Gbeta13F GLC embryos, Galpha was membrane associated. Therefore, ric-8, but not Gbeta13F, is crucial for the membrane targeting of Galpha in NBs and other cell types (Wang, 2005).
In ric-8 GLC NBs, Insc was cytosolic. Baz and aPKC localized non-uniformly/asymmetrically on the cortex, but with reduced intensity and often as broader crescents, indicating that residual polarity cues remained. Mira crescents were often mislocalized in metaphase ric-8 NBs; mitotic domain 9 cells failed to re-orient their spindle by 90°, indicating that ric-8 is required for spindle re-orientation in cells of mitotic domain 9. These defects are similar to those seen in Galphai mutant NBs. Ric-8 is also required for the asymmetric division of pI cells. In ric-8 mutant metaphase pI cells, Galpha and Pins did not form the anterior cortical crescents. Similarly, in Galphai metaphase pI cells, the anterior crescent of Pins did not form. In both ric-8 and Galpha mutants, the Pon crescent was undetectable or significantly reduced. Nevertheless, Pon localized at the anterior cortex in anaphase pI cells of both mutants (Wang, 2005).
Antibodies specific for Ric-8 were generated against the amino-terminal (aa 1-150) or carboxy-terminal (aa 425-573) region of Ric-8. Ric-8 was localized to the cytoplasm of NBs throughout the cell cycle, even though Galpha was seen as an apical crescent in mitotic NBs. However, interestingly, Ric-8 was also observed as 'spot'-like structures at the apical cortex of metaphase NBs, partially colocalizing with the Galpha, indicating that their interaction might occur on the cytosolic face of the plasma membrane or in the cytoplasm. Similarly, in pI cells, Ric-8 was also cytosolic throughout the cell cycle (Wang, 2005).
ric-8 GLCs also exhibit abnormal gastrulation, in addition to defects in asymmetric divisions. Since gastrulation defects were also seen in Gbeta13F and Ggamma1 GLC embryos but not in Galphai embryos, the relationship was examined between ric-8 and Gbeta13F. During cellular blastoderm formation, Gbeta13F is delocalized from the cortex and is largely cytosolic in ric-8 GLC embryos, indicating that ric-8 is required for cortical localization of Gbeta13F during these early stages. Consistently, Gbeta13F is also largely cytosolic in NBs throughout the various stages of the cell cycle in stage-10 embryos derived from ric-8 GLCs. Given that Galphai loss of function alone does not disturb Gbeta13F localization and Gbeta13F does not complex with Ric-8, it was hypothesized that Ric-8 mediates the cortical localization of Gbeta13F through its regulation of another Galpha subunit. To further explore this possibility, it was asked whether Ric-8 can complex with Pins in embryos devoid of maternal and zygotic Galphai. If there was another Galpha subunit involved, it might allow Ric-8 to complex with Pins by interacting with both, even in the absence of Galpha. Indeed, Ric-8 complexes with Pins in the absence of Galpha. Given that Ric-8 does not display a direct interaction with Pins, these data indicate that an, as yet unidentified, Galpha subunit that is also regulated by Ric-8 may act (possibly in conjunction with Galpha) to mediate Gbeta13F cortical localization (Wang, 2005).
Gbeta13F protein levels in ric-8 GLCs are significantly reduced compared with wild-type embryos; Galpha and Pins levels remain unaffected. By contrast, Galpha protein levels in Gbeta13F GLCs are reduced, whereas Ric-8 levels do not change in Galpha or Gbeta13F GLCs. Gbetagamma might normally be in excess; therefore, despite the reduction in Gbetagamma levels in ric-8 mutants, sufficient cytosolic levels may remain to stabilize normal levels of Galpha. These data indicate that Ric-8 is required only for membrane targeting of Galpha but not its stability; Gbeta13F is required for the stability of Galpha but not for its membrane targeting. In addition, Ric-8 is involved in both membrane association and the stability of Gbeta13F, possibly by acting through another Galpha subunit (Wang, 2005).
The requirement of Ric-8 for cortical localization and stability of Gbeta13F prompted an examination of whether NB spindle geometry and difference in daughter-cell size are severely disrupted in ric-8 mutants, as shown for Gbeta13F GLCs. In telophase NBs of wild-type stage-10 embryos, the ratio of ganglion mother cell (GMC) and NB (GMC/NB) diameter never exceeded 0.8 (average ratio = 0.42. By contrast, a hallmark of Gbeta13F or Ggamma1 loss is the high frequency of divisions that generate daughters of approximately equal size. These cells are telophase NBs in which the GMC diameter/NB diameter ratio was 0.8 or more (for Gbeta13F NBs, 64% of divisions were similar sized with an average GMC/NB ratio of 0.82. The residual size asymmetry which remained was shown to be due to the reduced levels of asymmetrically localized Par proteins. However, a surprising observation was that, although cortical Gbeta13F localization was disrupted in ric-8 mutant NBs, only 16% of telophase NBs divided into two similar-sized daughter cells, similar to those observed in Galphai mutant NBs. Thus, ric-8 GLC NBs did not display a phenotype similar to that of Gbeta13F loss-of-function mutants. Further removal of Baz (by RNA interference) in ric-8 GLCs resulted in similar-sized division in 94% of NBs, indicating that partially localized Baz (Par proteins) can provide some asymmetry cues in ric-8 mutant NBs. Therefore, Ric-8 probably acts in the same pathway as Galpha to redundantly regulate the difference in daughter-cell size in the Baz pathway. It was shown previously that in Gbeta13F mutants, the number of abdominal Even-skipped positive lateral (EL) neurons in stage-15 embryos was severely decreased, presumably because a high frequency of similar-sized divisions rapidly reduces the cell volume of daughter NBs, resulting in early cessation of divisions. It was found that wild-type embryos produced an average of 9.0 EL neurons per abdominal hemisegment at stage 15; both ric-8 GLCs and Galphai mutants showed a similar reduction of EL neurons. By contrast, Gbeta13F GLC embryos showed a more marked reduction in the numbers of EL neurons. These data indicate that, with respect to both numbers of EL neurons and NB daughter-cell size asymmetry, ric-8 and Galpha mutants exhibit similar phenotypes that are less severe than those seen in Gbeta13F mutants (Wang, 2005).
Two alternative explanations are envisioned for why ric-8 and Gbeta13F mutants have different effects on the asymmetric size of the daughter cells. (1) The generation of functional Gbetagamma may occur even in the absence of ric-8 function, despite the majority of the molecules being cytosolic. (2) Alternatively, the severe phenotypes seen in Gbeta13F or G gamma1 mutant NBs may be an indirect consequence caused by the uniform cortical distribution of Galpha (and Pins); the failure of ric-8 GLC NBs to exhibit a marked decrease in asymmetric daughter size would be because Galpha and Pins are both cytosolic in ric-8 mutants and presumably inactive. To distinguish between these possibilities, ric-8, Gbeta13F double mutant GLC embryos were made in which both ric-8 and Gbeta13F would be completely removed. Interestingly, the double mutant GLC NBs exhibited phenotypes similar to those of ric-8 GLC NBs rather than Gbeta13F GLC NBs. In double GLC NBs, Galpha and Pins are cytosolic, whereas Baz localized non-uniformly/asymmetrically on the cortex. Only 24% of NBs divided into two similar-sized daughter cells. These observations indicate that the cytoplasmic Gbetagamma in ric-8 GLC NBs is non-functional and further suggests that the marked decrease in the difference in daughter-cell size of Gbeta13F GLC NBs is an indirect consequence of the uniform cortical localization of Galpha (and Pins) (Wang, 2005).
These data indicate that ric-8 mutants mediate asymmetric division of NBs and SOPs by regulating heterotrimeric G-protein localization. ric-8 acts at the top of a hierarchy for the sequential membrane/cortical localization of the apical proteins Galphai-Pins-Insc. The role of Ric-8 in membrane targeting of Galpha is novel. Interestingly, Ric-8 also promotes cortical localization of Gbeta13F in Drosophila. These data raise the possibility that this may be mediated indirectly by additional substrate(s) of Ric-8, which are presumably additional Galpha subunit(s). Rat Ric-8A interacts with multiple brain membrane Galpha subunits, including Galpha13, Galphao, Galphaq and Galpha1,2. It is therefore speculated that Ric-8 may control the localization and stability of Gbeta13F by regulating multiple Galpha subunits. Precedence for a role of Galpha in Gbetagamma membrane localization has been reported in mammalian cells (Wang, 2005).
This analyses of ric-8, Galphai, Gbeta and ric-8;Gbeta mutants support the view that, in NBs, cortically localized Galpha mediates asymmetric spindle geometry and asymmetric daughter-cell size, which is positively regulated by Ric-8, and that an important role of Gbetagamma is to restrict Galpha from the basal cortex. In the absence of Gbetagamma, the GoLoco/Galpha complex expands from its normal apical localization, becomes uniformly cortical and can largely override the residual polarity cues that are provided by the asymmetrically localized, but drastically reduced levels of, Par proteins to greatly reduce spindle asymmetry and the difference in daughter size. The residual asymmetry that is present in the absence of Gbeta13F is lost following further removal of Par function. The negative regulation of Galphai by Gbeta13F in Drosophila NBs is similar to that in the C. elegans zygote, in which excess Galpha activity was observed following loss of function of Gbeta or Ggamma. The findings that ric-8 mutants are genetically epistatic to Gbeta mutants, both with respect to Galphai-Pins localization and to spindle geometry, are different from those reported in C. elegans embryos, in which inactivation of Gbetagamma alleviates the requirement for RIC-8 in asymmetric division. This indicates that different mechanisms of heterotrimeric G-protein regulation are present in the asymmetric division of nematode embryos and Drosophila NBs. These findings are consistent with a model in which Ric-8 has a crucial role in Galpha activity by localizing the GoLoco/Galpha complex onto the cortex and/or generating GTP-Galpha as a GEF to mediate spindle geometry. Ric-8 also regulates the cortical localization and activity of Gbeta, possibly through its regulation of multiple Galpha subunits; Gbeta acts to restrict Galpha localization only to the apical cortex. Galpha subunits that are asymmetrically localized at the apical cortex, in conjunction with Par proteins, mediate asymmetric spindle geometry and differences in daughter-cell size (Wang, 2005).
Cortical polarity regulates cell division, migration, and differentiation. Microtubules induce cortical polarity in yeast, but few examples are known in metazoans. Astral microtubules, kinesin Khc-73, and Discs large (Dlg) induce cortical polarization of Pins/Gαi in Drosophila neuroblasts; this cortical domain is functional for generating spindle asymmetry, daughter-cell-size asymmetry, and distinct sibling fates. Khc-73 localizes to astral microtubule plus ends, and Dlg/Khc-73 and Dlg/Pins coimmunoprecipitate, suggesting that microtubules induce Pins/Gαi cortical polarity through Dlg/Khc-73 interactions. The microtubule/Khc-73/Dlg pathway acts in parallel to the well-characterized Inscuteable/Par pathway, but each provides unique spatial and temporal information: The Inscuteable/Par pathway initiates at prophase to coordinate neuroblast cortical polarity with CNS tissue polarity, whereas the microtubule/Khc-73/Dlg pathway functions at metaphase to coordinate neuroblast cortical polarity with the mitotic spindle axis. These results identify a role for microtubules in polarizing the neuroblast cortex, a fundamental step for generating cell diversity through asymmetric cell division (Siegrist, 2005).
A current model for the establishment of neuroblast cortical polarity is that an unknown cue recruits Baz, aPKC, Par-6, and Insc to the apical cortex of the neuroblast just prior to prophase, which is closely followed by the apical recruitment of Pins/Gαi proteins, presumably via Insc-Pins direct interactions. This is termed the cortical 'Insc/Par pathway' of Pins/Gαi localization to distinguish it from the Insc-independent 'microtubule-based pathway' of Pins/Gαi localization that is the focus of this paper (Siegrist, 2005).
insc22 null mutant embryos (insc mutants) lack apical localization of the Insc/Par complex proteins (Insc, Baz, aPKC, and Par-6), but interestingly that Pins, Gαi, and Dlg still form robust crescents in the majority of insc mutant metaphase neuroblasts. Similar results were observed in mitotic neuroblasts from embryos homozygous for the TE35 deficiency in which insc is not transcribed. Although Pins/Gαi/Dlg crescents form in insc mutants, the timing and position of crescent formation differed from wild-type: (1) in wild-type neuroblasts Pins/Gαi/Dlg crescents always formed at the apical surface adjacent to the overlying ectoderm, whereas in insc mutant neuroblasts Pins/Gαi/Dlg crescents were found at all positions around the cortex; (2) in wild-type neuroblasts Pins/Gαi crescents formed by early prophase (94%), whereas in insc mutants Pins/Gαi crescents were not detected at prophase but only at metaphase (78%). These results suggest that there is an Insc/Par-independent pathway that is active at metaphase to induce formation of Pins/Gαi/Dlg cortical crescents (Siegrist, 2005).
A clue to the identity of the Insc/Par-independent pathway was the observation that Pins/Gαi/Dlg crescents were always colocalized over one spindle pole, which can be mispositioned relative to the overlying ectoderm in insc mutants. This observation suggested that either spindle microtubules induced cortical polarity, or cortical polarity formed spontaneously at a nonapical position and induced spindle alignment. To distinguish between these mechanisms, microtubules were depolymerized in insc mutant neuroblasts with Colcemid, and Pins/Gαi/Dlg cortical crescents were scored. Colcemid treatment of insc mutant neuroblasts resulted in a nearly complete loss of Pins/Gαi/Dlg crescents: Pins is mostly cytoplasmic and Gαi/Dlg are uniform cortical. In contrast, Colcemid treatment of wild-type neuroblasts had no effect on Pins/Gαi/Dlg crescent formation, likely due to the association of Pins/Gαi/Dlg with the apical Insc/Par complex. In fact, the Insc/Par pathway of Pins/Gαi/Dlg localization requires only Insc and Baz proteins, because aPKC mutants that lack aPKC/Par-6 protein localization but retain Baz/Insc localization still formed Pins/Gαi/Dlg crescents in the absence of microtubules. It is concluded that spindle microtubules have the ability to induce Pins/Gαi/Dlg cortical crescents over one spindle pole in the absence of an Insc/Par pathway (Siegrist, 2005).
Recent work has shown that microtubules can directly regulate cortical polarity in yeast during C. elegans meiosis and in migrating cells. An important question is the extent to which microtubules regulate cortical cell polarity in other contexts. This study identifies a microtubule/kinesin pathway for inducing cortical polarity in Drosophila neuroblasts. This pathway is sufficient to induce cortical polarization of the evolutionarily conserved Dlg, Pins, and Gαi proteins and is necessary for reliable spindle orientation relative to apical Insc/Par cortical proteins (Siegrist, 2005).
A model is presented for the microtubule/Khc-73/Dlg pathway, in the absence of the Insc/Par function.
Asymmetric localization of Pins/Gαi proteins can be induced by two distinct pathways in embryonic neuroblasts: a well-characterized cortical pathway involving the Insc/Par proteins and a microtubule-dependent Khc-73/Dlg pathway. Each pathway is regulated differently and has unique features that provide different temporal and spatial information for generating cortical polarity (Siegrist, 2005).
First, each pathway is initiated by a different mechanism and provides unique information for the timing of Pins/Gαi polarization. The Insc/Par pathway is initiated at late interphase in response to an unknown extrinsic cue and is required for the early prophase cortical polarization of Pins/Gαi. In contrast, the Khc-73/Dlg pathway is initiated later at prometaphase/metaphase by astral microtubules and is required for cortical polarization of Pins/Gαi only in the absence of Insc/Par complex proteins. Consistent with this timeline, asymmetric enrichment of Dlg normally occurs well after polarization of Insc/Par/Pins/Gαi during the prometaphase/metaphase transition, and this temporal progression of Dlg cortical enrichment is not affected in insc mutants. The temporal polarization of Dlg coincides precisely with the onset of Pins/Gαi cortical polarity at prometaphase/metaphase that occurs in the absence of the Insc/Par pathway (Siegrist, 2005).
Next, each pathway provides different spatial information for the cortical polarization of Pins/Gαi. The Insc/Par pathway recruits Pins/Gαi to the apical cortex of the neuroblast at a position just below the overlaying epithelium, thus coordinating neuroblast cortical polarity with the neuroblast environment. In the absence of this pathway (e.g., insc mutant neuroblasts), cortical polarity can be generated but is not linked to tissue polarity, resulting in mispositioning of neuroblast progeny. In contrast, the microtubule/Khc-73/Dlg pathway induces Pins/Gαi crescent formation over one spindle pole, thus coordinating the neuroblast cortical polarity with the spindle axis. In the absence of this pathway (e.g., dlg mutant or Khc-73 RNAi neuroblasts), Insc/Baz can still recruit Pins/Gαi to the apical cortex, yet the spindle is not always properly aligned with this cortical polarity. Together these two pathways ensure the correct temporal and spatial positioning of apical complex proteins relative to extrinsic and intrinsic landmarks (Siegrist, 2005).
Drosophila sense organ precursors (SOPs) divide asymmetrically to generate an anterior pIIb cell and a posterior pIIa cell. During this division, Pins, Gαi, Dlg, and Numb form cortical crescents over the anterior spindle pole, and Baz localizes over the posterior spindle pole. Cell division orientation is fixed along the anterior/posterior axis by planar polarity cues mediated by the seven pass transmembrane receptor Frizzled. However, Frizzled signaling is required only for the position of Dlg/Pins crescents, not for their formation. When both frizzled and microtubules were remove together, it was found about 10% of the mitotic SOPs lack Pins crescents. This mild phenotype suggests that while astral microtubules may contribute to Dlg/Pins polarization in SOPs, there must be an additional mechanism involved. The best candidates for this third mechanism are the Par proteins because Par crescents still form in frizzled mutant SOPs at metaphase (Siegrist, 2005).
There are many similarities between asymmetric division of fly neuroblasts and the C. elegans zygote, but there are also striking differences. One of the most noteworthy differences is that C. elegans par mutants undergo symmetrically sized embryonic cell divisions, whereas in Drosophila, par or insc mutants maintain sibling cell size asymmetry. This work provides an explanation for this discrepancy. It is shown that astral microtubules are capable of generating Pins/Gαi cortical polarity in the absence of localized Par proteins and that this microtubule-induced Pins/Gαi cortical polarity is fully functional for generating an asymmetric spindle, cell size, and unique daughter cell fates. It is likely that C. elegans lacks this 'microtubule-based pathway' for inducing GPR1/2 (Pins) and Gα cortical polarity, at least during the first embryonic cell division, because posterior cortical localization of GPR1/2 is absent in par mutants and the daughter cells are equal in size. Interestingly, an increase is observed in symmetrically dividing neuroblasts in neuroblasts lacking both Insc/Par and microtubule pathways, compared to loss of single pathways alone. It appears that either the Insc/Par or microtubule/Khc-73/Dlg pathway is sufficient to induce Pins/Gαi cortical polarity, which generates daughter cells of different sizes and fates (Siegrist, 2005).
The microtubule/kinesin-induced Dlg clustering pathway described in this study may be evolutionarily conserved. In mammals, hDlg and the Khc-73 ortholog GAKIN are coexpressed in T cells and coimmunoprecipitate, and T cell activation leads to recruitment of hDlg to the immunological synapse. Interestingly, GAKIN targets hDlg into ectopic cellular projections in MDCK cells, and this targeting depends on microtubules. This has lead to the hypothesis that GAKIN may use a microtubule-based mechanism to target hDlg to the T cell immune synapse, similar to the microtubule/Khc-73 pathway described in this paper (Siegrist, 2005 and references therein).
The asymmetric division of Drosophila neuroblasts involves the basal localization of cell fate determinants and the generation of an asymmetric, apicobasally oriented mitotic spindle that leads to the formation of two daughter cells of unequal size. These features are thought to be controlled by an apically localized protein complex comprised of two signaling pathways: Bazooka/Drosophila atypical PKC/Inscuteable/DmPar6 and Partner of inscuteable (Pins)/Galphai. In addition, Gß13F is also required, however, the role of Galphai and the hierarchical relationship between the G protein subunits and apical components are not well defined. This study describes the isolation of Galphai mutants and shows that Galphai and Gß13F play distinct roles. Galphai is required for Pins to localize to the cortex, and the effects of loss of Galphai or pins are highly similar, supporting the idea that Pins/Galphai act together to mediate various aspects of neuroblast asymmetric division. In contrast, Gß13F appears to regulate the asymmetric localization/stability of all apical components, and GßF loss of function exhibits phenotypes resembling those seen when both apical pathways have been compromised, suggesting that it acts upstream of the apical pathways. Importantly, these results have also revealed a novel aspect of apical complex function, that is, the two apical pathways act redundantly to suppress the formation of basal astral microtubules in neuroblasts (Yu, 2003).
This study reports the isolation and analysis of loss of function mutations in Galpha and show that the loss of Galpha and Gß13F have distinct effects on NB asymmetric cell divisions. Galphai is required for Pins cortical association and asymmetric localization; loss of Galphai causes Pins to localize to the cytosol, and mutant NBs exhibit phenotypes that are highly similar to those seen in pins mutants. Analyses of double mutant combinations confirm Galphai RNAi results showing that Pins/Galphai and Baz/DaPKC/Insc act in an redundant fashion to mediate the formations of an asymmetric mitotic spindle and the generation of NB daughters of unequal size. Importantly, these analyses also revealed a new aspect of apical complex function: that the two apical pathways also act redundantly to suppress the formation of astral microtubules from the basal centrosome of NBs. In contrast, Gß13F appears to act upstream of the apical components and is required for their asymmetric localization/stability. The defects associated with NBs lacking Gß13F function are highly similar to those seen when the function of both apical pathways have been compromised. In addition, it was shown that high level overexpression of two different Galpha subunits, which can bind/complex to Gß13F, results in similar phenotypes seen in Gß13F mutant NBs, suggesting that it is the depletion of free Gß13F, which is responsible for the mutant phenotypes (Yu, 2003).
Pins and Galphai apical localization are mutually dependent. In pins NBs, Galphai is evenly distributed to the NB cortex, and in Galpha mutant NBs, Pins localizes to the cytosol. Pins asymmetric localization to the apical cortex of the NBs is a two-step process: Pins needs to be targeted to the cortex first: this requires the COOH-terminal Goloco motifs that can bind Galphai before Galphai can be recruited to the apical cortex in a process which requires the Galphai NH2-terminal TPR that can interact with Insc. The current results therefore suggest that Pins cortical targeting is most likely mediated by Galphai, which not only binds Pins, but also is able to localize to the plasma membrane through lipid modifications (Yu, 2003).
However, in Gß13F mutant NBs, although the levels of Pins are drastically reduced, the residual Pins is localized both to the cytosol and to the cell cortex. This poses a problem since in the Gß13F mutant NBs not only is Gß13F absent but Galphai also is undetectable with an anti-Galphai antibody. One possible explanation is that although Galphai is undetectable, there is still some Galphai remaining in the Gß13F NBs: this may account for the low level residual uniform cortical distribution of Pins. Alternatively, the possibility cannot be ruled out that the cortical Pins in Gß13F NBs is due to some unknown molecule that can recruit Pins to cortex in the absence of both Galphai and Gß13F (Yu, 2003).
The analysis of Gß13F function is complicated by the fact that in the Gß13F mutant NBs, Galphai levels are also down-regulated presumably due to the instability of the protein in the absence of Gß13F. Although loss of either Galpha or Gß13F causes aberrations in localization of the basal components and orientation of the mitotic spindle, it is clear that at least some of the defects associated with the loss of Gß13F cannot be attributable solely to the depletion of Galphai. In the great majority of Galphai mutant NBs, DaPKC and Baz still localize asymmetrically to a subset of the cell cortex. And consistent with the proposal that spindle geometry and the size asymmetry of the NB daughters are mediated by two redundant apical pathways, Pins/Galphai and Baz/DaPKC, the great majority (79%) of the Galpha mutant NBs generate an asymmetric mitotic spindle and divide to produce unequal size daughters. In contrast, in Gß13F NBs not only do Pins/Galphai always fail to become asymmetrically localized but the majority of mutant NBs (71%) also fail to asymmetrically localize Baz/DaPKC; consequently ~65% of NBs fail to generate an asymmetric mitotic spindle and divide to produce equal size daughters. Therefore, at least formally, Gß13F acts upstream of the two apical pathways (Yu, 2003).
It is believed that the major reason for the phenotypes associated with loss of Gß13F function is due to the disruption of Gßgamma signaling. Overexpression of Galphai will cause a high frequency of equal size divisions. In addition, overexpression of Galphao, a Galpha subunit that interacts with Gß13F but is not itself required for asymmetric divisions in wt NBs, will also mimic the Gß13F loss of function phenotype. For both overexpression of Galphai and Galphao, the frequency of equal size divisions is significantly higher than that seen in Gß13F loss of function. This difference may be due to the existence of other Gß subunits which might also function in NB asymmetric divisions. Three Gß genes have been identified by the Drosophila genome project, and although one of these genes, concertina, appears not to be involved in the process, it is possible that overexpression of Galpha molecules may deplete not only Gß13F but also Gß76C. This possibility could be addressed by the analysis of double mutants of Gß genes. Nevertheless, these observations are consistent with the view that the depletion of free Gßgamma, and not Galphai, is the major cause for the symmetric divisions seen in Gß13F mutant NBs. Hence, although previous analysis of Gß13F loss of function did not report any effects on NB daughter size, the current data are consistent with the notion that Gß13F plays a major role in mediating the distinct size of NB daughter cells (Yu, 2003).
The apical centrosome associates with prominent astral microtubules, whereas the basal centrosome connects to few if any astral microtubules in wt NBs and in mutants in which one of the two apical pathways is compromised. In contrast, in NBs that lack both apical pathways a symmetric mitotic apparatus is established that features extensive arrays of astral microtubules at both centrosomes. Therefore, either of the two apical pathways appears sufficient to prevent formation of basal astral microtubules. It is not clear how this might be accomplished at a mechanistic level. However, one might speculate that there exists an asymmetrically localized molecule, which can act to promote the formation of astral microtubules. When either of the apical pathways is functional, this molecule is asymmetrically localized and promotes the formation of astral microtubules only over the centrosome it overlies. However, when both apical pathways are mutated, or when Gß13F is mutated or when all apical components become uniformly cortical, e.g., when Galphai is overexpressed, then the hypothetical molecule becomes uniformly cortical and can promote the formation of astral microtubules over both centrosomes. This type of model can readily explain why either loss or uniform cortical localization of both apical pathways leads to symmetric astral microtubule formation over both centrosomes (Yu, 2003).
In summary, the results demonstrate that for NB asymmetric divisions Galphai and Gß13F play distinct roles. Galphai and Pins are members of one of the two apical pathways and Baz/DaPKC/Insc forms the other. Loss of Galphai function results in defects in NB asymmetry that are essentially indistinguishable from those seen in pins mutants. Gß13F (Gßgamma) functions upstream of both Pins/Galphai and Baz/DaPKC/Insc pathways to mediate their stability and/or asymmetric localization (and function). Without Gß13F, the function of both apical pathways are attenuated; Galphai levels are dramatically reduced and Pins/Galphai pathway is defective; in addition, the asymmetric localization of members of the Baz/DaPKC/Insc pathway is often defective. Consequently, loss of Gß13F function yields phenotypes that are similar to those seen when both apical pathways are disrupted by mutations (Yu, 2003).
In Drosophila, distinct mechanisms orient asymmetric cell division along the apical-basal axis in neuroblasts and along the anterior-posterior axis in sensory organ precursor (SOP) cells. Heterotrimeric G proteins are essential for asymmetric cell division in both cell types. The G protein subunit Galphai localizes apically in neuroblasts and anteriorly in SOP cells before and during mitosis. Interfering with G protein function by Galphai overexpression or depletion of heterotrimeric G protein complexes causes defects in spindle orientation and asymmetric localization of determinants. Galphai is colocalized and associated with Pins, a protein that induces the release of the ßgamma subunit and might act as a receptor-independent G protein activator. Thus, asymmetric activation of heterotrimeric G proteins by a receptor-independent mechanism may orient asymmetric cell divisions in different cell types (Schaefer, 2001).
While a significant amount of Galphai coimmunoprecipitates with Insc and Pins, no Galphao can be detected in the immunoprecipitate. It is concluded that Galphai but not Galphao is part of the Insc/Pins complex in vivo. To determine the subcellular localization of Galphai, Drosophila embryos were stained for Galphai, DNA, and Insc or Bazooka. Before stage 12 of embryogenesis, Galphai is expressed in all cells and localizes to the cell cortex. Costaining for the apical marker Bazooka reveals that Galphai is concentrated basolaterally in epithelial cells. Upon neuroblast delamination, when the expression of Insc starts, Galphai concentrates in an apical stalk that extends into the epithelial cell layer and then colocalizes with Insc in a crescent along the apical cell cortex during interphase, prophase, and metaphase until anaphase, when Insc disappears and Galphai becomes delocalized. Galphai but not the associated ß subunit is asymmetrically localized in neuroblasts, suggesting that Gß13F is also bound to other Galpha subunits, possibly Galphao (Schaefer, 2001).
To test whether Insc is required for asymmetric Galphai localization in neuroblasts, inscP72 mutant embryos were stained for Galphai and DNA. During neuroblast delamination, Galphai fails to localize apically in insc mutants and in 87% of insc mutant metaphase neuroblasts, the protein is distributed around the whole cell cortex. To test whether ectopic expression of Insc is sufficient for the apical localization of Galphai, insc was ubiquitously expressed from a heat-inducible transgene. While Galphai is localized basolaterally in epidermal cells of heat-shocked control embryos, heat-shock-induced ectopic expression of insc in these cells results in apical concentration of Galphai. Thus, expression of insc is both required and sufficient for apical recruitment of Galphai (Schaefer, 2001).
Since Galphai directly binds to Pins, the subcellular localization of Galphai was tested in pins mutants. No apical localization of Galphai was observed in 100% of the pins mutant metaphase neuroblasts. This might be an indirect consequence of the defect in Insc localization in pins mutant metaphase neuroblasts. However, initiation of Galphai localization also fails in 88% of pins mutant delaminating neuroblasts. Insc is normally localized in pins mutants at this stage and so it is concluded that both Insc and Pins are required for the apical localization of Galphai in neuroblasts (Schaefer, 2001).
Genetic analysis of Galphai is complicated by the presence of another gene within the first intron and the lack of identified P-element insertions near the gene. However, a P-element inserted into the 5' untranslated region of the Gß13F gene was identified and this was used to generate mutants by imprecise excision. Two lethal imprecise excisions were isolated, one of which (Gß13FDelta1-96A) removes the entire coding region, can be rescued to viability by a transgene containing the Gß13F genomic region, and was used in all experiments (Schaefer, 2001).
Since Gß13F has a strong maternal contribution, all experiments were performed in embryos from Gß13F mutant germline clones (here called Gß13F mutants). Gß13F mutants have characteristic morphological defects during gastrulation that lead to the formation of anterior and posterior holes in the cuticle. The defects and cuticle phenotypes are similar to embryos mutant for concertina (conc). Conc is a heterotrimeric G protein alpha subunit and the phenotypic similarity suggests that Conc signals through Gß13F. Galphai protein levels and localization are unaffected in conc mutants. In Gß13F mutants, however, Galphai disappears during gastrulation and is undetectable by immunofluorescence in all cell types during stage 10 of embryogenesis when neuroblasts undergo their first round of asymmetric cell division. Thus, both Galphai and Gß13F are absent from neuroblasts of Gß13F mutant embryos (Schaefer, 2001).
Staining for the neuronal marker Asense has shown that neuroblasts are correctly specified, delaminate, and enter mitosis shortly after delamination both in conc and Gß13F mutants. Furthermore, staining for DmPar-6 reveals no defects in epithelial polarity. However, while 86% of the asymmetric cell divisions in conc mutant neuroblasts are oriented along the apical-basal axis, only 26% of the divisions in Gß13F mutant neuroblasts have this orientation, whereas the others are misoriented by more than 30 degrees. Miranda localizes into a basal cortical crescent in 100% of the conc mutant metaphase neuroblasts, but only in 6% of the Gß13F mutant neuroblasts. In 29% of the Gß13F mutant neuroblasts, crescents are misoriented, whereas in 65%, Miranda is largely cytoplasmic. Defects in asymmetric localization are also observed for Numb. Thus, Gß13F mutants have defects in asymmetric cell division similar to or stronger than those observed in insc mutants, and therefore Insc distribution was analyzed in these mutants. When neuroblasts delaminate from the neuroectoderm, Insc begins to accumulate in a stalk that extends into the epithelium, and this initial localization is unchanged in Gß13F mutants. In Gß13F mutants, cortical localization of the protein is progressively lost after delamination. Weak cortical Insc crescents were found in 11% of the metaphase neuroblasts, but in 25%, the protein was partially, and in 64% completely, localized into the cytoplasm. Thus, heterotrimeric G proteins are required for maintaining Insc localization and for directing spindle orientation and asymmetric protein localization during neuroblast division (Schaefer, 2001).
Heterotrimeric G proteins can interact with their downstream targets either via the Gßgamma subunit or the GTP-bound Galpha subunit. Overexpression of wild-type Galphai and GalphaiQ205L, a GTPase-deficient mutant form, should distinguish between these possibilities. Wild-type Galphai should bind and deplete free Gßgamma and inhibit its downstream interactions. GalphaiQ205L, in contrast, should be in the GTP-bound form that does not bind Gßgamma and should not interfere with Gßgamma signaling. Signaling via the alpha subunit, however, should be enhanced by GalphaiQ205L, but not be affected by the wild-type form (Schaefer, 2001).
Asymmetric cell division was therefore analyzed in control embryos or embryos overexpressing wild-type Galphai from a ubiquitous maternal promoter. While both Pins and Galphai localize apically in control metaphase neuroblasts, they are uniformly distributed around the cortex of neuroblasts overexpressing Galphai. The intensity of cortical Pins staining is higher in Galphai-overexpressing embryos, indicating that Pins is recruited from the cytoplasm to the cell cortex. Miranda localizes into a cortical crescent in control metaphase neuroblasts but in only 20% of the Galphai-overexpressing neuroblasts. Instead, the protein is uniformly cortical (6%) or localizes partially or completely into the cytoplasm. Defects in asymmetric localization are also observed for Numb, even though Numb does not relocalize to the cytoplasm. Mitotic spindles (visualized by gamma-Tubulin staining) are oriented along the apical-basal axis in controls, but are misoriented in 74% of the Galphai-overexpressing neuroblasts. Insc localization is initiated during neuroblast delamination both in control and in Galphai-overexpressing neuroblasts. In metaphase neuroblasts, however, Insc forms an apical crescent in the controls, but localizes partially (40%) or completely (60%) to the cytoplasm upon Galphai overexpression (Schaefer, 2001).
If the defects observed upon Galphai overexpression are due to depletion of free Gßgamma, overexpression of GalphaiQ205L should be without effect. Asymmetric cell division was therefore analyzed in neuroblasts after overexpression of GalphaiQ205L under the same ubiquitous maternal promoter. Like wild-type Galphai, GalphaiQ205L localizes around the cell cortex when overexpressed in neuroblasts. However, the mutant form fails to recruit Pins to the cell cortex and has no effect on Pins localization. No defects in spindle orientation or basal localization of Numb and Miranda were observed and Insc was still localized into an apical crescent in 91% of the metaphase neuroblasts. This suggests that the phenotypes caused by Galphai overexpression might be a consequence of Gßgamma depletion (Schaefer, 2001).
However, several observations indicate that the defects observed after Galphai overexpression are not caused only by depletion of Gß13F. In addition to the defects described above, Galphai overexpression also causes phenotypes that are not observed in Gß13F mutants. While the size difference between daughter cells is unaffected in most Gß13F mutant neuroblasts, staining of the cell cortex by anti-alpha-spectrin reveals that 80% of the Galphai-overexpressing neuroblasts produce two equal sized daughter cells. While in Gß13F mutants, Miranda localization fails during metaphase but is largely normal during late stages of mitosis (similar to insc mutants), Galphai overexpression causes defects throughout mitosis and incorrectly positioned Miranda crescents are often bisected by the cleavage furrow. Thus, even though some of the Galphai overexpression phenotypes may be caused by depletion of Gß13F, other mechanisms like depletion of another Gß subunit or signaling via the GDP-bound form of Galphai may contribute to these phenotypes (Schaefer, 2001).
The strong overexpression phenotypes caused by wild-type Galphai but not by GalphaiQ205L suggest that the GDP-bound form of Galphai may have a function in asymmetric cell division. To test whether Pins interacts preferentially with the GTP- or the GDP-bound form, Galphai was immunoprecipitated in the presence or absence of the slowly hydrolyzable GTP-analog GTPgammaS. While Pins can be readily coimmunoprecipitated with Galphai in the presence of GDP, only trace amounts of Pins can be coimmunoprecipitated in the presence of GTPgammaS, suggesting that Pins preferentially interacts with the GDP-bound form of Galphai (Schaefer, 2001).
The GDP-bound form of Galpha is thought to be inactive and tightly associated with its ßgamma subunit. To test whether Galphai in the Insc/Pins complex is bound to the ß subunit, the Insc/Pins/Galphai complex was immunoprecipitated using a ß-Gal-tagged version of the functional domain of Insc. No Gß13F can be found in the complex, even though a significant amount of Gß13F can be detected in a control experiment where equal amounts of Galphai are precipitated by anti-Galphai. Thus, Galphai is bound to Gß13F in vivo but is free of the ß subunit in the complex with Insc and Pins. To test whether Pins is responsible for the release of the ß subunit, Galphai was immunoprecipitated in the presence of recombinant Pins protein. A significant amount of Gß13F is bound to Galphai in control experiments, but addition of an MBP (maltose binding protein)-fusion of full-length Pins (MBP-Pins) or the Pins GoLoco domains (MBP-GoLoco) during the immunoprecipitation causes the release of the ß subunit. The same effect can be achieved by addition of a 38 aa peptide corresponding to the last GoLoco domain of the Pins protein, but not with a peptide in which a conserved phenylalanine had been mutated to arginine. Thus, the Pins GoLoco domains cause the dissociation of Gß13F from Galphai (Schaefer, 2001).
These results suggest that Galphai exists in an unusual form in Drosophila neuroblasts that is bound to GDP but free of the ß subunit. Furthermore, the observation that recombinant Pins triggers the release of the ß subunit from Galphai is consistent with the hypothesis that Pins activates heterotrimeric G proteins without nucleotide exchange on the alpha subunit in the absence of an extracellular ligand (Schaefer, 2001).
To test whether G proteins also function in Insc-independent asymmetric cell division, the distribution of Galphai was analyzed in SOP cells during pupal development. In interphase, when Numb is homogeneously distributed around the cell cortex, Galphai is asymmetrically localized to the anterior cell cortex in SOP cells. During metaphase, both Numb and Galphai are found at the anterior cell cortex and in telophase, they segregate into the same daughter cell. Similar results were obtained for Pins. Thus, Pins and Galphai localize asymmetrically in SOP cells but in contrast to neuroblasts, they are at the same side as Numb (Schaefer, 2001).
Asymmetric cell divisions in SOP cells are oriented along the anterior-posterior axis of epithelial planar polarity. To test whether planar polarity is required for Galphai localization, Galphai localization was analyzed in frizzled mutants where planar polarity is disrupted. As in wild-type, Galphai localizes asymmetrically in frizzled mutant interphase SOP cells and localizes to the same side as Numb in mitosis. However, both the Numb and the Galphai crescents are misoriented in these mutants, suggesting that planar polarity determines the position of Galphai accumulation but is not required for its asymmetric localization per se (Schaefer, 2001).
To determine whether G proteins are required for asymmetric cell division in SOP cells, Gß13F mutant clones generated by mitotic recombination in eye imaginal discs were analyzed. No Galphai protein could be detected on the cell cortex of Gß13F mutant cells but it is not possible to distinguish between delocalization and degradation of the protein. While Numb localizes asymmetrically and Gß13F is uniformly cortical in mitotic SOP cells outside the clones, no asymmetric localization of Numb is seen within the clone where Gß13F cannot be detected. To directly test a requirement of Galphai in SOP cells, heritable RNAi was used to disrupt Galphai function. Expression of double-stranded Galphai RNA significantly reduces Galphai protein levels in all SOP cells. Eleven percent of the SOP cells no longer stained for Galphai and in these cells, Pins no longer localizes to the cell cortex. Numb is distributed around the cell cortex in metaphase, leading to cell fate transformations in the bristle lineage. Mitotic spindles are misoriented in the SOP cells that have lost Galphai, but their low frequency makes a quantitative analysis of the spindle orientation phenotype difficult. Similar defects are observed in SOP cells in mitotic clones mutant for the strong allele pins83. Neither Galphai nor Numb are asymmetrically localized in these cells, indicating that Pins and Galphai are codependent for their asymmetric localization in SOP cells. It is concluded that Galphai and Pins are also required for Insc-independent asymmetric cell divisions in SOP cells (Schaefer, 2001).
In neuroblasts, Galphai function does not seem to involve the GTP-bound form of Galphai. To test whether this is also the case in SOP cells, wild-type Galphai and GalphaiQ205L were overexpressed in SOP cells. Upon overexpression, Galphai is no longer asymmetrically localized and Numb is uniformly distributed around the cell cortex. Thirty-eight percent of the Galphai overexpressing SOP cells (n = 149) but only 9% of the controls divided at an angle that deviated more than 45° from the anterior-posterior axis. However, unlike in neuroblasts, in this case similar defects can be generated by overexpression of the activated GalphaiQ205L mutant form. The different effects of GalphaiQ205L overexpression in neuroblasts and SOP cells suggest that distinct pathways might function downstream of G proteins in the two cell types (Schaefer, 2001).
In neuroblasts, the Insc protein is critical for the asymmetric localization of Galphai and its binding partner Pins. Neuroblasts arise from epithelial cells in which Insc is not expressed and Galphai is localized basolaterally. When neuroblasts delaminate, Insc expression starts and the protein functions as an adaptor that links the Pins/Galphai complex to the Bazooka/DmPar-6/DaPKC complex that is inherited from the apical cortex of the epithelial cells. Neither Pins nor Galphai are required for Insc localization during this stage. In delaminated neuroblasts, however, Insc, Pins, and Galphai become codependent for their apical localization. At this point, their subcellular localization in various mutants can no longer be explained simply by protein-protein interactions of the known components. When Galphai is overexpressed, for example, Pins is recruited to the cell cortex whereas Insc relocalizes into the cytoplasm, suggesting that the two proteins no longer interact. Thus, events that happen downstream of Galphai seem to be involved in maintaining the colocalization of the more upstream components. The simplest model is that G proteins establish a positional cue at the apical cell cortex during neuroblast delamination -- this cue is needed for maintaining apical protein localization in delaminated neuroblasts and ultimately, for orienting asymmetric cell division. In Drosophila, this downstream activity remains to be identified, but a similar feedback loop for asymmetric protein localization is found in yeast and here its molecular components are well understood. Local activation of a heterotrimeric G protein in response to the pheromone alpha-factor recruits Cdc24 to the site of G protein activation. Cdc24 is an exchange factor that locally activates the small G protein Cdc42 and activated Cdc42, in turn, is needed to maintain Cdc24 localization. Thus, the initiation of an autoregulatory feedback loop at a particular position may be a common theme in cell polarity (Schaefer, 2001).
The function of heterotrimeric G proteins in directing cell polarity and asymmetric cell division is not restricted to Drosophila. In C. elegans, a Gßgamma subunit is required for correct orientation of mitotic spindles during early development and two Galpha subunits function redundantly in asymmetric spindle positioning and generation of different daughter cell sizes. Since the role of the Bazooka/DmPAR-6/DaPKC complex is also conserved from C. elegans to Drosophila, a homologous machinery may direct asymmetric cell division in the two organisms. RNAi experiments so far have failed to reveal a function for the C. elegans Pins homolog, but recently, two other proteins containing a GoLoco domain have been found to be required for asymmetric cell division in a chromosome-wide RNAi screen. G proteins are not asymmetrically localized and not required for the asymmetric segregation of determinants in C. elegans, but it is possible that asymmetric activation of G proteins by GoLoco domain proteins is a conserved mechanism to orient mitotic spindles in the two organisms (Schaefer, 2001).
The Hedgehog (Hh) signalling pathway has an evolutionarily conserved role in patterning fields of cells during metazoan development, and is inappropriately activated in cancer. Hh pathway activity is absolutely dependent on signalling by the seven-transmembrane protein smoothened (Smo), which is regulated by the Hh receptor patched (Ptc). Smo signals to an intracellular multi-protein complex containing the Kinesin related protein Costal2 (Cos2), the protein kinase Fused (Fu) and the transcription factor Cubitus interruptus (Ci). In the absence of Hh, this complex regulates the cleavage of full-length Ci to a truncated repressor protein, Ci75, in a process that is dependent on the proteasome and priming phosphorylations by Protein kinase A (PKA). Binding of Hh to Ptc blocks Ptc-mediated Smo inhibition, allowing Smo to signal to the intracellular components to attenuate Ci cleavage. Because of its homology with the Frizzled family of G-protein-coupled receptors (GPCR), a likely candidate for an immediate Smo effector would be a heterotrimeric G protein. However, the role that G proteins may have in Hh signal transduction is unclear and quite controversial, which has led to widespread speculation that Smo signals through a variety of novel G-protein-independent mechanisms. This study presents in vitro and in vivo evidence in Drosophila that Smo activates a G protein to modulate intracellular cyclic AMP levels in response to Hh. The results demonstrate that Smo functions as a canonical GPCR, which signals through Gαi to regulate Hh pathway activation (Ogden, 2008).
To examine whether a G protein is involved in Hh signalling, a series of G proteins was targetted by double-stranded RNA (dsRNA)-mediated knockdown. Drosophila clone-8 (Cl8) cells were treated with control or Gα-subunit-specific dsRNA and assayed for changes in Hh-mediated induction of a ptc-luciferase reporter construct. Whereas Gαs (also called G-sα60A) and Gαo (also called G-oα47A) dsRNAs do not significantly alter Hh-induced reporter activation knockdown is able to trigger a decrease in Hh-dependent reporter gene expression. Although not as effective as Smo knockdown in silencing Hh reporter gene activation, Gαi (also called G-iα65A) dsRNA specific to the coding sequence, or 3' untranslated region (UTR), reduces Hh-induced reporter activity by approximately 70%, supporting a role for Gαi in the Hh pathway. To confirm the specificity of Gαi dsRNA effects attempts were made to rescue reporter activity through ectopic expression of wild-type Gαi or constitutively active GαiQ205L. Hh-stimulated reporter activity can be restored by both wild-type and constitutively active Gαi, confirming the specificity of the Gαi dsRNA-mediated effects. Western blot analyses of Cl8 lysates reveal that cells treated with Gαi dsRNA show attenuated stabilization of Ci and decreased Fu phosphorylation in response to Hh. Hh-induced Smo phosphorylation is maintained in the presence of Gαi dsRNA, suggesting that Gαi functions downstream of Smo and upstream of Fu and Ci (Ogden, 2008).
To determine whether Gαi can modulate Hh pathway activity in vivo, Gαi constructs were expressed in wing imaginal discs using MS1096-Gal4 or C765-Gal4. Expression of an inactive Gαi mutant (GαiG204A) or wild-type Gαi has little effect on wing vein patterning. However, expression of constitutively active GαiQ205L results in widening of longitudinal vein LV3-LV4 spacing and ectopic vein material on LV2 and LV3. The severity of this phenotype is dose-dependent, as higher-level expression of UAS-GαiQ205L triggers more severe ectopic vein material anterior to LV3, and further widening of LV3-LV4 spacing. Expression of GαiQ205L in wing imaginal discs also results in over-growth of the wing pouch, along with expansion of full-length Ci. This Ci expansion triggers ectopic expression of the Hh target gene decapentaplegic (dpp) in the wing pouch, as shown by a dpp-lacZ reporter gene. Gαi-mediated ectopic expression of dpp is consistent with the ectopic veins observed in wings expressing GαiQ205L. Taken together, these results support a role for activation of Gαi in regulating the stability of Ci, and link Gαi to regulation of a known Hh target gene (Ogden, 2008).
To determine whether Gαi functions downstream of Smo in vivo, the ability of Gαi to modulate Hh pathway activity was analysed in a smo sensitized background. As previously demonstrated, expression of a dominant-negative smo transgene, UAS-Smo5A, results in severe disruption of LV3-LV4 wing patterning. Expression of wild-type Gαi in this smo sensitized background allows for partial rescue of wing vein structures in the LV3/LV4 zone. Expression of constitutively active GαiQ205L results in a more complete rescue of the Hh loss-of-function phenotype, allowing for near total restoration of LV3/LV4 patterning. As a control, UAS-GFP was co-expressed with Smo5A, and found to have no effect on the Smo5A-induced phenotype (Ogden, 2008).
To examine the ability of GαiQ205L to modulate Ci stability and Hh target gene activation in the smo sensitized background, wing imaginal discs were immunostained with antibodies that recognize full-length Ci and the target gene product Ptc. UAS-Smo5A expression results in decreased ptc expression and disruption of the Ci gradient. Expression of constitutively active GαiQ205L in this smo sensitized background results in partial restoration of the Ci gradient and a near-complete rescue of ptc expression at the anterior/posterior border. These results support the model that Gαi contributes to the regulation of Hh target gene expression and Ci stability. Furthermore, the fact that this regulation occurs when Smo function is compromised suggests that Gαi affects Hh signalling at a level downstream of Smo (Ogden, 2008).
To determine whether Gαi is required for Hh signalling in vivo, Hh target gene expression was examined in clones of cells homozygous for Gαi mutation. The null allele GαiP20 removes the entire coding region of the Gαi gene, and is homozygous lethal. GαiP8 is a putative hypomorph, which removes the bulk of exons 1 and 2, but leaves the transcriptional start site intact and produces a transcript. Flies that are homozygous for the GαiP8 mutation are viable, but weak. Mosaic analysis reveals that expression of the Hh target gene dpp is decreased in both GαiP20 and GαiP8 mutant clones, supporting a role for Gαi in activation of Hh target genes in vivo. To confirm that the effects on dpp expression are due to loss of Gαi, attempts were made to rescue GαiP20 null clones with UAS-Gαi. Ectopic expression of Gαi is able to rescue dpp reporter gene expression in GαiP20 clones, consistent with decreased dpp expression resulting from disruption of Gαi (Ogden, 2008).
To determine whether compromised Gαi activity alters Hh-dependent patterning, the viable mutant allele GαiP8 and an additional viable allele described to be a null or strong hypomorph, Gαi57 were used. Whereas homozygous GαiP8 and Gαi57 mutants do not have vein fusions that are typical of strong Hh loss of function, their wings are smaller than wild-type wings. Small wing size might result from altered dpp expression in anterior cells of the wing pouch, as Dpp regulates wing blade size. Additionally, both GαiP8 and Gαi57 mutant flies demonstrate varying degrees of incomplete thorax closure, as shown by mild to severe thoracic clefts. This phenotype is also consistent with decreased dpp expression, in that Dpp, in conjunction with JNK signalling, controls spreading of the anterior edge of wing imaginal discs to initiate thorax closure. To confirm that this phenotype results from decreased Hh signalling, ptc was expressed in the notum and dorsal compartment of the wing imaginal disc. ptc expression triggers the formation of a thoracic cleft when expressed under control of pannier and apterous promoters, suggesting that the thoracic phenotype observed in Gαi flies results from compromised Hh signalling. Because GαiP20 null mutant animals are not viable, their wings or thoraces could not be examined. However, attenuation of Hh signalling by expressing dominant-negative Smo5A is enhanced in GαiP20 heterozygotes, as shown by disruption of LV3 (Ogden, 2008).
in vitro and in vivo data suggest that loss of Gαi might compromise Ci stabilization in Hh-receiving cells. When Ci and Smo levels were examined in Gαi mutant clones, both appeared to be increased in a cell-autonomous manner. These results are consistent with the modest stabilization of Smo and Ci on in vitro Gαi knockdown in non-Hh-treated cells. Although these results are unexpected, as Gαi loss is predicted to increase PKA activity and Ci degradation, previous studies have demonstrated that PKA functions to regulate Hh signalling both positively and negatively. Phosphorylation of Smo by PKA has a positive role in pathway activation, and might account for the modest stabilization of Ci that was observed (Ogden, 2008).
If Smo signals through Gαi it should be able to induce Gαi activation rapidly in response to Hh stimulation. To assay for Hh-mediated activation of Gαi, Cl8 cells were treated with conditioned media containing the amino-terminal Hh signalling molecule (HhN) or control conditioned media, then assayed for Hh-induced changes in intracellular cAMP. Within 5-10 min, HhN treatment reduces the basal intracellular cAMP concentration by approximately 50%. To confirm that the Hh-induced decrease in intracellular cAMP is dependent on Hh signalling through Smo and Gαi, cells were treated with smo, Gαi or control dsRNA, then assayed for a Hh-induced decrease in cAMP. Whereas cells transfected with control dsRNA maintain the ability to decrease intracellular cAMP in response to HhN, cells transfected with either smo or Gαi dsRNA are attenuated in their ability to do so. Taken together, these results support the idea that Gαi is activated rapidly, in a Smo-dependent manner, in order to modulate cAMP levels in response to Hh (Ogden, 2008).
To determine whether modulation of cAMP can alter Hh signalling in vivo, a hypomorphic mutant allele of the cAMP-specific phosphodiesterase dunce (dnc1) was used to raise intracellular cAMP levels in a Hh-independent manner. Hemizygous dnc1 animals are viable with no obvious Hh defects. However, introduction of the dnc1 mutation into a smo sensitized background enhances the Smo loss-of-function phenotype, resulting in wings with near complete elimination of wing vein patterning. This enhanced Hh loss-of-function phenotype is similar to the phenotype obtained on decreasing smo gene dosage by one-half in the same smo sensitized background. Along with in vitro cAMP assays, these results indicate that Hh activates Smo to modulate intracellular cAMP, via Gαi, and that this function is important for proper pathway activity in vivo (Ogden, 2008).
Cos2 associates with membranes, microtubules, PKA, Smo, Fu and Ci. To determine whether Cos2 facilitates the coupling of Gαi with these Hh signalling components, lysates were prepared from cells expressing HA-Gαi, and then immunoprecipitated Cos2. It was found that Gαi associates with the Cos2 complex, and that this association is enriched in response to Hh. The binding of Fu to Cos2 is not altered by Hh, suggesting that the recruitment of Gαi to this protein complex is regulated. This result suggests that Cos2 facilitates the coupling of Smo with Gαi and additional downstream effectors necessary to transduce the Hh signal (Ogden, 2008).
This study has shown a requirement for Gαi in the Hh signalling pathway. Hh-mediated recruitment and activation of Gαi results in decreased intracellular cAMP, indicating that Hh may regulate PKA through modulation of the intracellular cAMP concentration. It was also demonstrated that Gαi can modulate Hh pathway activity in vitro and in vivo, and seems to do so at a level downstream of Smo. Furthermore, loss of Gαi alters Hh signalling in vivo, supporting the idea that Gαi is a requisite member of the Hh pathway (Ogden, 2008).
The blood-brain barrier of Drosophila is established by surface glia, which ensheath the nerve cord and insulate it against the potassium-rich hemolymph by forming intercellular septate junctions. The mechanisms underlying the formation of this barrier remain obscure. The G protein-coupled receptor (GPCR) Moody, the G protein subunits Gαi and Galphao, and the regulator of G protein signaling Loco are required in the surface glia to achieve effective insulation. The data suggest that the four proteins act in a complex common pathway. At the cellular level, the components function by regulating the cortical actin and thereby stabilizing the extended morphology of the surface glia, which in turn is necessary for the formation of septate junctions of sufficient length to achieve proper sealing of the nerve cord. This study demonstrates the importance of morphogenetic regulation in blood-brain barrier development and places GPCR signaling at its core (Schwabe, 2005).
The Drosophila nerve cord is ensheathed by a thin single-layer epithelium, which in turn is surrounded by an acellular layer of extracellular matrix material. Ultrastructural analysis has revealed that septate junctions (SJs) between the epithelial cells are responsible for the insulation of the nerve cord. Fate-mapping studies have shown that the nerve cord is enveloped by glia expressing the glial-specific marker Repo, but to date there has been no direct proof that it is these surface glia that form intercellular SJs and thus the insulating sheath. Moreover, the time course for the formation of the sheath and of the SJ-mediated seal has not been established (Schwabe, 2005).
Several assays were developed to follow the morphogenesis of the surface glial sheath. Due to the onset of cuticle formation, immunohistochemistry becomes unreliable after 16 hr of development. Live imaging of GFP-tagged marker proteins was therefore used to visualize cell shapes, in particular the actin cytoskeleton marker GFP/RFP-Moesin and the SJ marker Neuroglian (Nrg)-GFP. Nrg-GFP expressed under its own promoter and RFP-Moesin driven by repo-Gal4 are colocalized in the same cells, establishing that the SJ-forming cells are repo positive and thus conclusively demonstrating the insulating function of the surface glia. To probe the permeability of the transcellular barrier, fluorescent dye was injected into the body cavity and dye penetration into the nerve cord was quantified by determining mean pixel intensity in sample sections (Schwabe, 2005).
The surface glia are born in the ventrolateral neuroectoderm and migrate to the surface of the developing nerve cord, where they spread until they touch their neighbors (17 hr of development). The glia then join to form a contiguous sheet of square or trapezoidal cells, tiled to form three-cell corners. SJ material is visible as a thin contiguous belt by 18 hr but continues to accumulate until the end of embryogenesis. Similar to other secondary epithelia, the surface glia do not form a contiguous adherens-junction belt (zonula adherens), but only spotty, inconsistent adherens junctions were seen, as visualized by Armadillo-GFP (driven by own promoter). At 16 hr, the fluorescent dye freely penetrates into the nerve cord, but by 20 hr the nerve cord is completely sealed. The completion of the seal thus coincides with the onset of visible movements in the late embryo (Schwabe, 2005).
To further gauge the dye-penetration assay, embryos mutant for known septate-junction components were examined: Neurexin IV, which is required for blood-nerve barrier formation in the PNS, Neuroglian, and the sodium-pump component Nervana 2, for which only a role in the earlier formation of the ectodermal seal has been demonstrated. In all three mutants, severe penetration of dye was found, well after the nerve cord is sealed in wild-type (22 hr). These findings provide further evidence that the sealing of the nerve cord is achieved by SJs and suggest that the components of the ectodermal SJs are required for the function of surface glial SJs as well (Schwabe, 2005).
In a genome-wide screen for glial genes, using FAC sorting of GFP-labeled embryonic glia and Affymetrix microarray expression analysis, two novel GPCRs, Moody (CG4322) and Tre1 (CG3171: Trapped in endoderm-1) were identified. Both are orphan receptors belonging to the same novel subclass of Rhodopsin-family GPCRs. Their expression was examined by RNA in situ hybridization; different subtypes of glia in the embryonic nerve cord can be distinguished based on their position and morphology. In the CNS, moody is expressed in surface glia from embryonic stage 13 onward (10 hr); in addition to cells surrounding the nerve cord (subperineurial glia), this includes cells lining the dorsoventral channels (channel glia). moody is also expressed in the ensheathing glia of the PNS (exit and peripheral glia). Both CNS and Peripheral nervous system expression of moody are lost in mutants for the master regulator of glial fate, glial cells missing (gcmN17), confirming that they are indeed glial. tre1 is expressed in all longitudinal glia and a subset of surface glia, as well as in cells along the midline. As expected, the (lateral) glial expression is lost in gcm mutants, while midline expression is not. Both moody and tre1 are also expressed outside the nervous system in a largely mutually exclusive manner, specifically in the germ cells, the gut, and the heart (Schwabe, 2005).
Several additional G protein signaling components are found in the surface glia. The six extant Gα genes show broad and overlapping expression in embryogenesis, with three of them (Go, Gq, and Gs) expressed throughout the nervous system and Gi expressed more specifically in surface glia. Gβ13F and Gγ1 are ubiquitously expressed during embryogenesis. Finally, the RGS loco is uniformly expressed in early embryos due to a maternal contribution but is then transcriptionally upregulated in surface and longitudinal glia, as well as in other tissues outside the nervous system. The nervous-system expression of loco is lost in gcm mutants. The presence of both Moody and Loco protein in the surface glia is confirmed using immunohistochemistry, but at 17 hr of development, when staining is feasible, the protein levels are still quite low (Schwabe, 2005).
In sum, the GPCR Moody, the RGS Loco, and Gi are differentially expressed in surface glia. This expression precedes and accompanies the morphogenesis and sealing of the surface glial sheath (Schwabe, 2005).
To examine protein expression and distribution of the GPCR signaling components in greater detail, third-instar larval nerve cords were examined. By this stage, the surface glia have doubled in size and show robust protein expression of GPCR signaling and SJ components (Schwabe, 2005).
Moody immunostaining is found at the plasma membrane, where it shows strong colocalization with the SJ marker Nrg-GFP. Loco immunostaining is punctate and more dispersed throughout the cytoplasm, with some accumulation at the plasma membrane, where it colocalizes with Moody. To avoid fixation and staining artifacts, fluorescent-protein fusions (Moody-mRFP; Loco-GFP) were generated and expressed using moody-Gal4, which drives weak surface glial expression. In the live nerve-cord preparations, Loco-GFP is much less dispersed and shows strong colocalization with Moody-mRFP at the plasma membrane (Schwabe, 2005).
In the absence of a known ligand, the coupling of G proteins to receptors is difficult to establish, but their binding to RGS proteins is readily determined. Loco physically binds to and negatively regulates Gi, and vertebrate Loco homologs (RGS12/14) have been shown to negatively regulate Gi/Go. In S2 tissue-culture assays, it was found that Loco binds to Gi and Go, but not to Gs and Gq. Double-label immunohistochemistry confirms that both Gi and Go are expressed in the surface glia (Schwabe, 2005).
Thus, Loco physically interacts with Gi and Go and shows subcellular colocalization with Moody, suggesting that the four signaling components are part of a common molecular pathway (Schwabe, 2005).
Using dye penetration as the principal assay, whether the GPCR signaling components that are expressed in surface glia play a role in insulation was examined. moody genomic (Δ17; Bainton, 2005) and RNAi mutants show similar, moderate insulation defects. The embryos are able to hatch but show mildly uncoordinated motor behavior and die during larval or pupal stages. The dye-penetration defect of moodyΔ17 is completely rescued by genomic rescue constructs containing only the moody ORF. Both moody splice forms (α and β; (Bainton, 2005) are able to rescue the defect independently, as well as in combination. tre1 genomic (Kunwar, 2003) and RNAi mutants show no significant dye-penetration defect and no synergistic effects when combined with moody using RNAi. Thus, despite the close sequence similarity of the two GPCRs and their partially overlapping expression in surface glia, only moody plays a significant role in insulation. Overexpression of moody causes intracellular aggregation of the protein (Schwabe, 2005).
loco is expressed both maternally and zygotically. loco zygotic nulls are paralytic and, on the basis of an ultrastructural analysis, a disruption of the glial seal, has been suggested. In a dye-penetration assay, loco zygotic null mutants show a strong insulation defect, which can be rescued by panglial expression of Loco in its wt or GFP-tagged form. The extant null allele of loco (Δ13) did not yield germline clones; therefore loco RNAi was used to degrade the maternal in addition to the zygotic transcript. In loco RNAi embryos, dye penetration is indeed considerably more severe. Overall, insulation as well as locomotor behavior is affected much more severely in loco than in moody and is close in strength to the SJ mutants. Overexpression of loco is phenotypically normal (Schwabe, 2005).
Thus, positive (moody) and negative (loco) regulators of G protein signaling show qualitatively similar defects in loss of function, suggesting that both loss and gain of signal are disruptive to insulation. Such a phenomenon is not uncommon and is generally observed for pathways that generate a localized or graded signal within the cell (Schwabe, 2005).
Both Gi and Go have a maternal as well as a zygotic component. Gi zygotic null flies survive into adulthood but show strong locomotor defects. In Gi maternal and zygotic null embryos show a mild dye-penetration defect, which is markedly weaker than that of moody, suggesting redundancy among Gα subunits. To further probe Gi function, the wt protein (Gi-wt) as well as a constitutively active version (Gi-GTP) were overexpressed in glia using repo-Gal4; such overexpression presumably leads to a masking of any local differential in endogenous protein distribution. Expression of Gi-wt results in very severe dye penetration, while overexpression of Gi-GTP is phenotypically normal. Only Gi-wt but not Gi-GTP can complex with Gβγ; overexpression of Gi-wt thus forces Gβγ into the inactive trimeric state. This result therefore suggests that the phenotypically crucial signal is not primarily transduced by activated Gi but rather by free Gβγ. Similar results have been obtained in the analysis of Gi function in asymmetric cell division (Schwabe, 2005).
Go null germline clones do not form eggs and do not survive in imaginal discs, indicating an essential function for cell viability (Katanaev, 2005). Therefore animals with glial overexpression of constitutively active (Go-GTP), constitutively inactive (Go-GDP), and wt (Go-wt) Go (Katanaev, 2005) were examined. Overexpression of Go-GDP, which cannot signal but binds free Gβγ, leads to severe dye penetration, again pointing to a requirement for Gβγ in insulation. However, Go-GTP and Go-wt show a moderate effect, suggesting that signaling by active Go does contribute significantly to insulation, in contrast to active Gi (Schwabe, 2005).
Overall, it was found that all four GPCR signaling components expressed in surface glia are required for insulation, further supporting the notion that the four components are part of a common pathway. The phenotypic data suggest that this pathway is complex: two Gα proteins, Gi and Go, are involved, but with distinct roles: activated Go and Gβγ appear to mediate most of the signaling to downstream effectors, while activated Gi seems to function primarily as a positive regulator of Gβγ. The loss of moody appears much less detrimental than the loss of free Gβγ (through overexpression of Gi-wt or Go-GDP); this is inconsistent with a simple linear pathway and points to additional input upstream or divergent output downstream of the G proteins. Finally, it was consistently observed that both loss (moody, Gi null, and Go-GDP) and gain (loco and Go-GTP) of signal are disruptive to insulation, suggesting that the G protein signal or signals have to be localized within the cell (Schwabe, 2005).
These complexities of G protein signaling in insulation preclude an unambiguous interpretation of genetic-interaction experiments and thus the linking of moody to Gi/Go/loco by genetic means. Double-mutant combinations between moody and loco were generated using genomic mutants as well as RNAi, with very complex results: in moody loco genomic double mutants, the insulation defect is worse than that of loco alone, while in moody loco RNAi double mutants the insulation defect is similar to that of moody alone. This strong suppression of loco by moody is also observed in the survival and motor behavior of the RNAi-treated animals. Thus the phenotype of the double-mutant combination is dependent on the remaining levels of moody and loco, with moody suppressing the loco phenotype when loco elimination is near complete (Schwabe, 2005).
To understand how the GPCR signaling components effect insulation at the cellular level, the distribution of different markers in the surface glia was examined under moody and loco loss-of-function conditions and under glial overexpression of Gi-wt. To rule out cell fating and migration defects, the presence and position of the surface glia were determined using the panglial nuclear marker Repo. In all three mutant situations, the full complement of surface glia is present at the surface of the nerve cord, with the positioning of nuclei slightly more variable than in wt (Schwabe, 2005).
In the three mutants, the SJ marker Nrg-GFP still localizes to the lateral membrane compartment, but the label is of variable intensity and sometimes absent, indicating that the integrity of the normally continuous circumferential SJ belt is compromised. Notably, the size and shape of the surface glia are also very irregular. While qualitatively similar, the phenotypic defects are more severe in loco and under Gi-wt overexpression than in moody, in line with the results of functional assays. When examining the three mutants with the actin marker GFP-Moesin, it was found that the cortical actin cytoskeleton is disrupted in varying degrees, ranging from a thinning to complete absence of marker, comparable to the effects observed with Nrg-GFP. However, GFP-positive fibrous structures are present within the cells, indicating that the abnormalities are largely restricted to the cell cortex. The microtubule organization, as judged by tau-GFP marker expression, appears normal in the mutants. The light-microscopic evaluation thus demonstrates that, in the GPCR signaling mutants, the surface glia are positioned correctly and capable of forming a contiguous epithelial sheet as well as septate junctions. Instead, the defects occur at a finer scale -- abnormally variable cell shapes and sizes, and irregular distribution of cortical actin and SJ material (Schwabe, 2005).
The changes in cell shape and actin distribution that were observed in the three mutants might simply be a secondary consequence of abnormalities in the SJ belt; to test this possibility, how a loss of the SJ affects the morphology and the actin cytoskeleton of the surface glia was examined. SJ components are interdependent for the formation and localization of the septa, and lack of a single component, such as Nrg, leads to nearly complete loss of the junction and severe insulation defects. In Nrg mutants, the surface glial cell shape and cortical actin distribution show only mild abnormalities. Thus, in contrast to the GPCR signaling mutants, the complete removal of the SJ causes only weak cytoskeletal defects, strongly arguing against an indirect effect. It is concluded that GPCR signaling most likely functions by regulating the cortical actin cytoskeleton of the surface glia, which in turn affects the positioning of SJ material along the lateral membrane (Schwabe, 2005).
More detailed insight into the nature of the defects in GPCR signaling mutants is afforded by electron microscopy. The surface glia in nerve cords of first-instar wild-type and mutant larvae were examined. Initially, dye penetration into the nerve cord was tested using ruthenium red. In wild-type, the dye diffuses only superficially into the surface glial layer, while in moody and loco mutants the dye penetrates deep into the nerve cord, in concordance with light-microscopic data. Tissue organization and SJ morphology were examined under regular fixation in randomly selected transverse sections. It has been reported that the surface glial sheath is discontinuous in loco mutant nerve cords, but this analysis was carried out at 16 hr of development, i.e., at a time when, even in wild-type, SJs are not yet established and the nerve cord is not sealed. In contrast to these findings, in the current study it was observed that, in loco as well as moody mutants, the glial sheath is in fact contiguous at the end of embryonic development. The ultrastructure of individual septa and their spacing also appear normal, indicating that moody and loco do not affect septa formation per se. However, the global organization of the junctions within the glial sheath appears perturbed: in wild-type, the surface glia form deep interdigitations, and the SJs are extended, well-organized structures that retain orientation in the same plane over long distances. In moody and loco mutants, the SJs are much less organized; they are significantly shorter in length and do not form long planar extents as in wild-type (Schwabe, 2005).
Taken together, the light- and electron-microscopic evaluations of the GPCR signaling mutants both show defects in the organization of the surface glial epithelium. The reduction in SJ length is consonant with the variability and local disappearance of the Nrg-GFP marker. Since the sealing capacity of the junction is thought to be a function of its length, the reduction in mean SJ length in the mutants provides a compelling explanation for the observed insulation defect (Schwabe, 2005).
Therefore, in addition to a reduction of the insulating SJs, this analysis of the GPCR signaling mutants revealed irregular cell shape and size, as well as weaker and variable accumulation of cortical actin in the surface glia. These data suggest that the primary defect in the mutants lies with a failure to stabilize the cortical actin, whose proper distribution is required for the complex extended morphology of the glia, which then affects SJ formation as a secondary consequence. Several lines of evidence exclude the reverse chain of causality, that is, a primary SJ defect resulting in destabilization of cortical actin and cell-shape change. Surface glia coalesce into a contiguous sheath and show strong accumulation of cortical actin before SJ material accumulates and sealing is completed. In the GPCR signaling mutants, there is misdistribution of SJ material along the cell perimeter, but the junctions do form. Finally, the GPCR signaling mutants show cell-shape and cortical actin defects that are much more severe than those observed in the near complete absence of SJ (Schwabe, 2005).
Compared to the columnar epithelia of the ectoderm and the trachea, the surface glial sheath is very thin. Compensating for their lack in height, surface glia form deep 'tongue-and-groove' interdigitations with their neighbors. This increases the length of the intercellular membrane juxtaposition and thus of the SJ, which ultimately determines the tightness of the seal. It is proposed that the surface glial interdigitations are the principal target of regulation by GPCR signaling. In GPCR signaling mutants, a loss of cortical actin leads to diminished interdigitation and thus to a shortening of the SJ, resulting in greater permeability of the seal. This model integrates all the observations at the light- and electron-microscopic levels (Schwabe, 2005).
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date revised: 10 August 2012
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