The orientation of the mitotic spindle relative to the cell axis determines whether polarized cells undergo symmetric or asymmetric divisions. Drosophila epithelial cells and neuroblasts provide an ideal pair of cells to study the regulatory mechanisms involved. Epithelial cells divide symmetrically, perpendicular to the apical-basal axis. In the asymmetric divisions of neuroblasts, by contrast, the spindle reorients parallel to that axis, leading to the unequal distribution of cell-fate determinants to one daughter cell. Receptor-independent G-protein signalling involving the GoLoco protein Pins is essential for spindle orientation in both cell types. This study identifies Mud as a downstream effector in this pathway. Mud directly associates and colocalizes with Pins at the cell cortex overlying the spindle pole(s) in both neuroblasts and epithelial cells. The cortical Mud protein is essential for proper spindle orientation in the two different division modes. Moreover, Mud localizes to centrosomes during mitosis independently of Pins to regulate centrosomal organization. It is proposed that Drosophila Mud, vertebrate NuMA4 and Caenorhabditis elegans Lin-5 have conserved roles in the mechanism by which G-proteins regulate the mitotic spindle (Izumi, 2006).
Drosophila neuroblasts delaminate from the epithelial cell layer and undergo asymmetric divisions to produce a chain of smaller ganglion mother cells (GMCs) on the basal side. These divisions are accomplished by localizing cell-fate determinants such as Numb, Prospero and its adaptor Miranda asymmetrically to the basal cortex, and rotating the spindle 90° to ensure unequal partitioning of the determinants. The atypical protein kinase C (aPKC)–Par complex (including Bazooka/Par-3, aPKC and Par-6) acts to create cell polarity in both epithelial cells and neuroblasts. The oriented division of those cells also requires heterotrimeric G-protein signalling, which involves the heterotrimeric G-protein subunit Galphai and guanine nucleotide dissociation inhibitors (GDIs) with the GoLoco motif (Pins and Loco), and can activate the Galpha and Gßgamma subunits independently of receptor signalling. Whereas in epithelial cells, Galphai and Pins localize to the lateral cortex, Inscuteable is expressed in neuroblasts. Inscuteable then recruits Pins–Galphai to the apical cortex by interacting with both Baz and Pins (Izumi, 2006).
Although a growing body of evidence indicates that the Pins–Galphai pathway is involved in the regulation of spindle orientation and spindle configuration in Drosophila, Caenorhabditis elegans and mammals, the underlying mechanisms are poorly understood. To address this question, molecules were sought that mediate the interactions of the Pins–Galphai complex with astral microtubules in Drosophila by coimmunoprecipitation with Pins. FLAG-tagged variants of Pins were overexpressed in embryos, and their extracts were subjected to immunoprecipitation with anti-FLAG antibody. A protein with a relative molecular mass of more than 200,000 was specifically coimmunoprecipitated with the amino-terminal region of Pins, PinsDelta5–FLAG. Mass spectrometry revealed this protein to be Mushroom body defect (Mud). The mud gene, which was previously identified from mutations affecting adult brain morphology, encodes several large coiled-coil proteins. Of the three characterized Mud isoforms, the longest isoform (2,501 amino acids) is mainly expressed in embryos. When wild-type embryos were subjected to immunoprecipitation with the anti-Pins antibody, the endogenous Mud protein coimmunoprecipitated with Pins. In in vitro binding assays, the Pins amino-terminal region directly interacts with a domain in the longest Mud isoform, which was found in the Mud fragment that was sufficient for the asymmetric distribution to the apical cortex. These results strongly indicate that Mud directly associates with Pins in vivo (Izumi, 2006).
Mud and Pins were compared in terms of their subcellular localization by generating several antibodies specific to different parts of Mud. In neuroblasts, Mud was detected at the apical cortex throughout the cell cycle, whereas it is barely detectable in the basal cortex. In addition, Mud emerged in both the apical and basal centrosomal regions during mitosis. Mud staining was stronger for the apical centrosome, reflecting the differential sizes of the two centrosomes. Mud and Pins colocalized at the apical cortex, although Pins was absent in the centrosomal regions. In epithelial cells, Mud and Pins colocalize along the lateral cortex throughout the cell cycle, whereas Mud (but not Pins) is also detected in the two centrosomal regions during mitosis. Both apical and centrosomal distributions of Mud were observed in dividing cells in mitotic domain 9 of the procephalic neuroepithelium, where cells divide perpendicular to the embryo's surface. These results indicate that the cortical domains where the two proteins colocalize are tightly correlated with spindle orientation in those three mitotic cell types (Izumi, 2006).
Next, how the subcellular localization of Mud is determined was examined. In both pins and Galphai mutant neuroblasts, Mud remains in the two centrosomal regions but fails to localize to the apical cortex during mitosis. Since the absence of Mud does not affect the asymmetric localization of Pins, this finding indicates that Pins recruits Mud to the apical cortex in mitosis via a direct molecular interaction. By contrast, during interphase, Mud localization is not affected in pins mutant cells, indicating that a secondary mechanism functions for Mud apical localization in interphase. By contrast, microtubules are required for the centrosomal, but not the cortical, localization of Mud; it distributes along microtubules near centrosomes during mitosis. When wild-type embryos were treated with colcemid to depolymerize the microtubules, Mud remained at the apical cortex in neuroblasts, but not in the centrosomes. Given this set of findings, it is concluded that Mud distributes in mitotic cells in two mutually independent ways: a Pins–Galphai-dependent mechanism for cortical localization, and a Pins-independent, microtubule-dependent mechanism for centrosomal accumulation (Izumi, 2006).
To investigate the role of Mud in spindle orientation, embryos homozygous for strong or null mud alleles were examined, since germline clone embryos that are both maternally and zygotically homozygous for any available mud mutation do not develop. From embryonic stage 11–12 onwards, Mud immunoreactivity becomes virtually undetectable, indicating that such embryos are in strongly hypomorphic states. During mitosis, these mutant neuroblasts localize Pins (and aPKC) and Miranda (and Prospero) in opposite cortical crescents. In wild-type neuroblasts, the asymmetric localization of these components represents cortical polarity that is perpendicular to the overlying epithelium. The orientation of the Miranda crescent in wild-type and mud neuroblasts is essentially indistinguishable during metaphase, indicating that mud neuroblasts retain cortical polarity with normal orientation. However, spindle orientation is severely affected in the mutant neuroblasts. In wild-type neuroblasts, the mitotic spindle orients along the apical–basal axis, tightly aligning with the polar distribution of Miranda (and Pins) from metaphase onwards. The spindle in mud neuroblasts, however, frequently fails to orient in the apical–basal direction, which results in its poor coordination with the basal Miranda crescent. 'Spindle coupling' is defined as how the spindle axis aligns in respect to the Miranda crescent or cortical polarity. The spindle uncoupling that is observed in mud neuroblasts continues until the completion of cytokinesis. It is concluded that Mud is required for the coupling of spindle orientation to cortical polarity (Izumi, 2006).
A similar failure in spindle coupling has been observed in pins-mutant neuroblasts; pinsp62 germline clone embryos were used to examine pins-null phenotypes, which are designated as 'pins-' or 'pins mutant' hereafter). However, pins mutants differ from mud mutants in two aspects: first, during metaphase and anaphase, both the Miranda crescent and the spindle misorient from the apical–basal axis in pins neuroblasts, whereas Miranda is oriented normally in mud metaphase neuroblasts. This indicates that Mud acts with Pins in spindle coupling with cortical polarity, but not in a separate role of Pins in maintaining the orientation of cortical polarity. Second, spindle coupling in pins mutants is, nevertheless, recovered to a large extent during telophase, a phenomenon that is termed 'telophase rescue'. Telophase rescue does not occur in mud mutants, indicating that Mud has a Pins-independent role at telophase (Izumi, 2006).
The size difference between two daughter cells is also affected in mud neuroblast divisions: the more tilted the spindle orientation, the less different the two daughter cells tend to be in stage-11 embryos. The aPKC–Par complex and Galphai–Pins function redundantly to make the two daughter cells unequal in size, presumably by regulating spindle organization. In mud-mutant neuroblasts, these components normally localize as apical crescents, generating normal cortical polarity. It is speculated that spindles that are oblique to the apical–basal axis would decrease differential effects of the apical signals on their two poles (or asters), which in turn reduces asymmetric spindle organization. When neuroblasts divide perpendicular to the apical–basal axis, spindles are indeed nearly symmetric. In these extreme cases, neuroblasts undergo centric divisions into two equal-sized daughters, both of which inherit Prospero. Although it is unclear how those daughter cells retain the properties of the neuroblast or the GMC, neuronal fate defects and/or loss of progeny neurons may occur. As expected, aberrant neuronal progeny were observed in mud-mutant embryos (Izumi, 2006).
In addition to the spindle uncoupling phenotype, supernumerary centrosomes are often observed in mud-mutant mitotic cells. These extra centrosomes are not accompanied by the formation of multipolar spindles, although infrequently a faint microtubule array emanates from an extra centrosome. Instead, a virtually normal bipolar spindle with astral microtubules is formed from a pair of centrosomes in those neuroblasts. Centrosome amplification may arise from abnormal assembly of centrosomes or cytokinesis defects. The absence of observable multinuclear figures or polyploidy in mud mutants indicates that cytokinesis occurs normally. Thus, in centrosomes, Mud seems to function in centrosome assembly or maintenance (Izumi, 2006).
Spindle uncoupling in mud mutants may be due to the loss of cortical Mud or, alternatively, result from the selection of two abnormally positioned centrosomes from supernumerary centrosomes to form the spindle. To distinguish between these two possibilities, spindle orientation was compared relative to the Miranda crescent in metaphase neuroblasts with two centrosomes and those having three or more centrosomes. The spindle orientation relative to the Miranda crescent was indistinguishable in the two neuroblast populations, indicating that spindle uncoupling occurs independently of the centrosome number in mud mutants. It is inferred from these results that cortical Mud is required for spindle coupling with cortical polarity. Mud in the centrosome may also contribute to a Pins-independent role of Mud in spindle coupling, which is suggested by the absence of telophase rescue in mud mutants, although other possibilities have not been ruled out (Izumi, 2006).
Unlike neuroblasts, proliferating epithelial cells orient the mitotic spindle parallel to the epithelial plane; this spindle alignment requires laterally distributing Pins. In mud-mutant epithelia, the spindle occasionally fails to orient in this direction, as has been described for pins mutants. The Mud–Pins complex therefore acts in spindle orientation in both neuroblasts and epithelial cells, such that the spindle orients towards the cortical domain where this complex resides (Izumi, 2006).
To further investigate the functional relationship between Mud and Pins, the effect of ectopic expression of Inscuteable was examined in epithelial cells, which do not normally express this protein. Ectopic Inscuteable relocalizes Pins from the lateral cortex to the apical cortex due to the ability of Inscuteable to bind both Bazooka and Pins, and rotates the division axis 90° to reorient along the apical–basal axis. The misexpression of Inscuteable also results in the relocalization of Mud to the apical cortex throughout the cell cycle, indicating that the Pins–Inscuteable complex can, in a dominant fashion, recruit Mud to the cortical region where the complex distributes. When Inscuteable is expressed in pins-mutant epithelial cells, the spindle does not rotate 90° due to the failure to localize Insc cortically, and frequently orients randomly, as observed in pins-mutant epithelial cells. The apical localization of Mud is never observed in these cells. These observations and the requirement of Pins for Mud localization in neuroblasts indicate that Pins is both necessary and sufficient to determine Mud cortical localization during mitosis (not interphase), and that the cortical position of the Mud–Pins (and Galphai) complex determines spindle orientation. Thus, cortical Mud probably acts downstream of Pins–Galphai to couple the spindle with cortical polarity (Izumi, 2006).
This study has shown that Mud forms a cortical complex with Pins–Galphai to act in spindle orientation during both the symmetric division of epithelial cells and the asymmetric division of neuroblasts. In vertebrates, NuMA associates with a Pins homologue LGN, which in turn binds Galphai/Galphao through its GoLoco motif and regulates spindle movement. C. elegans Lin-5 forms a complex with GoLoco proteins (GPR-1/2) and Galpha (GOA-1/GPA-16) to generate the pulling force for astral microtubules in one-cell zygotes. Therefore, NuMA, Lin-5 and Mud seem to have similar roles in the cortical processes that regulate spindle positioning. A short sequence shared by Mud, NuMA and Lin-5 is found within the respective GoLoco GDI-binding regions. This shared sequence may be important for the association with their GoLoco GDI partners (Izumi, 2006).
How then does Mud regulate spindle orientation at the cell cortex? The cortical Mud–Pins–Galphai complex probably interacts with the plus end of astral microtubules, either directly with tubulin or with microtubule plus-end-binding proteins (+TIPs). NuMA, indeed, interacts with the Dynein–Dynactin complex, which localizes to the plus-end of microtubules. Mud, therefore, links astral microtubules with Pins–Galphai, which is in turn connected with the aPKC–Par complex by Inscuteable. This sequential association between the apical components seems to achieve coupling of the spindle with cortical polarity in dividing neuroblasts. A recent study indicates that Pins apical localization is dictated by astral microtubules via +TIP Kinesin Khc-73 and the cortical Discs large protein. Mud may interact with this kinesin to affect spindle coupling (Izumi, 2006).
Another feature that is common to Mud, NuMA and Lin-5 is their localization to the centrosomal regions. The centrosomal localization of Mud is Pins-independent, and centrosome assembly is abnormal in mud mutants, but not in pins mutants. Mud function in the centrosome therefore seems to be independent of G-protein signalling. The Dynein/Dynactin complex cooperates with NuMA in the coalescence of spindle poles, as in the cell cortex. Drosophila mutants for Lis1 and glued (components of the Dynein–Dynactin complex) show defects in assembling microtubule minus ends at the pole. These observations raise the possibility that Mud may also function with the Dynein–Dynactin–Lis1 complex in centrosomal organization. Interestingly, a genome-wide two-hybrid analysis indicated that Mud binds Centrosomin, a protein that is necessary for centrosomal organization. Centrosomin may bind Mud at the centrosome to localize Mud (Izumi, 2006).
These findings suggest that the cortical complex of Galpha, GoLoco proteins and a coiled-coil protein (Mud, NuMA or Lin-5) functions in an evolutionarily conserved, receptor-independent mechanism that regulates spindle orientation. The 'search and capture' mechanism that is driven by molecules such as APC and EB1 has been proposed as another general mechanism for orienting the spindle. This mechanism is also thought to involve the Dynein–Dynactin complex. How the receptor-independent G-protein pathway and the search and capture system are related is an open question (Izumi, 2006).
To test whether the sequence similarity results in conserved protein interactions, Mud was examined to see if it is part of a ternary complex with Pins and Gαi. Similar to previous studies (Du, 2004; Du, 2001; Du, 2002), C-terminal truncations of Mud (Mud-C) were used, containing the putative Pins and microtubule binding regions. Gαi complexes were immunoprecipitated from S2 cells transfected with myc-tagged Mud-C. In untransfected cells, Gαi can coprecipitate Pins. Upon transfection of Myc-Mud-C, immunoprecipitation of Gαi coprecipitates Pins and Myc-Mud-C. This suggests that Mud-C is in a complex with Pins and Gαi. To determine whether Mud-C binds directly to Pins, these proteins were tested in an in vitro binding assay. In vitro-translated Mud-C can bind to bacterially produced GST-Pins, but not to GST alone, indicating that a Pins-Mud-C complex can form in vitro without additional cofactors. Consistent with this, Mud coimmunoprecipitates with Pins from wild-type embryo extracts. It is concluded that Mud binds to Pins by using a C-terminal region and is part of a ternary complex with Gαi (Bowman, 2006).
Because NuMA binds to the N-terminal TPR repeats of mammalian Pins (Du, 2001), whether Mud behaves similarly in Drosophila was investigated. For this, the N-terminal TPR and C-terminal GoLoco repeats of Drosophila Pins were GFP tagged and expressed in S2 cells with Myc-Mud-C. Immunoprecipitation of GFP shows that Myc-Mud-C binds to Pins-TPR-GFP, but not to GFP-Pins-GoLoco, indicating that Pins binds to Mud by using the TPR repeats. Gαi does not bind to Mud-C in vitro, but earlier work shows that Gαi directly binds to the C-terminal GoLoco repeats of mammalian and Drosophila Pins (Du, 2004; Schaefer, 2001). Because Mud, like NuMA, binds to the N terminus of Pins, this suggests that the geometry of the heterotrimeric NuMA-Pins-Gαi complex is conserved in Drosophila (Bowman, 2006).
The conserved C-terminal fragment of human NuMA can interact with the TPR repeats of Drosophila Pins (Du, 2004), so whether Mud could bind to human Pins was tested. For this, His-HsPins-TPR and GST-Mud-C fusion proteins were produced in bacteria and used in an in vitro binding assay. His-HsPins-TPR binds to GST-Mud-C, but not to GST alone. The binding of human Pins to Drosophila Mud argues for the evolutionary conservation of this interaction. Taken together, these results demonstrate that Mud is part of a heterotrimeric complex that is highly conserved from insects to vertebrates (Bowman, 2006).
NuMA binds to microtubules and can stimulate their polymerization (Du, 2002). To find out if Mud has similar biochemical qualities, whether Mud binds microtubules in a microtubule sedimentation assay was tested. In this experiment, a soluble protein extract was created from S2 cells transfected with Myc-Mud-C. Polymerization of microtubules with GTP and taxol, followed by high-speed centrifugation, separated microtubules and microtubule binding proteins from the supernatant. As expected, α-tubulin and the microtubule binding protein Eb1 remain soluble in the absence of GTP and taxol. When microtubules are stabilized, however, these proteins can be found in the microtubule pellet along with Pins and Myc-Mud-C. It is concluded that Mud and Pins can associate with microtubules. To test whether Mud, like NuMA, can stimulate microtubule polymerization, a solution microtubule formation assay was performed. Tubulin subunits labeled with rhodamine were incubated in an energy-regenerating system with GST, with the GST-Mud-C fusion protein, or in buffer alone. After fixation of this preparation to coverslips, the number of microtubules generated was counted in ten random fields. The average number of microtubules per field formed with GST or buffer alone is less than 20, but when tubulin is incubated with GST-Mud-C, the average number of microtubules formed increases to over 100 per field. This shows that the interaction of Mud-C with tubulin is direct, and, like NuMA-C (Du, 2002), Mud-C can stimulate microtubule formation in vitro. The interaction of Mud with microtubules together with its membership in a ternary complex with Pins and Gαi strongly suggest that Mud is the functional homolog of NuMA in Drosophila (Bowman, 2006).
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. 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, 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, 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. 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 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. 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. 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 our 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).
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