par-6: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References
Gene name - par-6

Synonyms -

Cytological map position - 16C5--6

Function - scaffolding protein

Keywords - oocyte, neuroblast, asymmetric division

Symbol - par-6

FlyBase ID: FBgn0026192

Genetic map position - 1-

Classification - PDZ domain protein

Cellular location - cytoplasmic

NCBI link: Entrez Gene
par-6 orthologs: Biolitmine
Recent literature
Whitney, D. S., Peterson, F. C., Kittell, A. W., Egner, J. M., Prehoda, K. E. and Volkman, B. F. (2016). Binding of Crumbs to the Par-6 CRIB-PDZ module is regulated by Cdc42. Biochemistry 55: 1455-1461. PubMed ID: 26894406
Par-6 is a scaffold protein that organizes other proteins into a complex required to initiate and maintain cell polarity. Cdc42-GTP binds the CRIB module of Par-6 and alters the binding affinity of the adjoining PDZ domain. Allosteric regulation of the Par-6 PDZ domain was first demonstrated using a peptide identified in a screen of typical carboxyl-terminal ligands. Crumbs, a membrane protein that localizes a conserved polarity complex, was subsequently identified as a functional partner for Par-6 that likely interacts with the PDZ domain. This study shows by nuclear magnetic resonance that Par-6 binds a Crumbs carboxyl-terminal peptide and reports the crystal structure of the PDZ-peptide complex. The Crumbs peptide binds Par-6 more tightly than the previously studied carboxyl peptide ligand and interacts with the CRIB-PDZ module in a Cdc42-dependent manner. The Crumbs:Par-6 crystal structure reveals specific PDZ-peptide contacts that contribute to its higher affinity and Cdc42-enhanced binding. Comparisons with existing structures suggest that multiple C-terminal Par-6 ligands respond to a common conformational switch that transmits the allosteric effects of GTPase binding.
Nunes de Almeida, F., Walther, R. F., Presse, M. T., Vlassaks, E. and Pichaud, F. (2019). Cdc42 defines apical identity and regulates epithelial morphogenesis by promoting apical recruitment of Par6-aPKC and Crumbs. Development 146(15). PubMed ID: 31405903
Cdc42 regulates epithelial morphogenesis together with the Par complex (Baz/Par3-Par6-aPKC), Crumbs (Crb/CRB3) and Stardust (Sdt/PALS1). However, how these proteins work together and interact during epithelial morphogenesis is not well understood. To address this issue, this study used the genetically amenable Drosophila pupal photoreceptor and follicular epithelium. During epithelial morphogenesis active Cdc42 accumulates at the developing apical membrane and cell-cell contacts, independently of the Par complex and Crb. However, membrane localization of Baz, Par6-aPKC and Crb all depend on Cdc42. Although binding of Cdc42 to Par6 is not essential for the recruitment of Par6 and aPKC to the membrane, it is required for their apical localization and accumulation, which was found to also depend on Par6 retention by Crb. In the pupal photoreceptor, membrane recruitment of Par6-aPKC also depends on Baz. This work shows that Cdc42 is required for this recruitment and suggests that this factor promotes the handover of Par6-aPKC from Baz onto Crb. Altogether, it is proposed that Cdc42 drives morphogenesis by conferring apical identity, Par-complex assembly and apical accumulation of Crb.

The par genes (partitioning defective) are required to establish embryonic polarity and direct asymmetric cell division in the C. elegans embryo. Drosophila Bazooka, a homolog of C. elegans Par-3, is required for establishing apical-basal polarity in epithelial cells and for orchestrating asymmetric cell division in neuroblasts. The PDZ-domain protein Par-6, the Drosophila homolog of C. elegans Par-6, cooperates with Bazooka for both of these functions. Par-6 colocalizes with Bazooka at the apical cell cortex of epithelial cells and neuroblasts, and binds to Bazooka in vitro. Par-6 localization requires Bazooka, and mislocalization of Bazooka through overexpression redirects Par-6 to ectopic sites of the cell cortex. In the absence of Par-6, Bazooka fails to localize apically in neuroblasts and epithelial cells, and is distributed in the cytoplasm instead. Epithelial cells lose their apical-basal polarity in Par-6 mutants and asymmetric cell divisions in neuroblasts are misorientated (Petronczki 2001). Par-6 and Bazooka also required to maintain oocyte fate. Germline clones of mutants in either gene give rise to egg chambers that develop 16 nurse cells and no oocyte (Huynh, 2001). These results indicate that homologous protein machineries direct asymmetric cell division in worms and flies (Petronczki, 2001; Huynh, 2001).

Asymmetric cell divisions, in which a cell produces two daughter cells of unequal size, protein content or developmental potential, are used to generate cell diversity during development in many organisms. In Drosophila embryos, neuroblasts delaminate from a polarized epithelium and divide asymmetrically into a smaller, basal ganglion mother cell (GMC) and a larger, apical daughter cell that retains neuroblast characteristics. Both apical-basal orientation of the mitotic spindle and correct basal localization of these determinants require a protein complex that assembles at the apical cell cortex during neuroblast delamination. Inscuteable, a key component of this complex, starts to be expressed during neuroblast delamination and accumulates in an apical stalk that extends into the epithelial cell layer. During interphase and most of mitosis, Inscuteable forms an apical cortical crescent until it disappears in anaphase. In the absence of Inscuteable, mitotic spindles in neuroblasts fail to rotate into an apical-basal orientation (Petronczki, 2001 and references therein).

The PDZ domain protein Bazooka binds directly to Inscuteable. Bazooka has an important function in epithelial cells where it localizes to the apical cell cortex and is required for maintaining apical-basal polarity. When neuroblasts delaminate, the apical localization of Bazooka is maintained. The protein colocalizes with Inscuteable in the apical stalk and then at the apical cell cortex; like Inscuteable, it disappears in anaphase. In the absence of Bazooka, Inscuteable fails to localize asymmetrically and is found in the cytoplasm instead. Thus, Bazooka seems to be part of a protein pathway that connects asymmetric cell division in neuroblasts to apical-basal polarity in epithelial cells (Petronczki, 2001 and references therein).

The primary sequence of Bazooka is highly similar to the C. elegans protein PAR-3. In C. elegans, the first cell division of the zygote is asymmetric and generates a larger, anterior AB and a smaller, posterior P1 cell. The genes par-1 (see Drosophila Par-1), par-2, par-3, par-4 , par-5 and par-6, the atypical protein kinase C pkc-3 (see Drosophila atypical protein kinase C) and the non-muscle myosin nmy-2 (see Drosophila Zipper) are all required to generate this morphological and developmental asymmetry. Whereas PAR-1 and PAR-2 localize to the posterior cell cortex during the first cell division, PAR-3, PAR-6 and PKC-3 colocalize at the anterior cell cortex and have a strikingly similar phenotype, suggesting that they have a very close functional connection (Petronczki 2001 and references therein). Drosophila Par-6 was identified by Hung and Kemphues (1998) in their characterization of C. elegans PAR-6 (Petronczki, 2001).

A connection with epithelial polarity was established by following Par-6 localization in epidermal cells, where the development of epithelial polarity is well understood. During stage 5 of embryogenesis, before the establishment of epithelial polarity, Par-6, which is maternally expressed, is localized in the cytoplasm. After the establishment of apical and basolateral membrane domains during gastrulation, Par-6 becomes localized exclusively at the apical cortex of epithelial cells. After germband retraction, Par-6 is concentrated in two apicolateral spots. Co-staining with Armadillo, a marker for adherens junctions, shows that Par-6 is concentrated in adherens junctions and in the cortical area located just apical to these junctions. It is concluded that Par-6 is apically localized in polarized epithelial cells (Petronczki, 2001).

Drosophila neuroblasts arise from polarized epithelial cells. Par-6 localization was followed during neuroblast delamination and through neuroblast cell division. Par-6 is localized in an apical stalk that extends into the epithelium during neuroblast delamination, and in an apical cortical crescent in delaminated interphase and metaphase neuroblasts. During telophase, the crescent becomes wider and weaker, indicating that the protein becomes delocalized and finally disappears. This subcellular localization is reminiscent of Bazooka and, indeed, double staining for Par-6 and Bazooka shows colocalization of the two proteins in epithelial cells and neuroblasts. Thus, Par-6 and Bazooka colocalize at the apical cell cortex of epithelial cells and neuroblasts. In neuroblasts, colocalization of Par-6 and Inscuteable is also observed. Par-6 has also been shown to physically associate with Bazooka in vitro (Petronczki, 2001).

Par-6 colocalizes with Bazooka in epithelial cells and neuroblasts. Whether there is a functional connection between the two proteins was tested by analysing Par-6 localization in RNA interference (RNAi) mutants of bazooka (bazookaRNAi) in which both maternal and zygotic bazooka function are disrupted. Whereas Par-6 is apically localized in epithelial cells and forms an apical cortical crescent in 93% of metaphase neuroblasts in control-injected embryos, Par-6 was homogeneously distributed in the cytoplasm of bazookaRNAi mutant embryos. In these embryos, 100% of metaphase neuroblasts that had lost Bazooka protein showed cytoplasmic localization of DmPAR-6. Thus, both apical and cortical localization of Par-6 require bazooka. Whether mislocalization of Bazooka can redirect Par-6 localization was tested by overexpressing Bazooka in Drosophila embryos using the UAS-GAL4 system. Overexpression of bazooka perturbs epithelial polarity and results in accumulation of Bazooka protein at ectopic sites of the cell cortex. Co-staining of Bazooka overexpressing embryos for Bazooka and Par-6 has revealed that the two proteins colocalize at these ectopic positions, indicating that Bazooka is not only required but also sufficient for localization of Par-6 (Petronczki, 2001).

The function of Bazooka in neuroblasts, at least in part, is to localize Inscuteable to the apical cortex. Bazooka is strictly required for Inscuteable localization, but Inscuteable is dispensable for Bazooka localization even though Bazooka crescents become weaker in Inscuteable mutants. Therefore Par-6 localization was analyzed in inscuteable mutants. Whereas 88% of metaphase control neuroblasts showed a strong apical crescent, normal localization of Par-6 was only observed in 14% (n = 42) of inscuteableP72 mutant neuroblasts. In 52% of these neuroblasts, Par-6 was localized into an apical crescent that was weaker and extended further to the lateral cortex than in control embryos and in 33% of the metaphase neuroblasts, Par-6 was not asymmetrically localized. Thus, although Bazooka is strictly required for Par-6 localization, absence of Inscuteable only causes a partially penetrant defect in Par-6 localization (Petronczki, 2001).

Therefore Drosophila Par-6 has an important function in both maintaining apical-basal polarity of epithelial cells and directing asymmetric cell division of neuroblasts in Drosophila. Physical interaction, colocalization and functional similarity of Par-6 with Bazooka, the Drosophila PAR-3 homolog, all indicate that these two proteins may cooperate closely in these functions. In neuroblasts Inscuteable may be a functional part of this complex and is recruited into this complex through direct interaction with Bazooka (Petronczki, 2001 and references therein).

In C. elegans, PAR-3 and PAR-6 colocalize at the anterior cell cortex during the first cell division of the zygote. Like their Drosophila homologs Bazooka and Par-6, PAR-3 and PAR-6 are co-dependent for asymmetric and cortical localization and essential for asymmetric cell division. However, their mutant phenotypes show characteristic differences to Drosophila: whereas mitotic spindles are misorientated in bazooka or Par-6 mutant Drosophila neuroblasts, no defect in spindle orientation during the first cell division has been reported for the corresponding C. elegans mutants. During the second cell division, PAR-3 and PAR-6 actually act as inhibitors of spindle orientation: the anterior daughter cell of the zygote that inherits PAR-3 and PAR-6 inappropriately rotates its mitotic spindle by 90° in par-3 or par-6 mutants. Moreover, the size difference between the two daughter cells of the C. elegans zygote is lost in these mutants, but bazooka or Par-6 mutant Drosophila neuroblasts still form two daughter cells of unequal size. Thus, the function of PAR-3 and PAR-6 in cell polarity seems to be conserved between worms and flies, but they may interact differently with the molecular machineries that orient and position mitotic spindles and determine daughter cell sizes (Petronczki, 2001 and references therein).

In fact, recent experiments have identified characteristic differences in the way size difference between the two daughter cells is achieved in the two organisms. In C. elegans the mitotic spindle moves posteriorly during the first cell division, leading to a more posterior positioning of the cleavage furrow. In Drosophila neuroblasts, the mitotic spindle itself becomes asymmetric. It forms a larger apical and a smaller basal aster, and the cleavage furrow does not form midway between the two centrosomes. This has been attributed to differences between the two centrosomes and thus may be independent of the cortical asymmetries mediated by PAR-3 and PAR-6 (Petronczki, 2001 and references therein).

Complex formation occurs between mammalian PAR-3 and PAR-6 homologs. These proteins bind by a direct interaction of the PAR-6 PDZ domain with the first PDZ domain of PAR-3. Consistent with this observation, Drosophila Par-6 binds to MBP-Bazooka but does not bind when the amino terminus including the first PDZ domain is deleted. In addition to PAR-6 and PAR-3, the mammalian complex also includes the atypical protein kinase C (PKC)-zeta (Drosophila homolog Atypical protein kinase C). In C. elegans, the atypical PKC, PKC-3, colocalizes with PAR-3/PAR-6, and disruption of PKC-3 by RNAi causes a PAR-3/par6-like phenotype, suggesting that atypical PKC is an additional functional part of the complex. Drosophila aPKC binds to Bazooka in vivo and can be co-immunoprecipitated with Par-6 from Drosophila embryos, suggesting that this third component is also conserved in flies. Together with the fact that human PAR-6 can actually stimulate PKC-zeta kinase activity, this offers the intriguing possibility that PAR-3/PAR-6 functions by localizing or locally activating an atypical PKC at the apical cell cortex of epithelial cells and neuroblasts. Human PAR-6 also interacts specifically with the GTP bound form of the small GTPases Cdc42 and Rac1, and might function as an effector of these small GTPases. In C. elegans, Cdc42 is required for asymmetric cell division, suggesting a functional connection to PAR-6. In Drosophila imaginal discs, absence of cdc42 or expression of a dominant-negative version results in epithelial defects, but a requirement in embryos or in asymmetrically dividing neuroblasts has not been investigated (Petronczki, 2001 and references therein).

Although many aspects of Par-6 function are conserved, no structural homolog of Inscuteable has been identified in other organisms and therefore the principal downstream effector seems to be unique to Drosophila. These results suggest, however, that Inscuteable is not the only downstream target of Par-6. Par-6 functions in epithelial cells where inscuteable is not expressed. In neuroblasts, Numb and Miranda are still asymmetrically localized in the absence of Inscuteable, even though their crescents form at random positions. In Par-6 germline clones, however, 80% of all neuroblasts show no asymmetric localization of Miranda, even though Inscuteable is only completely delocalized in 32% of these neuroblasts. Thus, Par-6 either interacts directly with the Numb and Miranda localization pathways or functions through other downstream targets. Whether atypical PKC and Cdc42 are components of such alternative pathways remains to be determined (Petronczki, 2001).

Par complex cluster formation mediated by phase separation

The evolutionarily conserved Par3/Par6/aPKC complex regulates the polarity establishment of diverse cell types and distinct polarity-driven functions. However, how the Par complex is concentrated beneath the membrane to initiate cell polarization remains unclear. This study shows that the Par complex exhibits cell cycle-dependent condensation in Drosophila neuroblasts, driven by liquid-liquid phase separation. The open conformation of Par3 undergoes autonomous phase separation likely due to its NTD-mediated oligomerization. Par6, via C-terminal tail binding to Par3 PDZ3, can be enriched to Par3 condensates and in return dramatically promote Par3 phase separation. aPKC can also be concentrated to the Par3N/Par6 condensates as a client. Interestingly, activated aPKC can disperse the Par3/Par6 condensates via phosphorylation of Par3. Perturbations of Par3/Par6 phase separation impair the establishment of apical-basal polarity during neuroblast asymmetric divisions and lead to defective lineage development. It is proposed that phase separation may be a common mechanism for localized cortical condensation of cell polarity complexes (Li, 2020).

How the conserved Par (Par3/Par6/aPKC) complex is selectively recruited and concentrated on membranes for polarity establishment remains unclear. In this study, different from previously reported crescent localization patterning, the endogenous Par complex is revealed to exhibit cell cycle-dependent discrete puncta formation on the apical cortex in Drosophila NBs. The condensed Par puncta emerge from prophase, further condensate and enlarge as a clustered puncta structure in metaphase, then subsequently disassemble into scattered small puncta from anaphase. The cell cycle-dependent clustering of Par proteins in Drosophila NBs were also observed by two recent studies. In vitro biochemical data together with heterologous cell-based studies showed that the Par3/Par6 complex can undergo liquid-liquid phase separation (LLPS) at very low protein concentrations, and mutations of Par3 or Par6 that impair LLPS were found to alter asymmetric cell division (ACD) in Drosophila NBs. It has been recently shown that the basal condensation of Numb in dividing NBs is also regulated by LLPS of the Numb/Partner of numb complex (Shan, 2018). Thus, LLPS may be a common mechanism for the local condensation of apical and basal polarity determining protein complexes (Li, 2020).

It is important to note that the Par proteins, each at their endogenous level, can form clustered puncta via LLPS on the cortex. Though the measured endogenous Baz level in Drosophila NBs was too low to induce its LLPS in the cytoplasm, two-dimensional membrane attachment was expected to locally enrich the protein and lead to its LLPS. In return, LLPS-mediated Par complex condensates formation acts as an effective way for cells to further concentrate limited amount of Par proteins to specific cell cortices for polarity establishment. It is proposed that apical Baz/Par3 localization is a balanced result of apical anchoring and LLPS-mediated local condensation (via multivalent protein-protein interaction, self-association, protein-membrane interaction, etc.). Thus, for the knock-in mutant Baz ΔNTD, partially impaired LLPS ability due to its defective oligomerization led to its less condensed localization and significant cytoplasmic diffusion. However, the situation was different for the overexpressed Baz NTDmu (driven by UAS/GAL4) in the rescue assay, which is ectopically localized. As LLPS is very sensitive to concentrations of biological components, an overexpression of Par proteins especially Baz/Par3, the core driving factor of LLPS, may cause artificial promotion of the Par complex condensation via LLPS. Whereas the apical anchoring capacity of NBs seems to have a limitation. In UAS/GAL4-based rescue assay, the overexpressed Baz WT phase condensates may just have reached the threshold of apical anchoring capacity, whereas the LLPS deficient, overexpressed Baz NTDmu broke the balance, and led to its cortical and cytoplasmic diffusion. If the expression level goes higher, even Baz WT can not be afforded apically. Consistent with this notion, high Flag-Baz expression in a WT background (driven by insc-gal4), has a dominant-negative effect and leads to ectopic localization of endogenous Par complex throughout the cortex, and consequently disrupts localization of basal proteins. Similarly, ectopic Baz localization was observed when exogenous Baz is forcedly expressed in embryonic NBs. It was recently shown that overexpression of Par3-induced cell polarity in apolar S2 cells by forming concentrated Par-dots that further fused into amorphous Par-islands. According to a study of protein LLPS on lipid membrane bilayers, protein clusters gradually grew and fused into larger ones with irregular shapes, and finally coalesced into a mesh-like network. Thus, the amorphous structure of Par-islands in S2 cells may arise from the overexpression and overaccumulation of Par3 in the membrane region. Therefore, caution should be taken in interpreting the overexpression phenotypes of Par3 (Li, 2020).

Another key finding in this study is that aPKC can be recruited and concentrated in Par3/Par6 condensates as an inactive client. Such condensed phase droplets could be an efficient mechanism for local condensation of aPKC. Spatiotemporal activation of aPKC (e.g., by Cdc42) and consequent phosphorylation on Par3 CR3 leads to disassembly of the Par complex condensates. Another cell cycle regulator that might play a role in Par LLPS regulation is Plk1, which inhibits the oligomerization of Par3 by phosphorylating NTD in C. elegans. A critical but currently unknown point is how the autoinhibition of Par3 is relieved, as the open conformation of Par3 is critical for the Par complex condensate formation. Nonetheless, it is likely that multilayered regulatory mechanisms can act concertedly to control the spatiotemporal assembly and disassembly of the Par complex phase separation, and hence the cell polarity regulations (Li, 2020).

It is increasingly recognized that LLPS is a common strategy for cells to form membrane-less compartments by selectively recruiting and condensing proteins/RNAs/lipids. In a broader sense, the local condensation of other master polarity complexes, such as the conserved Lgl/Dlg/Scribble complex in the apical-basal polarity, and the Prickle/Vangl and Frizzled/Disheveled/Diego complexes in the planar cell polarity, may adopt a similar LLPS-driven mechanism to establish cell polarity in different tissues. Like the Par complex proteins, all these complexes share several common features: (1) these proteins contain multiple domains, which mutually interact with each other or self-associate in vitro to form complex platform, which further recruits other binding partners to assemble into higher order protein interaction network; (2) these complexes are found to form condensed patches or puncta attached to the inner surface of plasma membranes in vivo; and (3) proteins within these condensed patches or puncta are highly dynamic and rapidly exchange with corresponding proteins in the cytoplasm. The multivalent interaction-induced LLPS theory can perfectly explain above phenomena, allowing the stable existence of large concentration gradients of the proteins within the local protein condensates and those in the cytoplasm, and at the same time, keeping the proteins in the condensed phase highly dynamic. Such dynamic association may be essential for the fast assembly/disassembly of these polarity complexes in responding to extrinsic/intrinsic cues/signals to rearrange the cell polarity. It is postulated that LLPS of polarity protein complexes induced by multivalent interactions is a general mechanism for the cell polarization (Li, 2020).

Glial-secreted Netrins regulate Robo1/Rac1-Cdc42 signaling threshold levels during Drosophila asymmetric neural stem/progenitor cell division

Asymmetric stem cell division (ASCD) is a key mechanism in development, cancer, and stem cell biology. Drosophila neural stem cells, called neuroblasts (NBs), divide asymmetrically through intrinsic mechanisms. This study shows that the extrinsic axon guidance cues Netrins, secreted by a glial niche surrounding larval brain neural stem cell lineages, regulate NB ASCD. Netrin-Frazzled/DCC signaling modulates, through Abelson kinase, Robo1 signaling threshold levels in Drosophila larval brain neural stem and progenitor cells of NBII lineages. Unbalanced Robo1 signaling levels induce ectopic NBs and progenitor cells due to failures in the ASCD process. Mechanistically, Robo1 signaling directly impinges on the intrinsic ASCD machinery, such as aPKC, Canoe/Afadin, and Numb, through the small GTPases Rac1 and Cdc42, which are required for the localization in mitotic NBs of Par-6, a Cdc42 physical partner and a core component of the Par (Par-6-aPKC-Par3/Bazooka) apical complex (de Torres-Jurado, 2022).

A precise regulation of ASCD is critical in development, tissue homeostasis, and tumorigenesis. Extrinsic signals from specialized microenvironments, the niches, importantly contribute to that regulation by promoting the stem cell fate in the daughter cell that receives those signals, whereas the other daughter cell enters a differentiation program. Intriguingly, the ASCD of some stem cells, including the Drosophila CNS stem cells called NBs, the subject of this study, seems to depend exclusively on intrinsic regulatory mechanisms. Then, are those stem cells completely independent of their surrounding environment? Also, how general is the requirement of niches and the signals secreted from them for maintaining the stem cell fate in an ASCD (de Torres-Jurado, 2022)?

This study shows that the ASCD of Drosophila NBs, a traditional paradigm for studying intrinsic ASCD regulatory mechanisms, do also depend on extrinsic cues secreted by a glial niche, which are in close contact with those neural stem cell lineages in the larval brain. However, these extrinsic signals are not required to maintain the stem cell fate. They ultimately impact on the regulation of intrinsic factors to induce differentiation in one daughter cell, repressing the self-renewal 'basal state' in this cell. Different studies have shown the possibility of growing in culture isolated larval NBs, which are able to form crescents and divide asymmetrically without any additional extrinsic signal. However, most of these experiments were performed using central brain type I NB lineage (NBI) NBs, which do not require Fra and Robo1 signaling for their correct development, or included type II NB lineage (NBII,) but only particular markers were analyzed (i.e., Baz and Pon). This is relevant as, for example, it was observed that the localization of some ASCD regulators were not affected without extrinsic signals (i.e., Insc, Mira, and Brat). In the experiments in culture, some of the latter regulators are frequently used. It is also key to properly quantify the cases of crescent formation at metaphase, as the phenotypes are never fully penetrant and can be even totally rescued at telophase. For example, in the system used in this study, aPKC showed about 50% localization failures at metaphase, implying that there are NBIIs that show aPKC crescents. Nevertheless, finding that glia-secreted cues are specifically required in NBII lineages was indeed very intriguing. NBII lineages are larger than NBI, as they undergo an additional proliferation phase through INPs and hence are more prone to induce tumor-like overgrowth when ASCD fails. Thus, additional levels of regulation might have evolved in these lineages to ensure the correct division of the NB and INPs to avoid overgrowths. This issue will be further examined in the future (de Torres-Jurado, 2022).

This work has unveiled a novel function for the axon guidance cues Netrins and their Fra/DCC-like receptor in regulating self-renewal versus differentiation in neural stem and progenitor cells of larval NBII lineages. The cortex glial niche that surrounds those lineages secretes Netrins, which modulate Robo1 signaling threshold levels through Fra and Abl kinase in stem and progenitor cells. Whereas Robo1 signaling is activated by its ligand Slit, also secreted by the glial niche, Abl kinase represses this signaling, and these balanced Robo1 signaling levels appear to be critical for the cell-fate commitment of the daughter cell prone to differentiate. The cortex glia dynamically undergoes remodeling through larval stages; only at late third instar larval stages (L3) the glia chamber enwrapping each NB lineage forms completely. This study already observed the presence of the secreted ligands Slit and NetA in the cortex glia at L2, suggesting that these cues are being secreted from the glia since the reactivation of dormant NBs at early L2 (de Torres-Jurado, 2022).

Slit-Robo signaling regulates progenitor cell proliferation in the mammalian CNS and promotes the terminal asymmetric division of a differentiation-committed cell in the Drosophila CNS. Likewise, in mammary stem cells, Robo1 favors their asymmetric mode of cell division. Slit-Robo signaling is also required in other stem or progenitor cells to regulate their lineage specification, identity, or their adhesion/anchoring to the niche. No role for Netrin-Fra/DCC signaling has been previously described in all those contexts (de Torres-Jurado, 2022).

Ultimately, a transcriptional control has been pointed out as the most common way of action by which Robo signaling regulate the above-mentioned cellular processes. This study shows a novel, transcription-independent mechanism by which Robo1 signaling regulates ASCD. Robo1 signaling would be required to activate the small GTPases Rac1 and Cdc42 by repressing its inhibitor RhoGAP93B as well as by recruiting the Dock-Pak complex, which, through Pak, can also bind activated forms of both Rac1 and Cdc42 Rac1 and Cdc42 downregulation directly impacted on the intrinsic machinery that modulate ASCD in neural stem and progenitor cells. Specifically, compromising those small GTPases led to defects in the localization of the ASCD regulators, Par-6, aPKC, Cno, and Numb, and the concomitant formation of ectopic NBs (eNBs) within brain neural lineages, a phenotype that recapitulates that of Slit-Robo1 signaling impairment. The overexpression of robo1 caused similar defects than the loss of robo1. It was also a similar phenotype than for the loss of fra, the downregulation of Abl, or the expression of a kinase dead form of Abl (unable to repress Robo1). Hence, based on all those experiments, a working model proposes that Netrin-Fra signaling would be modulating, through Abl kinase, the Robo1 signaling threshold levels necessary to regulate in turn the correct activity of the small GTPases Rac1 and Cdc42. In fact, and according to this, the expression of Rac1V12 within NBII lineages caused the formation of eNBs and led to defects in the localization of aPKC in NB and progenitor cells, a similar phenotype than that observed after overexpressing robo1 in these NBII lineages. It would be interesting to determine whether this novel function of Netrin-Fra/DCC signaling regulating ASCD is also conserved in vertebrates, and whether Robo/Rac1-Cdc42 signaling threshold levels in the above-mentioned contexts are also critical for and dependent on Netrin-DCC signaling, as was have found in Drosophila (de Torres-Jurado, 2022).


cDNA clone length - 2110 bases

Bases in 5' UTR - 274

Exons - 3

Bases in 3' UTR - 780


Amino Acids - 351

Structural Domains

Protein database searches reveal that C. elegans PAR-6 contains one PDZ domain. PDZ domains are protein motifs of approximately 100 amino acids that are found in a growing number of proteins and mediate protein-protein interactions. The PDZ domain of C. elegans PAR-6 shares most similarity to the PDZ domain of Tax clone 40, a human protein that interacts with Tax protein of Human T-CELL Leukemia virus (HTLV). The alignment of C. elegans PAR-6 PDZ with PSD-95 PDZ3 shows that the amino acids forming the b-sheets and a-helix structures in PSD-95 PDZ3 are well conserved in C. elegans PAR-6 PDZ, suggesting the overall secondary structure of the PDZ domain of PAR-6 would be similar to PSD-95 PDZ3. In database searches, Drosophila melanogaster and Mus musculus EST cDNA clones have been identified. These cDNA clones have been completely sequenced and it is found that the fly and mouse cDNAs show 47% and 45% overall similiarity with C. elegans PAR-6, respectively. The conservation is greatest among these homologs over a 115 amino acid region containing the PDZ domain; worm PAR-6 is 88% and 80% identical to the fly and mouse proteins, respectively. In addition to the PDZ domain, the N-terminal portions are also quite similar among the three proteins. When compared with the worm PAR-6 sequence over amino acids 14-96, the fly and mouse protein are 52% and 43% identical (Hung, 1999).

par-6: Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

date revised: 30 June 2001

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