The Interactive Fly

Zygotically transcribed genes

Protein targeting, Asymmetric cell division and apical/basal polarity

  • Drosophila Ric-8 regulates Gαi cortical localization to promote Gαi-dependent planar orientation of the mitotic spindle during asymmetric cell division
  • Inscuteable regulates the Pins-Mud spindle orientation pathway
  • A role for a novel centrosome cycle in asymmetric cell division
  • Asymmetric centrosome inheritance maintains neural progenitors in the mammalian neocortex
  • Drosophila neuroblasts retain the daughter centrosome
  • Drosophila Hey is a target of Notch in asymmetric divisions during embryonic and larval neurogenesis
  • Apical-basal polarity in Drosophila neuroblasts is independent of vesicular trafficking
  • Rap1 and Canoe/afadin are essential for establishment of apical-basal polarity in the Drosophila embryo
  • A NudE/14-3-3 pathway coordinates dynein and the kinesin Khc73 to position the mitotic spindle
  • The conserved Discs-large binding partner Banderuola regulates asymmetric cell division in Drosophila
  • Aurora A triggers Lgl cortical release during symmetric division to control planar spindle orientation
  • Uninflatable and Notch control the targeting of Sara endosomes during asymmetric division
  • Dlg5 maintains apical polarity by promoting membrane localization of Crumbs during Drosophila oogenesis
  • Role of Tau, a microtubule associated protein, in Drosophila photoreceptor morphogenesis
    Genes involved in protein targeting, Asymmetric cell division, apical/basal polarity

    Drosophila Ric-8 regulates Gαi cortical localization to promote Gαi-dependent planar orientation of the mitotic spindle during asymmetric cell division

    Localization and activation of heterotrimeric G proteins have a crucial role during asymmetric cell division. The asymmetric division of the Drosophila sensory precursor cell (pl) is polarized along the antero-posterior axis by Frizzled signalling and, during this division, activation of G&alpha:i depends on Partner of Inscuteable (Pins). This study establishes that Ric-8, which belongs to a family of guanine nucleotide-exchange factors for Gαi, regulates cortical localization of the subunits G&alpha: and Gβ13F. Ric-8, Gαi and Pins are not necessary for the control of the anteroposterior orientation of the mitotic spindle during pl cell division downstream of Frizzled signalling, but they are required for maintainance of the spindle within the plane of the epithelium. On the contrary, Frizzled signalling orients the spindle along the antero-posterior axis but also tilts it along the apico-basal axis. Thus, Frizzled and heterotrimeric G-protein signalling act in opposition to ensure that the spindle aligns both in the plane of the epithelium and along the tissue polarity axis (David, 2005).

    Analysis of spindle orientation in epithelial cells revealed that division takes place in the plane of the epithelium in both wild-type and pins mutant cells. This demonstrates, first, that the requirement for Pins/Gαi to maintain the planar orientation of the spindle is specific to pI cells and, second, that a pI-specific activity tilts the spindle in the absence of Pins/Gαi signalling. Fz signalling was an obvious candidate for this pI-specific activity for two reasons. First, Fz signalling is still active in the pins and Gαi mutants as the spindle was correctly oriented along the antero-posterior axis in these mutants. Second, Fz accumulates at the posterior apical cortex of pI cells and this accumulation of Fz is maintained in Gαi pI cells. It is therefore envisaged that, although orienting the spindle along the antero-posterior axis, Fz signalling may also be responsible for tilting the spindle along the apico-basal axis in the absence of Pins/Gαi signalling. fz,pins double mutants were analyzed to test this hypothesis. Strikingly, in the absence of both Fz and Pins, the spindle was parallel to the plane of the epithelium. Therefore, in the absence of Pins/Gαi signalling, the activity tilting the spindle along the apico-basal axis is Fz-dependent. Intriguingly, in fz,pins pI cells, the spindle was even less tilted than in wild-type cells, indicating that Fz may also tilt the spindle in wild-type cells along their apico-basal axis. To test this, spindle orientation was analyzed in the fz mutant. In the absence of Fz, division takes place within the plane of the epithelium, the spindle being less tilted than in wild-type cells. Together, these results demonstrate that in pI cells, a Fz-dependent activity tends to tilt the spindle along the apico-basal axis. This activity is counterbalanced by a Ric-8a/Pins/Gαi-dependent one that maintains the spindle in the plane of the epithelium. Orientation of the spindle in wild-type cells arises from this balance. Finally, the analysis of spindle orientation in baz mutant pI cells revealed that Fz exerts its activity on the spindle independently of Baz, and hence probably independently of the Par complex. The tight control of the spindle apico-basal orientation probably regulates the morphogenesis of the pIIb cell and of the differentiated sensory organs (David, 2005).

    In C. elegans, ric-8 regulates spindle positioning in anaphase, downstream of the par genes and upstream or downstream of the GPR-Gαi complex, which is the homologue of the Pins-Gαi complex. These data demonstrate that, in the dividing pI cell, Ric-8a is required for asymmetric localization of Pins, Baz and Numb and for mitotic-spindle positioning. It is proposed that these activities of Ric-8a depend on an unexpected function of Ric-8a: localizing Gαi and G<β13F at the plasma membrane. This study of ric-8a also revealed that, in the pI cell, ric-8a, pins, Gαi and Gγ1 are all required for orientation of the spindle within the plane of the epithelium. The milder apico-basal phenotype that was observed in ric-8a pI cells could be accounted for by some persistence of the Ric-8a protein in somatic clones. Alternatively, an intriguing possibility is that ric-8a may also affect Gαo activity, which has recently been proposed to act downstream of Fz signalling. ric-8a loss of function would thereby affect both the Fz- and Gαi-dependent activities exerted on the spindle, resulting in a milder apico-basal tilt (David, 2005).

    Importantly, developmental processes ranging from gastrulation, neural-tube closure, neurogenesis and retina formation to asymmetric segregation of cell-fate determinants require that spindle orientation is controlled in two directions: along the polarity axis of the tissue (antero-posterior, animal-vegetal, central-peripheral, etc) and parallel to the plane of the epithelium. This study shows that, in dividing pI cells, these two orientations are controlled by different and opposing activities. A Fz-dependent activity orients the spindle along the antero-posterior axis but tends to tilt it along the apico-basal axis, and a Gαi-dependent activity maintains the spindle parallel to the plane of the epithelium. The Fz- and Gαi-dependent activities are likely to act through forces pulling on astral microtubules. Fz and heterotrimeric G signalling are implicated in mitotic-spindle positioning during both symmetric and asymmetric cell division. The elucidation of the molecular mechanisms underlying these forces in the pI cell might therefore generally contribute to understanding of the mechanisms that control mitotic-spindle positioning (David, 2005).

    Inscuteable regulates the Pins-Mud spindle orientation pathway

    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).

    A role for a novel centrosome cycle in asymmetric cell division

    Tissue stem cells play a key role in tissue maintenance. Drosophila melanogaster central brain neuroblasts are excellent models for stem cell asymmetric division. It has been shown that their mitotic spindle orientation is established before spindle formation. This study investigated the mechanism by which this occurs, revealing a novel centrosome cycle. In interphase, the two centrioles separate, but only one is active, retaining pericentriolar material and forming a 'dominant centrosome.' This centrosome acts as a microtubule organizing center (MTOC) and remains stationary, forming one pole of the future spindle. The second centriole is inactive and moves to the opposite side of the cell before being activated as a centrosome/MTOC. This is accompanied by asymmetric localization of Polo kinase, a key centrosome regulator. Disruption of centrosomes disrupts the high fidelity of asymmetric division. A two-step mechanism is proposed to ensure faithful spindle positioning: the novel centrosome cycle produces a single interphase MTOC, coarsely aligning the spindle, and spindle-cortex interactions refine this alignment (Rusan, 2007).

    By prophase, NBs contain two MTOCs that are almost fully separated and aligned along the NB/GMC axis, but analysis of fixed NBs revealed a single MTOC positioned opposite the GMCs before mitotic entry. Thus MTOC behavior was examined throughout the cell cycle as an initial approach to test the hypothesis that fixing the position of one MTOC through successive divisions helps ensure persistent spindle orientation. Live NBs expressing GFP-G147, an MT-associated protein were examined, revealing a striking temporal difference in MTOC activity. During interphase, a single detectable MTOC persists opposite the previous division site; this is referred to as the dominant MTOC. As NBs approach mitosis, this MTOC increases activity (matures; empirically judged by size and MT fluorescence intensity), forming an MT basket around the nucleus. This is referred to as preprophase; this stage is also seen in fixed samples stained for tubulin. Soon after, sometime before the dominant MTOC fully matures, something striking happens: a second MTOC appears distant from the first. This is the second MTOC and this stage is referred to as prophase onset. The second MTOC increases activity, maturing ~10 min before nuclear envelope breakdown (NEB). Using 4D imaging, the possibility was excluded that the second MTOC was present earlier in another focal plane. To further assess this, forming spindles were imaged end on. It is clear that the second MTOC did not emerge from the dominant MTOC or travel around the nucleus. Instead, the second MTOC appeared roughly opposite the dominant MTOC. MTOC separation began immediately, and by NEB, they were 146 ± 20° apart. Thus, NBs form two distinct MTOCs: an MTOC persisting from the previous division and another only activated at mitotic entry (Rusan, 2007).

    This distant activation of the second MTOC raised questions about the centrosome cycle. One possibility is that NBs have two MT nucleating centrosomes, but only one can retain MTs and act as an MTOC during interphase, whereas the second acquires MT retention ability during mitotic entry, explaining the second MTOC's sudden appearance. There is precedent for this: mouse L929 cells have two γtub-bearing centrosomes that can nucleate MTs, but only one contains Ninein and can retain MTs to form an MTOC (Rusan, 2007).

    To test this hypothesis in NBs, EB1-GFP was used. This binds growing MT plus ends and reliably identifies MT nucleation sites. Only one nucleation site was present in interphase and preprophase, and a new nucleation site appeared distant from the first, consistent with spatially and temporally distinct second MTOC activation. Thus, NBs regulate MT nucleation and not just MT retention (Rusan, 2007).

    To examine how the new nucleation center forms, centrosomes were imaged using a PCM protein, GFP-Cnn. NBs contain a single detectable centrosome during interphase. When NBs reenter mitosis, a second Cnn-positive centrosome appears distant from the first, mimicking activation of the second MTOC. To verify that these occur simultaneously, NBs expressing mCherry-Tubulin (chTub) and GFP-Cnn were imaged. This revealed perfect temporal and spatial correlation between the appearance of the second centrosome and activation of the second MTOC. Physical separation of two centrosomes/MTOCs was never seen. This is the first example of asynchronous and physically distant centrosome maturation, suggesting that NBs use a novel centrosome cycle (Rusan, 2007).

    Higher temporal/spatial resolution imaging revealed that two GFP-Cnn spots separate during mitotic exit. One GFP-Cnn spot persists as the NB interphase centrosome, forming the dominant MTOC, whereas the other spot disappears. The persistent Cnn spot (centrosome) remains relatively stationary in interphase, consistent with the hypothesis that coarse spindle alignment begins in interphase by anchoring the dominant centrosome (Rusan, 2007).

    Centrosome fate was examined in the two daughters (new NB and GMC). They differ dramatically in PCM retention, in contrast to mammalian cells, where both daughters' centrosomes retain PCM. The GMC centrosome sheds all PCM. The new NB centrosome (that becomes the dominant MTOC) retains PCM throughout interphase and further accumulates PCM during the next mitosis. The complete shedding of PCM in GMCs appears to be the normal behavior of interphase centrosomes in most fly cells, whereas in syncytial early embryos, both daughters retain PCM foci through the cell cycle. In contrast to both cell types, the NB daughters exhibit differential PCM retention (Rusan, 2007).

    These data suggest that NBs have a novel centrosome cycle in which the second centrosome matures distant from the dominant centrosome/MTOC. One hypothesis to explain this would be the distal positioning of a differentially regulated centriole that is blocked from recruiting PCM in interphase and thus cannot form an MTOC until 'activated' during mitosis. If this centriole is always inherited by the GMC, it might also explain complete PCM loss as GMCs exit mitosis (Rusan, 2007).

    Centrioles were examined live to test this hypothesis, using the centriole marker GFP-PACT and Histone-GFP Mother/daughter centrioles disengaged in late telophase, as in mammalian cells and fly embryos. Thus, two NB centrioles are present throughout interphase despite the presence of only one MTOC (Rusan, 2007).

    The two centrioles then exhibit different behaviors. One remains fairly stationary, whereas the second moves to roughly the other side of the nucleus. Disengagement perfectly correlates with separation of Cnn spots, suggesting that the stationary centriole retains PCM to form the dominant MTOC and the mobile centriole completely sheds PCM. To test this, NBs were imaged expressing chTub and GFP-PACT. The stationary centriole retained MTs, whereas the mobile centriole did not. Upon reentering mitosis, the mobile centriole regained nucleation activity, forming the second MTOC (Rusan, 2007).

    This suggests that full separation of the MTOCs that organize the spindle is biphasic. It begins in interphase, when one centriole retains PCM, remains stationary, and forms the dominant MTOC, whereas the second centriole sheds PCM and becomes mobile. Movement of the second centriole away from the dominant MTOC in interphase accounts for ~70% of the separation needed to form a spindle. Mechanisms of transporting the mobile centriole remain to be identified, but it is nonrandom, as in 26/30 NBs, the second MTOC emerged more than 90° from the dominant MTOC. After the second MTOC is activated, the two separate the last 30%, most likely via MT sliding forces. This might explain defects in lissencephaly1 mutants, where MTOCs are only separated by 124° at NEB. Perhaps interphase centriole movement is normal, but MT-based MTOC separation is defective (Rusan, 2007).

    These data suggest that NBs differentially regulate the activity of their two centrioles within the same cytoplasm. Interestingly, a similar observation was made in clam eggs, which have three centrosomes just after fertilization. The sperm centrosome is functionally inactivated, whereas female centrosomes organize the meiotic spindle (Rusan, 2007).

    Next NB centrosome regulation was examined. In preprophase, one centriole (marked by anti-DPLP) formed the dominant MTOC, whereas the second centriole had no associated MTs and was randomly positioned, confirming the live-cell data. Thus whether γtub is recruited asymmetrically was examined. Fixed preprophase NBs had two centrioles; only that opposite the GMCs accumulated γtub. Further, both γtub and Cnn are absent from the NB centriole nearest the GMCs in interphase/preprophase (Rusan, 2007).

    Polo kinase promotes centrosome maturation by promoting γtub recruitment during mitotic entry. Differences in Polo localization/activity might underlie differences in timing of NB centrosome maturation. NBs expressing Polo-GFP and the centriole marker mCherry-DSAS-6 were examined. Only the centriole pair that forms the dominant centrosome was Polo-GFP positive during preprophase. Polo-GFP accumulated on the mobile centriole pair as the NB entered mitosis, increased on both centriole pairs through prophase, and moved on to kinetochores. When Polo-GFP was examined in cells exiting mitosis, it was retained at low levels on the dominant centrosome. In the future, it will be interesting to examine the localization of Aurora A, another centrosome regulator (Rusan, 2007).

    Unlike the distal appendages of mammalian mother centrioles, fly mother and daughter centrioles have been thought to have no known ultrastructural or molecular differences. These data suggest that differences exist. It is unlikely that this differential regulation is a result of location, as both centrioles are initially adjacent after disengagement. The differences may be due to centriole age or procentriole maturation state (Rusan, 2007).

    The NB spindle is largely aligned by NEB. Based on the current data, the hypothesis was tested that the dominant centrosome helps define one spindle pole before prophase. The angle between the dominant centrosome/MTOC axis and the anaphase axis was calculated, using the nuclear centroid as a fixed reference. This revealed two phases in defining the future spindle axis. Through prophase onset, the dominant centrosome remains fairly stationary roughly opposite the GMCs, agreeing with fixed images, whereas the second centriole moves to a distal position (to within 46 ± 33° of the anaphase axis). This is consistent with the hypothesis. The dominant centrosome may be immobilized by aster–cortex interactions or by absence of an active displacement mechanism. In the second phase, alignment is refined in prophase and prometaphase (the angle between the NB centrosome and anaphase axes decreases from 31 ± 29° to 15 ± 12° (Rusan, 2007).

    To further test whether anchoring the dominant centrosome helps roughly align the spindle, asl mutant NBs were imaged live. They lack functional centrosomes and astral MTs. Mutant NBs lack a dominant interphase centrosome, allowing assessment of its role in spindle orientation and asymmetric cell division. Live imaging revealed robust chromatin-mediated MT nucleation and spindle assembly producing fairly normal spindles. Spindle poles emerge from a disorganized MT array near the chromosomes that focuses as the spindle lengthened. Spindles do not rotate during formation, always forming along the initial pole separation axis, but do rotate during metaphase, suggesting that rotation can occur without astral MTs or that asl mutants have a reduced astral array sufficient for rotation. Surprisingly, consecutive divisions in asl mutants usually produce adjacent or near-adjacent daughters, as in wild type. In a few cases, however, spindles form parallel to the GMC cap and, presumably, the polarity axis (2/13; ~15%); these NBs divide symmetrically. This suggests that the second phase of spindle alignment can occur without a dominant centrosome and can rescue misalignment, as long as it is not too extreme, but occasional atypical symmetric divisions occur. This results in defective brain anatomy, with ectopic paired, smaller NBs, presumably progeny of symmetric divisions (Rusan, 2007).

    These data reveal two new aspects of asymmetric division in this stem cell model. First, cells can adjust the canonical centrosome cycle to allow novel cell behaviors, as was observed during clam meiosis. Central brain NBs also alter this cycle: rather than both centrosomes maturing in synchrony and proximity, the two centriole pairs are differentially regulated, maturing asynchronously and distant from one another. One retains MT nucleating activity throughout the cell cycle, forming the dominant MTOC during interphase, whereas the second is initially inactive, only forming a functional centrosome and nucleating MTs at mitotic entry. One speculative possibility is that these are mother and daughter centrioles and that one is preferentially retained in the stem cell, a hypothesis that will now be tested. It is also of interest to ask whether other stem cells use this mechanism (Rusan, 2007).

    Second, the data suggest that this novel centrosome cycle helps ensure high-fidelity spindle positioning and thus asymmetric division. A model is proposed in which NB mitotic spindles are aligned in two phases to ensure that GMC daughters are born next to the previous GMC. Rough alignment is achieved by confining the dominant MTOC to a relatively fixed position from the previous division and moving the second centriole to the other side of the cell. As spindles form, a second process refines this initial alignment. In asl mutants, without centrosomes, the first mechanism is inactive, but the second mechanism can align the spindle unless initial alignment is wildly off axis. In mud mutants, centriole separation must occur normally, as prophase MTOCs are nearly fully separated, but alignment of spindle poles to cortical polarity cues is defective. The normal two-step process is a robust mechanism ensuring successful asymmetric divisions and reproducible brain anatomy (Rusan, 2007).

    Asymmetric centrosome inheritance maintains neural progenitors in the mammalian neocortex

    Asymmetric divisions of radial glia progenitors produce self-renewing radial glia and differentiating cells simultaneously in the ventricular zone (VZ) of the developing neocortex. Whereas differentiating cells leave the VZ to constitute the future neocortex, renewing radial glia progenitors stay in the VZ for subsequent divisions. The differential behaviour of progenitors and their differentiating progeny is essential for neocortical development; however, the mechanisms that ensure these behavioural differences are unclear. This study shows that asymmetric centrosome inheritance regulates the differential behaviour of renewing progenitors and their differentiating progeny in the embryonic mouse neocortex. Centrosome duplication in dividing radial glia progenitors generates a pair of centrosomes with differently aged mother centrioles. During peak phases of neurogenesis, the centrosome retaining the old mother centriole stays in the VZ and is preferentially inherited by radial glia progenitors, whereas the centrosome containing the new mother centriole mostly leaves the VZ and is largely associated with differentiating cells. Removal of ninein, a mature centriole-specific protein, disrupts the asymmetric segregation and inheritance of the centrosome and causes premature depletion of progenitors from the VZ. These results indicate that preferential inheritance of the centrosome with the mature older mother centriole is required for maintaining radial glia progenitors in the developing mammalian neocortex (Wang, 2009).

    Radial glia cells constitute a major population of neural progenitor cells that occupy the proliferative VZ in the developing mammalian neocortex. In addition to their well-characterized function as a scaffold in supporting neuronal migration, radial glia cells display interkinetic nuclear oscillation and proliferate extensively at the luminal surface of the VZ (that is, the VZ surface). During the peak phase of neurogenesis [around embryonic day 13-18 (E13-E18) in mice] they predominantly undergo asymmetric division to self-renew while simultaneously giving rise either directly to a neuron, or to an intermediate progenitor cell which subsequently divides symmetrically to produce neurons. Whereas differentiating progeny progressively migrate away from the VZ to form the cortical plate (CP) (the future neocortex) renewing radial glia progenitors remain in the VZ for subsequent divisions. The distinct migratory behaviour of radial glia progenitors and their differentiating progeny is fundamental to the proper development of the mammalian neocortex; however, little is known about the basis of these behavioural differences (Wang, 2009).

    Centrosomes, the main microtubule-organizing centres in animal cells, have an important role in many cell processes, particularly during cell division. All normal animal cells initially inherit one centrosome, consisting of a pair of centrioles surrounded by an amorphous pericentriolar material. The two centrioles differ in their structure and function. The older 'mother' centriole, which is formed at least one-and-a-half generations earlier, possesses appendages/satellites that bear specific proteins, such as cenexin (also known as Odf2), and anchor microtubules and support ciliogenesis. In contrast, the younger 'daughter' centriole, which is formed during the preceding S phase, lacks these structures. Full acquisition of appendages/satellites by the daughter centriole is not achieved until at least one-and-a-half cell cycles later. During each cell cycle, the centrosome replicates once in a semi-conservative manner, resulting in the formation of two centrosomes: one of which retains the original old mother centriole (that is, the mother centrosome) while the other receives the new mother centriole (that is, the daughter centrosome). This intrinsic asymmetry in the centrosome has recently been demonstrated to be important for proper spindle orientation during the division of male germline stem cells and neuroblasts in Drosophila, although female germline stem cells appear to divide normally in the absence of centrioles/centrosomes. These studies indicate a critical role for the differential behaviour of centrosomes with differently aged mother centrioles in asymmetric division of the progenitor/stem cells, although it remains unclear whether proper behaviour and development of the progenitor/stem cells and their differentiating daughter cells depend on centrosome asymmetry. Asymmetric division of radial glia progenitors accounts for nearly all neurogenesis in the developing mammalian neocortex. Three out of four autosomal recessive primary microcephaly (MCPH) genes identified so far encode centrosomal components, suggesting that proper neocortical neurogenesis and development entail a tight regulation of the centrosome, which is so far poorly understood. To address these issues, centrosome regulation during the peak phase of mammalian neocortical neurogenesis was investigated (Wang, 2009).

    To examine centrosome behaviour, a plasmid encoding centrin 1, a central component of the centriole, fused with enhanced green fluorescent protein (EGFP-CETN1) was introduced into the developing neocortex of E13.5 mouse embryos by in utero electroporation. As expected, EGFP-CETN1 formed pairs of dots that co-localized with γ-tubulin, a centrosomal marker, suggesting that transient expression of EGFP-CETN1 reliably labels the two centrioles of individual centrosomes in the developing neocortex in vivo. Moreover, it was observed that at the onset of peak neurogenesis (E13-E14), EGFP-CETN1-labelled centrosomes were predominantly located at the VZ surface with a small subset located in the subventricular zone (SVZ) and the intermediate zone (IZ) (Wang, 2009).

    To identify the cell types harbouring EGFP-CETN1-labelled centrosomes, a plasmid encoding DsRedexpress (DsRedex), a red fluorescent protein that diffuses throughout cells and thereby reveals their morphology, was co-electroporated . In bipolar radial glia progenitors in the VZ the centrosome was located in their ventricular endfeet at the VZ surface, whereas in multipolar cells in the IZ and the SVZ the centrosome was harboured in their cell bodies. Moreover, dividing radial glia progenitors were observed that possess a pair of centrosomes together with condensed chromosomes at the VZ surface. Consistent with this differential centrosome localization between radial glia progenitors and their differentiating daughter cells, a progressive increase was observed in the appearance of EGFP-CETN1-labelled centrosomes in the IZ and the CP as development proceeded, in addition to some that remained at the VZ surface. This gradual increase in centrosome localization in the IZ and the CP coincided with the production and migration of differentiating cells such as neurons to these regions during this period (Wang, 2009).

    The distinct positioning of the centrosome in radial glia progenitors versus their differentiating progeny prompted asking whether the centrosomes in these two cell populations/types are different. To explore this, a plasmid encoding ninein, a mature centriole-specific protein that localizes to appendages/satellites, fused with EGFP (EGFP-Nin) together with a plasmid encoding CETN1 fused to DsRedex (DsRedex-CETN1) were electroporated into the developing mouse neocortex at E13.5. As expected, both EGFP-Nin and DsRedex-CETN1 formed dot-like structures and co-localized to the centrosomes, especially those at the VZ surface, as identified by an antibody to the integral centrosomal protein pericentrin 1. Notably, EGFP-Nin was preferentially concentrated at one of the two centrioles marked by DsRedex-CETN1 in individual centrosomes, suggesting that the two centrioles in interphase radial glia progenitors are not identical. Given that Nin specifically associates with mature centrioles, these results indicate that the centriole with abundant EGFP-Nin is the more mature mother centriole, whereas the one with little EGFP-Nin is the less mature daughter centriole. A similar inequity in the recruitment of EGFP-Nin by the duplicated centrosomes was also observed in dividing radial glia progenitors at the VZ surface, indicating that the duplicated centrosomes are not identical during late mitosis. The centrosome with abundant EGFP-Nin is probably the centrosome that retains the mature old mother centriole and the centrosome with little EGFP-Nin is probably the centrosome that bears the relatively immature new mother centriole (Wang, 2009).

    Having found that the centrosomes in dividing radial glia progenitors exhibit asymmetry in their maturity, it was next asked whether this centrosome asymmetry is related to the distinct behaviour of radial glia progenitors and their differentiating progeny in the developing neocortex during neurogenesis. To address this, the relative distribution of centrosomes labelled by DsRedex-CETN1 versus those labelled by EGFP-Nin in the developing neocortex were compared as development proceeded. Interestingly, whereas DsRedex-CETN1-labelled centrosomes progressively occupied the IZ and the CP, where differentiating cells are situated, EGFP-Nin-labelled centrosomes were mostly found in the VZ, where radial glia progenitors are located. Given that DsRedex-CETN1 labels all centrosomes whereas EGFP-Nin selectively labels mature centrosomes, these results point to an intriguing possibility that the duplicated centrosomes in dividing radial glia cells are differentially inherited depending on their age and maturity during neocortical neurogenesis. It is known that during each cell division one centrosome retains the old mature mother centriole and the other bears the new less mature mother centriole. Thus, these results suggest that centrosomes with differently aged mother centrioles are differentially inherited by the two daughter cells of asymmetrically dividing radial glia progenitors (Wang, 2009).

    To test this, an assay was developed to distinguish explicitly between the centrosome containing the old mother centriole and the centrosome containing the new mother centriole in the developing neocortex in vivo. The assay takes advantage of the photoconvertible fluorescent protein Kaede, which changes from green to red fluorescence on exposure to violet light. Centrioles in the developing neocortex were labelled by transient expression of CETN1 fused with Kaede (Kaede-CETN1). Photoconversion was then performed to switch labelled centrioles from green to red fluorescence. This green-to-red fluorescence conversion of Kaede proteins is irreversible and the red protein is very stable, thus allowing long-term tracking of the existing photoconverted proteins and the structures with which they are associated. Moreover, all newly synthesized Kaede proteins are green fluorescent. It is known that centriole duplication requires new protein synthesis of centrin. As a result, newly duplicated centrioles that contain newly synthesized Kaede-CETN1 are green fluorescent, whereas previously existing centrioles are red fluorescent. Hence, in the first cell cycle after photoconversion, both centrosomes contain a red fluorescent mother centriole and a green fluorescent daughter centriole. However, in the second and subsequent cell cycles, centrosomes with the new mother centriole contain only green fluorescent centrioles, whereas centrosomes retaining the original old mother centriole harbour both red and green fluorescent centrioles, thus distinguishing between centrosomes with differently aged mother centrioles (Wang, 2009).

    To carry out this assay in the developing neocortex in vivo, an in utero photoconversion procedure was developed and and combined with in utero electroporation. Kaede-CETN1, which localized specifically to the centrosomes, was introduced into the developing mouse neocortex at E13.5. One day later, that is, E14.5, the forebrain of electroporated embryos was treated with a short exposure of violet light while still in the uterus, which effectively converted nearly all Kaede-CETN1 proteins and their labelled centrosomes from green to red fluorescence [E13.5-E14.5(PC)]. The uterus was replaced and the embryos continued to develop in vivo. The localization and inheritance of centrosomes were analysed at different developmental stages thereafter (Wang, 2009).

    It was found that one day after photoconversion [E13.5-E14.5(PC)-E15.5], around 95% of centrosomes contained both red and green fluorescent centrioles (indicated by yellow colour in the merged image), consistent with the notion that the labelled cells have undergone one round of division and have duplicated their centrioles during the 24-h period after photoconversion. This was shown directly by imaging centrosomes at high magnification, revealing that each centriole was mostly only red or green fluorescent. This also demonstrates that there is little diffusion of centrin proteins between duplicated centrioles, or between the centrioles and a cytoplasmic pool which was confirmed by fluorescence recovery after photobleaching (FRAP) experiments. Moreover, it was found that more than 30% of centrosomes possessed only green fluorescent centrioles 2 days after photoconversion [E13.5-E14.5(PC)-E16.5]. The appearance of the solely green fluorescent centrosomes 48 h after photoconversion indicates that the initially labelled radial glia progenitors have undergone two rounds of division during this period; this is consistent with the previous observation that the duration of the neocortical progenitor cell cycle is about 12 to 20 h around this developmental stage. The ongoing division of labelled radial glia cells at a normal rate suggests that expression of Kaede-CETN1 and the photoconversion procedure had no effect on their cell cycle. In addition, no obvious DNA damage or cell death was induced by the photoconversion treatment. Besides the green and yellow fluorescent centrosomes, about 4% of solely red fluorescent centrosomes were observed, indicating that a few labelled cells do not undergo cell division during this period (Wang, 2009).

    Having successfully distinguished the centrosomes with differently aged mother centrioles in the developing neocortex in vivo, their distribution was examined to determine whether they are asymmetrically segregated. Remarkably, it was found that more than 76% of centrosomes with the new mother centriole (that is, only green fluorescent) were located in the IZ and the CP, whereas around 78% of centrosomes with the old mother centriole (that is, both green and red fluorescent) were located in the VZ in addition to the SVZ. These results demonstrate that the centrosomes with differently aged mother centrioles are asymmetrically segregated in the developing neocortex during the peak phase of neurogenesis. It is worth noting that a small fraction of both green and red (that is, yellow) fluorescent centrosomes was found in the IZ and the CP and that these centrosomes probably originated from the first cell cycle after photoconversion (Wang, 2009).

    The asymmetric segregation of centrosomes suggests differential regulation of the duplicated centrosomes in dividing radial glia progenitors. To gather further evidence for this, time-lapse imaging experiments were carried out to monitor the behaviour of centrosomes with differently aged mother centrioles in dividing radial glia progenitors at the VZ surface in situ. Kaede-CETN1 was introduced into radial glia cells together with mPlum, a far-red fluorescent protein, to label cell morphology. Around 24 h later, cortical slices were prepared. Photoconversion of the existing Kaede-CETN1 proteins was then performed in individual slices, which were then cultured for another 24 h before being subjected to time-lapse imaging. Labelled dividing radial glia cells with enlarged and rounded cell bodies possessing a pair of centrosomes at the VZ surface were monitored at 10-min intervals over a period of 5 to 8 h. In six out of seven dividing radial glia cells that proceeded through mitosis at the VZ surface and reached the two-cell stage, the centrosome retaining the old mother centriole in both red and green fluorescence stayed at the VZ surface, whereas the centrosome containing the new mother centriole in solely green fluorescence migrated away from the VZ surface. These results demonstrate that the centrosomes with differently aged mother centrioles in dividing radial glia progenitors exhibit distinct behaviour during the peak phase of neurogenesis (Wang, 2009).

    The distinct behaviour of the centrosomes suggests that they are differentially inherited by the two daughter cells embarking on different routes of fate specification and development. On the basis of their behaviour, it is postulated that the centrosome with the new mother centriole is largely inherited by differentiating cells, such as neurons, whereas the centrosome with the old mother centriole that remains located at the VZ is mostly inherited by radial glia progenitors. Indeed, it was found that 2 days after photoconversion [E13.5-E14.5(PC)-E16.5] the centrosomes with the new mother centriole, marked by green fluorescence alone, were mostly associated with cells expressing TUJ1, a differentiating neuronal marker, in the CP and the IZ. In contrast, the centrosomes that retained the old mother centriole in yellow (that is, both green and red) fluorescence at the VZ were largely associated with cells expressing Pax6, a radial glia progenitor marker. These results show that the centrosomes with differently aged mother centrioles in dividing radial glia cells are asymmetrically inherited by the two daughter cells: whereas the renewing radial glia progenitor inherits the centrosome with the old mother centriole, the differentiating daughter cell inherits the centrosome with the new mother centriole (Wang, 2009).

    These data thus far show that centrosomes with differently aged mother centrioles are differentially inherited by the two daughter cells of asymmetrically dividing radial glia progenitors in the developing neocortex. Next, whether the selective inheritance of the centrosome with the old mature mother centriole by radial glia progenitors is necessary for their maintenance at the VZ was tested. Should this be the case, given that Nin is an essential component of the appendage/satellite structures specific to the mature centriole, it was predicted that removal of Nin, which prevents centriole maturation, would disrupt asymmetric segregation of centrosomes with differently aged mother centrioles and impair the maintenance of radial glia progenitors in the developing neocortex (Wang, 2009).

    To test this, short hairpin RNA (shRNA) sequences were developed against Nin, that effectively suppressed its expression (Nin shRNAs). Consistent with the prediction, expression of Nin shRNA, but not control shRNA, disrupted asymmetric segregation of centrosomes with differently aged mother centrioles labelled with Kaede-CETN1 in the developing neocortex, suggesting that Nin is necessary for centriole maturation, thereby generating asymmetry between duplicated centrosomes. The presence of solely green fluorescent centrosomes in Nin shRNA-expressing cortices indicates that centrosome duplication and segregation and cell division are not severely affected by removal of Nin. More importantly, it was found that removal of Nin caused a premature depletion of cells from the VZ, where radial glia progenitors reside. This effect of Nin shRNAs correlated with their efficacy in suppressing Nin protein expression and was rescued by a shRNA-insensitive Nin plasmid, suggesting that the effect of the Nin shRNA is due to a specific depletion of the endogenous Nin protein. A similar reduction in cells in the VZ was observed when Nin expression was suppressed using small interfering RNA (siRNA) (Wang, 2009).

    To characterize further the extent to which removal of Nin leads to a depletion of radial glia progenitors, the fate specification was examined of cells expressing either control or Nin shRNA. When compared with the control, expression of Nin shRNA led to a marked reduction in the percentage of cells positive for Pax6 and glutamate transporter (GLAST), two radial glia progenitor markers, and a significant increase in the percentage of cells positive for TUJ1, a differentiating neuronal marker. These results suggest that removal of Nin leads to a depletion of radial glia progenitors and a concomitant increase in differentiating neurons. Consistent with this, a significant reduction was observed in phospho-histone 3 (P-H3)-labelled mitotic cells at the VZ surface and a marked increase in cell cycle exit. No obvious change in the cleavage plane orientation of late stage mitotic cells at the VZ surface was observed (Wang, 2009).

    Previous studies showed that the carboxy-terminus of Nin is responsible for its localization to the centriole and expression of this region displaces endogenous protein at the centriole. Interestingly, it was found that, similar to removal of Nin, expression of the carboxy-terminus of Nin (Nin-Cter) led to a premature depletion of radial glia progenitor cells from the VZ, suggesting that centriolar Nin is critical for maintaining radial glia progenitor cells in the VZ. Taken together, these results indicate that preferential inheritance of a centrosome containing the mature mother centriole is required for the maintenance of radial glia progenitors in the proliferative VZ of the developing neocortex (Wang, 2009).

    The results presented in this study suggest that the centrosomes with differently aged centrioles in asymmetrically dividing radial glia progenitors exhibit different behaviour and are differentially inherited by the two daughter cells during the peak phase of mammalian neocortical neurogenesis. Whereas the centrosome with the less mature new mother centriole migrates away from the VZ surface and is largely inherited by differentiating cells, the centrosome with the more mature old mother centriole stays at the VZ surface and is predominantly inherited by renewing radial glia progenitors. Recently, asymmetric behaviour of centrosomes has been observed during asymmetric division of Drosophila male germline stem cells and neuroblasts. The findings of this study suggest that this type of asymmetric centrosome regulation may be a general feature of asymmetric cell division across species. Furthermore, the findings provide new insight into centrosome regulation in the developing mammalian neocortex, which has been linked to the pathogenesis of human microcephaly (Wang, 2009).

    Centrosomes with differently aged mother centrioles differ in their protein composition and thereby in their biophysical properties, such as microtubule anchorage activity. In this study, it was found that Nin, an appendage/satellite-specific protein required for centriole maturation, localized differently to the duplicated centrosomes in radial glia progenitors in late mitosis. Notably, another appendage/satellite-specific protein cenexin was recently found to be asymmetrically localized to centrosomes in sister cells after mitosis; moreover, the cell receiving the more mature old mother centriole usually grew a primary cilium first (Anderson, 2009). The asymmetric inheritance of centrosomes with distinct biophysical properties may thereby differentially regulate the behaviour and development of the daughter cells that receive them. For example, given that primary cilia have essential roles in a number of signal transduction pathways, including Sonic hedgehog (Shh) and platelet-derived growth factor (PDGF) signalling, the asynchrony in cilium formation could differentially influence the ability of the two daughter cells to respond to environmental signals and thereby their behaviour and fate specification. Furthermore, the strong microtubule anchorage activity associated with the centrosome retaining the older mother centriole would facilitate its anchorage to a specific site (for example, the VZ surface), thereby tethering the cell that inherits it. Indeed, it was found that disruption of centriole maturation by removing Nin not only impairs asymmetric segregation of centrosomes, but also depletes radial glia progenitors from the VZ, a proliferative niche in the developing mammalian neocortex. Aside from their participation in microtubule organization and ciliogenesis, centrosomes associate with messenger RNAs (mRNAs) and membrane-bound organelles such as the Golgi and recycling endosomes and regulate protein degradation, thereby raising the possibility that asymmetric centrosome inheritance might contribute to proper segregation of cell fate determinants to the two daughter cells of asymmetrically dividing progenitor/stem cells (Wang, 2009).

    Drosophila neuroblasts retain the daughter centrosome

    During asymmetric mitosis, both in male Drosophila germline stem cells and in mouse embryo neural progenitors, the mother centrosome is retained by the self-renewed cell; hence suggesting that mother centrosome inheritance might contribute to stemness. This hypothesis was tested in Drosophila neuroblasts (NBs) tracing photo converted centrioles and a daughter-centriole-specific marker generated by cloning the Drosophila homologue of human Centrobin. This study shows that upon asymmetric mitosis, the mother centrosome is inherited by the differentiating daughter cell. These results demonstrate maturation-dependent centrosome fate in Drosophila NBs and that the stemness properties of these cells are not linked to mother centrosome inheritance (Januschke, 2011).

    The correlation between mother versus daughter centrosome segregation and asymmetric cell fate was first documented in Drosophila male germline stem cells (mGSCs), in which the mother centrosome is anchored near the niche and is retained by the stem cell, whereas the daughter centrosome migrates to the opposite side of the cell and is segregated into the differentiating gonial cell after mitosis (Yamashita, 2007). More recently, mother centrosome retention has also been demonstrated during asymmetric mitosis in radial glia progenitors in the developing mouse neocortex. In Drosophila mGSCs, the possible functional relevance of such stereotyped centrosome behaviour is still unclear, although tantalizing hypotheses have been put forward. In the developing mouse neocortex, centriole maturation has been shown to be required for maintaining radial glia progenitors (Januschke, 2011 and references therein).

    Asymmetric centrosome behaviour has also been documented in Drosophila neuroblasts (NBs). NBs are stem-cell-like precursors that generate the fly's central nervous system through a series of asymmetric divisions giving rise to a self-renewed NB and a differentiating ganglion mother cell (GMC), which typically divides only once into a pair of neurons or glia. NB asymmetric division is largely driven by cortical polarity. It starts by the apical accumulation of a number of protein complexes including the Par complex (Par-3, Par-6, aPKC) as well as Mushroom body defect, recruited by Partner of Inscuteable, which associates with Gαi, Inscuteable and Par-3. Apical complexes control the clustering at the basal cortex of cell fate determinants, such as Numb, Prospero and Brain Tumour, through their adaptor proteins Partner of Numb and Miranda. The apical cortex also controls the orientation of the mitotic spindle along the apico-basal axis, which is essential to position the cytokinesis furrow, such that cleavage segregates the apical and basal sides of the cortex into the newly formed NB and GMC, respectively. NB asymmetric division is also controlled by other auxiliary modulators (Januschke, 2011 and references therein).

    Mosaic clones mutant for any of the known cell fate determinants overgrow in the larval brain and develop as malignant neoplasms upon transplantation into wild-type adult flies, showing that correct execution of the asymmetric division programme in Drosophila NBs is crucial to prevent tumour growth. Centrosomes are important in this process and larval brains that have cells without centrosomes or with more than two centrosomes per cell are highly prone to developing tumours (Januschke, 2011 and references therein).

    NB asymmetry is not limited to cortical polarity at mitosis. Early in interphase, the NB centrosome splits in two, which display significant structural and functional differences. One of the resulting centrosomes retains most of the pericentriolar material (PCM) and remains at the apical cortex organizing the main microtubule network. The other centrosome has little, if any, PCM and microtubule organizing activity, and migrates extensively across the cytoplasm for most of the interphase. At mitosis, the apical centrosome is retained by the NB and the other centrosome is inherited by the GMC. Remarkably, at the time of splitting, the GMC-fated centrosome does contain a significant amount of PCM, as revealed by centrosomin fused to GFP which is shed off just before it starts to move. These centrosome asymmetries are thought to have a main role in the orientation of cortical polarity and mitotic spindle alignment (Januschke, 2011 and references therein).

    Unlike centrioles in vertebrate cells where centriolar satellites, appendages and cartwheel span correlate with centriole maturation, no ultrastructural dimorphism has been reported between mother and daughter centrioles in Drosophila. Mother or daughter centriole-specific molecular markers are also lacking in Drosophila. Consequently, it is still unclear if the conspicuously unequal size, activity and fate of Drosophila NBs' centrosomes correlate with centriole maturation. It is also unclear if at the time of splitting each centrosome contains a full diplosome or a single centriole, as is the case in Drosophila embryos and in some human cell lines (Januschke, 2011 and references therein).

    This study shows by tracing photo-converted centrioles that centrosome splitting occurs before centriole duplication and that the centrosome that loses PCM is motile during interphase. This centrosome is also inherited by the differentiating GMC and contains the mother centriole. The cloning of the Drosophila homologue of Centrobin is also reported. In vertebrate cell lines, Centrobin specifically accumulates on the daughter centriole. It was found that at the time of centrosome splitting, a yellow fluorescent protein (YFP) fusion to CNB stays on the centrosome that remains at the apical cortex, which is eventually inherited by the NB, and is excluded from the centrosome that breaks away and is fated to the GMC. From these observations, it is concluded that centrosome maturation and fate are tightly correlated during asymmetric mitosis in Drosophila NBs, in which the cell that retains stemness inherits the daughter centriole. It is also concluded that in spite of the lack of ultrastructural dimorphism, molecular dimorphism exists between mother and daughter Drosophila centrioles (Januschke, 2011).

    To investigate if mother-versus-daughter centriole differences determine centriole fate during NB asymmetric division, a direct approach was devised based on photo-conversion of a pancentriolar marker in the apical centrosome. There are two expected outcomes of such an experiment (see Photo-conversion of a centriolar marker in the apical centrosome). If at the time of photo-conversion, once the basal centrosome has moved away, the apical centrosome contains two centrioles, then, in the next cell cycle, both the apical and basal centrosomes will carry the converted signal). Maturation-dependent centrosome fate will become apparent in the following cycle by the consistent segregation of the converted signal, which traces the mother centriole to either the apical centrosome, which is retained in the NB, or the basal centrosome, which is fated to the GMC. Alternatively, if at the time of photo-conversion the apical centrosome contains only one centriole, the hypothetical maturation-dependent centrosome fate will be revealed in the next cell cycle when, upon centrosome splitting, only the apical or the basal centrosome will carry converted signal. In such case, the mother centriole will be systematically fated to either the NB or the GMC, respectively (Januschke, 2011).

    A time-lapse series including the most representative time points of a centrosome photo-conversion experiment using the pancentriolar marker PACT-d2Eos is presented. After the basal centrosome has started to move away from the apical centrosome, a pulse of 405 nm light is shed on a manually defined rectangular region of interest which overlaps and slightly exceeds the size of the apical centrosome, causing the photo-conversion of the centriole marker from green to red. The converted signal remains stably associated with the apical centrosome, which is retained by the NB after the ensuing mitosis, and migrates to the apical cortex in early interphase. When the basal centrosome breaks away and starts its characteristic migration through the cytoplasm, the photo-converted signal, which traces the mother centriole, goes with it and is subsequently delivered into the GMC at mitosis. Identical results were obtained in a total of 20 photo-conversion experiments (Januschke, 2011).

    Three main conclusions can be derived from these observations. First, at the time when apical and basal centrosomes split in Drosophila NBs, the non-motile apical centrosome contains only one centriole. Second, segregation of mother and daughter centrosomes is highly correlated with asymmetric cell fate. Finally, these results show that NBs retain the daughter rather than the mother centriole (Januschke, 2011).

    To independently corroborate these conclusions, it was decided to circumvent the current lack of molecular markers by cloning the Drosophila homologue of the human daughter-centriole-specific protein Centrobin (Jeong, 2007; Zou, 2005). By homology search, open reading frame CG5690, predicted to encode a 78.7 kDa protein of 689 aa (Flybase), was identified as the putative Drosophila centrobin (cnb) gene. Transgenic flies were generated constitutively co-expressing YFP-CNB, and a fusion between mKATE and the pancentriolar marker ASL47, and the localization of both fluorescent probes was followed in larval NBs. Although, as reported for other centriolar proteins, one or two randomly located YFP-CNB aggregates are often found in these cells, a clear pattern of YFP-CNB signal segregation emerges. Centrosome-bound YFP-CNB can be observed without interruption from telophase, up to the time of centrosome splitting, early in the following interphase. Then, when apical and basal centrosomes break apart, YFP-CNB remains bound to the apical non-motile centrosome, and cannot be detected over the migrating basal centrosome. Identical results were obtained in a total of 16 NBs (Januschke, 2011).

    Retention of the daughter-centriole marker YFP-CNB by the apical centrosome and the lack of YFP-CNB signal in the motile centrosome corroborates further the conclusions derived from the photo-conversion experiments. In addition, the age-dependent centriole-specific localization of YFP-CNB provides the first instance of molecular asymmetry between mother and daughter Drosophila centrioles (Januschke, 2011).

    Tracing mother and daughter centrioles with photo-converted PACT-d2Eos and YFP-CNB, respectively, it was shown that apical and basal Drosophila NB centrosomes do not have fully formed diplosomes when they split, and that mother-versus-daughter centriole segregation tightly correlates with cell fate; contrary to what has been generally assumed, the daughter centrosome is fated to be retained by the NB and the mother centrosome is fated to the GMC (Januschke, 2011).

    The functional relevance of specific centrosome retention by the stem cell in Drosophila remains to be ascertained. Both in mGSCs and in NBs, a microtubule-dependent mechanism hooks one centrosome to the region of the cortex, proximal to the hub in the germ line and apical in NBs38; that is, retained by the stem cell after mitosis. Preferential retention of the mother centrosome in the germ line might therefore simply be the inescapable consequence of carrying more PCM and having greater microtubule-organizing activity. This is less likely in NBs where the daughter centriole is retained, while the mother centriole breaks away carrying little, if any, PCM. That implies the removal of PCM from around the mother centriole and the retention of PCM by the daughter centriole. The shedding of PCM by the GMC-fated centrosome as it splits from the apical centrosome has been previously documented (Januschke, 2011).

    Precedence for centrosome transmitted cell fate information exist in other species, but not in Drosophila. There are, however, published data on the effect of switching centrosome fate by transient microtubule depolymerization. Upon disassembly of the interphase aster of the NB by treatment with microtubule poisons, the apical centrosome loses connection with the cortex and moves deeper inside the cell so that at mitosis onset, the position of both centrosomes is essentially randomized. Upon recovery of microtubule dynamics, mitosis resumes and asymmetric cell division takes place. In some cases, centrosome fate is switched so that the centrosome originally destined to the GMC is retained by the NB, and vice versa, yet mitosis generates an NB and a GMC that are morphologically normal, strongly suggesting that the identity of the resulting NB and GMC is not severely compromised. It is unknown, however, whether such switch in centriole fate might have long-term developmental consequences (Januschke, 2011).

    Centriole-maturation-dependent segregation in Drosophila NBs is somewhat reminiscent of spindle pole body (SPB) segregation in budding yeast. Similar to centrioles in animals, the SPB duplicates once per cell cycle. The 'new' SPB remains connected to the 'old' one by the bridge, which is cleaved in S phase. The two SPBs then separate to form the opposite poles of the spindle. In unperturbed cells, the 'old' SPB always migrates into the bud. 'Old' and 'new' SPBs are also functionally and biochemically distinct. For instance, the Bfa1p-Bub2p GTPase-activating protein complex, which is an integral part of the spindle position checkpoint, specifically binds to the 'old' SPB. The SPC inhibits the mitotic exit network) until the nucleus has migrated into the bud. Also the antigen-presenting cell-related molecule Kar9 localizes only to the 'old' SPB due to the activity of the 'new' SPB-resident Clb4/Cdc28 kinase. Interestingly, SPB fate can also be artificially switched, in which case, Bfa1p still localizes to the SPB that enters the bud independently of whether it is the 'old' or the 'new'. These data show that in budding yeast biochemical differences between the 'old' and 'new' SPBs are functionally relevant (Januschke, 2011 and references therein).

    As in yeast where the 'old' SPB is inherited by the long-lived bud, the mother centriole in Drosophila is inherited in testes by the mGSC, which is the most long-lived of the two mGSC daughters, and in larval brains by the GMC whose daughters outlive the NB. Thus, what appears to be a diametrically different behaviour between mGSCs and NBs using stemness as criteria is consistent, when the expected lifespan of each cell type is considered (Januschke, 2011).

    Drosophila Hey is a target of Notch in asymmetric divisions during embryonic and larval neurogenesis

    bHLH-O proteins are a subfamily of the basic-helix-loop-helix transcription factors characterized by an 'Orange' protein-protein interaction domain. Typical members are the Hairy/E(spl), or Hes, proteins, well studied in their ability, among others, to suppress neuronal differentiation in both invertebrates and vertebrates. Hes proteins are often effectors of Notch signalling. In vertebrates, another bHLH-O protein group, the Hey proteins, have also been shown to be Notch targets and to interact with Hes. The single Drosophila Hey orthologue is primarily expressed in a subset of newly born neurons that receive Notch signalling during their birth. Unlike in vertebrates, however, Hey is not expressed in precursor cells and does not block neuronal differentiation. It rather promotes one of two alternative fates that sibling neurons adopt at birth. Although in the majority of cases Hey is a Notch target, it is also expressed independently of Notch in some lineages, most notably the larval mushroom body. The availability of Hey as a Notch readout has allowed the study of Notch signalling during the genesis of secondary neurons in the larval central nervous system (Monastirioti, 2010).

    Among the superfamily of basic-helix-loop-helix (bHLH) transcription factors, several structurally distinct classes are discerned. One of these, the bHLH-Orange (bHLH-O) class, is characterized by the 'Orange' domain, a protein interaction domain perhaps serving as an extended dimerization surface. bHLH-O proteins are important developmental and physiological regulators in processes ranging from neurogenesis to circadian rhythm control (Monastirioti, 2010).

    In a number of invertebrate and vertebrate species, bHLH-O repressors are known to inhibit neural differentiation. In Drosophila, the seven E(spl) bHLH-O proteins are expressed in the neuroectoderm, where they inhibit cells from differentiating as neuroblasts (NBs). In vertebrates, a number of Hes bHLH-O proteins, notably Hes1, Hes3 and Hes5 in the mouse, are also expressed in the neuroectoderm; in this case it is the neural stem cells that express the Hes genes, which are subsequently downregulated in the differentiating neuronal progeny. Triple Hes1, Hes3, Hes5 knock-out causes premature neural differentiation, disruption of the neuroepithelium and a hypoplastic nervous system owing to stem cell depletion. In Drosophila, loss of the entire E(spl) locus results in supernumerary NB specification from the neuroectoderm and a hyperplastic nervous system. Despite these differences, owing to the different mode of neural precursor specification between vertebrates and insects, the generalization can be made that E(spl)/Hes proteins antagonize neuronal differentiation. At most developmental settings across metazoan phylogeny, neural expression of E(spl)/Hes genes is a direct response to Notch signalling (Monastirioti, 2010).

    Expression of another subfamily of bHLH-O genes has been detected in the progenitor cell zones of the developing vertebrate central nervous system (CNS). These genes encode the three Hey proteins, so named after a characteristic tyrosine residue in their C-terminal domain (Hairy/enhancer-of-split like with a Y); they are also known as Hrt, Herp, Hesr, Chf or Gridlock. Although neural defects are minor in Hey knock-out mice, overexpression studies have suggested that Hey and Hes proteins might synergize with each other in suppressing neural differentiation and maintaining the neural stem cell fate. Hey1 has even been linked to the pathogenesis and aggressiveness of gliomas. Hey knock-out mice have highlighted their roles in developmental processes outside the nervous system, in particular, heart and vasculature development. In these contexts, all three mammalian Hey genes appear to respond to Notch signalling, similar to E(spl)/Hes genes in neurogenesis. Biochemical data support Hes-Hey heterodimer formation, raising the possibility that these two subclasses of bHLH-O proteins might synergize in some developmental contexts as Notch effectors (Monastirioti, 2010).

    The Drosophila genome contains a single Hey orthologue (Kokubo, 1999), which had not been studied to date. This study characterized it in the hope of better understanding the process of neural precursor specification, based on the assumption that, by analogy to vertebrates, Hey might display protein-protein interactions with E(spl). Surprisingly, Hey was not co-expressed with the E(spl) proteins in the neuroectoderm, rather was restricted to differentiating neurons, suggesting a radically different role in neurogenesis than was assumed. Once NBs are specified in Drosophila, they undergo cycles of asymmetric cell divisions that give rise to a secondary precursor, called a ganglion mother cell (GMC), in addition to self-renewing. GMCs divide once to give rise to two neurons or, less often, glia. The majority of GMC divisions are asymmetric, with the fates of the two daughters dictated by unequal levels of Notch signalling. The 'A' sibling neuron requires high Notch signalling, whereas the 'B' sibling neuron downregulates Notch reception, which is usually achieved by asymmetric segregation of a Notch inhibitor, Numb, into the nascent 'B' neuron. This study describes a complex pattern of Hey expression in relation to these divisions during both neurogenic phases of the animal, early embryogenesis and larval life, where thousands of new neurons are added to generate the adult CNS. In all sibling pairs that were identified, Hey was expressed in the 'A' neuron. Genetic analysis confirmed that Hey is a Notch target gene in most instances. These results extend the Hey-Notch relationship to Drosophila in support of an ancient connection between bHLH-O genes and Notch activity and, for the first time, implicate a bHLH-O protein in the process of GMC asymmetric division (Monastirioti, 2010).

    A full-length Hey cDNA was amplified from a Drosophila cDNA library, which was used as a probe for in situ hybridization, and for cloning in prokaryotic expression vectors. Bacterially expressed full-length Hey protein was used to raise anti-Hey antibodies. There were no obvious differences between the RNA and protein patterns. Hey protein showed nuclear accumulation, as expected for a transcription factor, and was primarily detected in a segmentally repeated pattern within the CNS starting at stage 10. Later, more Hey-positive cells gradually appear in the CNS. The neuroectodermal epithelium, where the related E(spl) bHLH-O proteins are expressed already starting at stage 8, is devoid of Hey expression, which instead is detected at deeper levels overlapping with the GMC/immature neuron marker Pros. From double-staining with the neuronal antigen Elav it was clear that the vast majority of Hey-positive cells represent neurons rather than GMCs, confirmed as lack of colocalization with the NB/GMC marker Asense. Besides neurons, Hey expression was detected in a subset of Repo-positive glia of the CNS and peripheral nervous system (PNS). Of note, Eve staining, which was used to visualize particular neurons, also marks the dorsally located pericardial cells. No Hey immunoreactivity was detected within or near these heart precursors, contrary to the strong expression of mammalian Hey genes during cardiogenesis. Finally, a few Hey-positive cells per segment were detected in the embryonic PNS. Most of these were also neurons, by virtue of being Elav-positive, but were not characterized further (Monastirioti, 2010).

    Lineage-specific markers were used to characterize Hey expression in more detail. One was Even skipped, which marks a subset of neurons: the aCC/pCC sibling pair, the RP2 motoneuron, the cluster of U motoneurons and the cluster of EL interneurons. Another was the AJ96-lacZ enhancer trap, which marks the MP2 precursor and its progeny, the dMP2/vMP2 neurons. With AJ96-lacZ, strong Hey accumulation was detected in vMP2 but not in dMP2. Weak Hey expression was detected shortly before mitosis of the MP2 progenitor during late stage 10. Among the Eve-positive neurons, pCC and the U neurons expressed Hey. aCC, RP2 and the EL neurons were Hey-negative. At stage 11, the sibling of RP2, RP2sib, a smaller cell, which only transiently expresses Eve, was Hey-positive. Hey expression in all these neurons appeared transient. For example, whereas immunoreactivity in vMP2 was strong at stage 12, it was downregulated and barely detectable by stage 14. Similarly, by stage 14 no Hey could be detected in pCC cells, although it was still expressed strongly in some of the later-born U motorneurons. Transient Hey expression was also observed in the two identical progeny of MP1, a midline precursor, which are marked by Odd (Monastirioti, 2010).

    Most of the neurons described above belong to well-characterized lineages, in which sibling fates arise through differential Notch signalling. In each of the RP2/RP2sib, aCC/pCC and dMP2/vMP2 pairs, the second cell requires Notch signalling in order to acquire the 'A' fate, distinct from that of its sibling cell ('B' fate). Also in the U lineages, which arise from sequential GMCs from neuroblast NB7-1, the U neurons require Notch, whereas their Eve-negative Usib neurons do not. All Notch-requiring cells, namely RP2sib, pCC, vMP2 and the U cells, robustly express Hey, whereas none of their 'B'-fate siblings do so. This raises the possibility that Hey is expressed in response to Notch (Monastirioti, 2010).

    Thus Hey was detected almost exclusively in the CNS in young postmitotic neurons and glia, specifically those that receive a Notch signal at birth. It has long been appreciated that Notch signalling plays an important role in the acquisition of neuronal/glial cell fate after GMC division, with most GMCs producing two different progeny, an 'A' cell with high Notch activity and a 'B' cell with no Notch activity. Still, no Notch target genes had been identified in this process. This study shows that Hey is such a target gene in many, and perhaps all, GMC asymmetric divisions. These conclusions are based on the expression pattern of Hey, its response to Notch pathway perturbation and on the ability of ectopic Hey to block development of RP2 and dMP2, two 'B'-type neurons (Monastirioti, 2010).

    Although these is good evidence that Hey expression can recapitulate the effect of Notch signalling, Hey loss-of-function has only a mild phenotype. The trivial possibility that the transposon insertion allele used has residual activity is unlikely as (1) no Hey protein is detectable in homozygous mutants and (2) the Heyf06656 allele results in recessive lethality. Nevertheless, the issue will be permanently decided with the generation and analysis of more Hey alleles. The alternative hypothesis, which seems more probable, is that one or more additional factors besides Hey can also act as nuclear effectors downstream of Notch in the 'A' GMC progeny. No Hey paralogues exist in the D. melanogaster genome, but structurally divergent proteins, even outside the bHLH-O family, could share similar functional characteristics. At the moment, there are no good candidate for such a factor; however, a number of bHLH-O factors have been excluded that do not seem to be co-expressed with Hey in neurons, namely E(spl)mγ and m8, Hairy and Dpn (Monastirioti, 2010).

    Besides GMCs, a number of other neural progenitors, namely NBs, sensory organ precursors (SOPs) and SOP progeny cells, all undergo asymmetric cell divisions with Notch involvement. No Hey expression was detected in either the NB/GMC pair or in the SOP progeny cells of external sensory organs, suggesting that Hey expression is turned on exclusively in GMC asymmetric divisions. Hey-positive glia could also be the progeny of asymmetrically dividing GMCs. It is yet unclear which cells might be the immediate progenitors of the few Hey-positive PNS neurons (Monastirioti, 2010).

    Until the present work and the recent paper by Krejci (2009), the only Drosophila bHLH-O genes known to be targets of Notch were the seven of the E(spl) complex. Hey and two other bHLH-O genes, dpn and Her, had been predicted as candidate Notch targets based on nearby clustering of putative Su(H) binding sites, the DNA elements via which activated Notch is tethered to its target genes. Although HES-related (Her) does not seem to be a true Notch target have shown that dpn is a Notch target in the muscle-progenitor-like Drosophila DmD8 cell line; an in vivo context for such a response has yet to be determined. Together with Hey, this makes a total of 9 out of 13 bHLH-O genes in the Drosophila genome which are regulated by Notch. It should be stressed that Notch has a number of additional (non-bHLH-O) targets, depending on the species and cellular context, but few, if any, show such widespread association as the bHLH-O genes. The latter are activated by Notch in a multitude of unrelated contexts, such as neuroectoderm, mesoderm, wing epithelium, leg segmentation and now GMC asymmetric cell divisions in Drosophila, and in neural progenitors, presomitic mesoderm, cardiogenesis and vasculogenesis in vertebrates (Monastirioti, 2010 and references therein).

    In addition to its widespread Notch-dependent expression, this study detected a clear instance of Notch-independent expression of Hey within the GMCs and neurons of the MB precursors. Other examples where Hey expression does not correlate with known events of Notch signalling are the MP2 NB and the two MP1 midline neurons. It is also clear that in embryos with severe Notch signalling defects, a small number of Hey-positive cells is still seen in the CNS, suggesting that there are additional neural lineages, where Hey is likely to be expressed independently of Notch. Analysis of the cis regulatory regions of Hey should shed light on Notch-dependent and Notch-independent enhancer elements (Monastirioti, 2010).

    The bHLH-O family has undergone considerable diversification during evolution. Although sequence analysis can unambiguously assign genes to this family, it cannot identify orthologues in distantly related species. A classic example is the Drosophila to mammals comparison, where no clear orthologue relationships exist between Hairy, Dpn and the seven E(spl) in Drosophila and Hes1, 2, 3, 5, 6 and 7 in mammals, suggesting that the diversification of these proteins occurred separately after divergence of protostomes and deuterostomes. Hey proteins are the singular exception, being particularly well conserved. The bHLH domain of Drosophila Hey shows 97-98% similarity to that of its mammalian counterparts. This might lead one to expect substantial conservation of Hey function, which, strangely enough, was not observed (Monastirioti, 2010).

    First, mammalian Hey genes have a very broad expression pattern, including presomitic mesoderm, embryonic heart, vascular precursors, developing brain and spinal cord, neural crest etc (Kokubo, 1999; Leimeister, 1999). Fly Hey, by contrast, seems confined within the CNS and PNS. Although there is complexity in its expression, as documented in this study with its contextual Notch dependence/independence, the great majority of its expression pattern seems to be in the newly born Notch-dependent 'A'-type neurons. The absence of Hey expression from the developing Drosophila heart is most striking, given the foremost importance of Hey genes in vertebrate cardiogenesis. A second indicator of functional non-conservation comes from comparing the role of Hey within the nervous systems of mammals versus Drosophila. In the former, Hey has been proposed to act in the maintenance of progenitor fate and to antagonize neuronal differentiation, similar to Hes proteins. In fact, it has been proposed that Hey-Hes heterodimers mediate these effects. In the fly, Hey expression was not detected within progenitor cells, with the few rare GMC exceptions, noted above. Hey-E(spl) or Hey-Dpn co-expression could not be detected, although all seven E(spl) genes were not tested for lack of specific reporter lines. To overcome any doubt, functional tests were made by ectopically expressing Hey. Instead of suppressing sensory organ formation, it mildly increased the number of bristles, showing an opposite phenotype from that of E(spl) or hairy ectopic expression. It can therefore be confidently said that Hey does not antagonize neural differentiation in the fly (Monastirioti, 2010).

    This leaves a puzzle of why Hey is so strongly conserved. Perhaps some yet uncharacterized molecular aspect of its role in chromatin recognition/transcriptional regulation is conserved, despite considerable diversification in cellular and developmental contexts. These contexts have diverged greatly between insects and vertebrates, the only unifying theme being their regulation by Notch signalling. A homologous function might be that of promoting gliogenesis, as Hey2 was shown to promote Müller glia formation in the murine retina. Further comparative studies encompassing more species will no doubt shed light on the function of this highly conserved bHLH-O protein (Monastirioti, 2010).

    Apical-basal polarity in Drosophila neuroblasts is independent of vesicular trafficking

    The possession of apical-basal polarity is a common feature of epithelia and neural stem cells, so-called neuroblasts (NBs). In Drosophila, an evolutionarily conserved protein complex consisting of atypical protein kinase C and the scaffolding proteins Bazooka/PAR-3 and PAR-6 controls the polarity of both cell types. The components of this complex localize to the apical junctional region of epithelial cells and form an apical crescent in NBs. In epithelia, the PAR proteins interact with the cellular machinery for polarized exocytosis and endocytosis, both of which are essential for the establishment of plasma membrane polarity. In NBs, many cortical proteins show a strongly polarized subcellular localization, but there is little evidence for the existence of distinct apical and basolateral plasma membrane domains, raising the question of whether vesicular trafficking is required for polarization of NBs. This study analyzed the polarity of NBs mutant for essential regulators of the main exocytic and endocytic pathways, including exocyst component Exo84, shibire, sec5, sec6, sec15, rab5, Hrs, Vps25 and erupted/TSG101. Surprisingly, none of these mutations affected NB polarity, demonstrating that NB cortical polarity is independent of plasma membrane polarity and that the PAR proteins function in a cell type-specific manner (Halbsgut, 2011).

    The data strongly indicate that vesicle trafficking is not involved in polarization of NBs, in contrast to epithelia, where it is essential for polarity. In epithelial cells vesicle trafficking controls cell polarity mainly by regulating the levels of the transmembrane proteins Crumbs and DE-Cad at the membrane. In NBs no asymmetrically localized transmembrane protein has been described so far, except for one: Numb-interacting protein (NIP) is a multipass transmembrane protein that colocalizes with Numb at the basal cortex of dividing NBs. In Drosophila Schneider cells, Numb and NIP colocalize at the plasma membrane, and RNA interference–mediated knockdown of NIP results in a release of Numb from the plasma membrane. Whether NIP is required for proper localization of Numb in dividing NBs is not known since no null mutation in moladietz, the gene encoding NIP, is available. It has also not been studied whether Numb may be required for the asymmetric localization of NIP in NBs (Halbsgut, 2011).

    So, how could cell polarity be established and maintained in NBs? Baz, PAR-6, and aPKC are all localized to the apical junctional region of the neuroectodermal epithelium at the time when NBs ingress from the epithelium. Thus the components of the PAR/aPKC complex are already apically enriched in NBs prior to their first division. It is furthermore known that Baz can associate with the plasma membrane by direct binding to phosphoinositide lipids. However, there is no evidence for an asymmetric distribution of phosphoinositides in NBs, which might cause the asymmetric localization of Baz. In analogy to the mechanism that operates in the Caenorhabditis elegans zygote, the hypothesis is favored that the apical localization of Baz is stabilized by a mutual repression mechanism involving phosphorylation of Baz by the basally localized kinase PAR-1 and phosphorylation of PAR-1 by aPKC. Although this mechanism may be sufficient to stably polarize an NB, extrinsic cues from adjacent neuroectodermal cells contribute to the positioning of the Baz crescent to the apical cortex (Halbsgut, 2011).

    In conclusion, this work shows for the first time that cortical polarity in NBs can be established even when intracellular vesicular trafficking is blocked, in striking contrast to the situation in epithelia. Although it cannot be completely rule out that the lack of polarity phenotypes in NBs homozygous for the mutations that were analyzed may be due to the perdurance of the respective wild-type protein in the clones, this possibility is considered unlikely. It has been shown that the same mutations that were analyzed in NBs cause strong polarity phenotypes when clones are induced in epithelia. Furthermore, in some of the experiments perdurance cannot be ruled out, for example in the experiments using the shi1 allele, and these also showed no polarity defects in NBs (Halbsgut, 2011).

    These findings imply that the PAR/aPKC complex can function in different ways, to polarize only the cortex, as in NBs or the C. elegans zygote, or the cortex and the plasma membrane, as in epithelia and probably also in neurons. In the future it will be important to dissect these different mechanisms at the molecular level in order to understand the function of the PAR proteins in a specific cellular context (Halbsgut, 2011).

    Rap1 and Canoe/afadin are essential for establishment of apical-basal polarity in the Drosophila embryo

    The establishment and maintenance of apical-basal cell polarity is critical for assembling epithelia and maintaining organ architecture. Drosophila embryos provide a superb model. In the current view, apically positioned Bazooka/Par3 is the initial polarity cue as cells form during cellularization. Bazooka then helps to position both adherens junctions and atypical protein kinase C (aPKC). Although a polarized cytoskeleton is critical for Bazooka positioning, proteins mediating this remained unknown. This study found that the small GTPase Roughened/Rap1 and the actin-junctional linker Canoe/afadin are essential for polarity establishment, as both adherens junctions and Bazooka are mispositioned in their absence. Rap1 and Canoe do not simply organize the cytoskeleton, as actin and microtubules become properly polarized in their absence. Canoe can recruit Bazooka when ectopically expressed, but they do not obligatorily colocalize. Rap1 and Canoe play continuing roles in Bazooka localization during gastrulation, but other polarity cues partially restore apical Bazooka in the absence of Rap1 or Canoe. The current linear model for polarity establishment was tested. Both Bazooka and aPKC regulate Canoe localization despite being 'downstream' of Canoe. Further, Rap1, Bazooka, and aPKC, but not Canoe, regulate columnar cell shape. These data suggest that polarity establishment is regulated by a protein network rather than a linear pathway (Choi, 2013).

    Polarity is a fundamental property of all cells, from polarized cell divisions in bacteria or fungi to the elaborate polarity of neurons. Among the most intensely studied forms of polarity in animal cells is epithelial apical-basal polarity. Polarity of epithelial sheets is key to their function as barriers between body compartments, and is also critical in collective cell migration and cell shape change during morphogenesis, as cytoskeletal and apical-basal polarity often go hand in hand. Loss of apical-basal polarity is a hallmark of metastasis. Significant advances have been made in defining the machinery required for cell polarity in many settings, but fundamental questions remain unanswered (Choi, 2013).

    Cadherin-catenin complexes, which assemble into adherens junctions (AJs) near the apical end of the lateral cell interface, are critical polarity landmarks that define the boundary between apical and basolateral domains. Studies in C.elegans and Drosophila identified other key regulators of apical-basal polarity. In the textbook view, the apical domain is defined by the Par3/Par6/aPKC and Crumbs/Stardust(Pals1)/ PATJ complexes, while Scribble, Dlg, Lgl, and Par1 define the basolateral membrane (Choi, 2013).

    Complex cross-regulatory interactions between apical and basolateral proteins maintain these mutually exclusive membrane territories. These proteins also regulate other types of polarity during morphogenesis; e.g., fly Par3 (Bazooka; Baz), aPKC, and AJ proteins are planar-polarized during fly convergent extension, thus regulating polarized cell movements (Choi, 2013).

    Polarized cytoskeletal networks also play key roles in establishing and maintaining apical-basal and planar polarity. These networks are thought to be physically linked to apical junctional complexes. The earlier model suggesting that cadherin-catenin complexes link directly to actin via α-catenin is now viewed as over-simplified. Instead, different proteins are thought to mediate this connection in different tissues and at different times (Choi, 2013).

    Among the linkers is Canoe (Cno)/Afadin, an actin-binding protein that binds transmembrane nectins via its PDZ domain. While originally hypothesized to be essential for cell adhesion, subsequent work supports a model in which afadin modulates adhesive and cytoskeletal machinery during cell migration in vitro and the complex events of mouse gastrulation. Afadin has two N-terminal Ras association domains for which the small GTPase Rap1 is the major binding partner, and Afadin and Rap1 are functionally linked in both flies and mice. Rap1, Cno, and the Rap1 GEF Dizzy/PDZGEF are all essential for maintaining effective linkage between AJs and the apical actomyosin cytoskeleton during apical constriction of Drosophila mesodermal cells during fly gastrulation. Rap1 regulates Cno localization to the membrane. Cno plays a related role during convergent extension, though its role is planar polarized during this process. Cno also regulates collective cell migration, signaling, and oriented asymmetric divisions. The Rap1/Cno regulatory module is also important in disease, as Afadin and Rap1 are implicated in congenital disorders of the cardiovascular system and cancer metastasis. It remains unclear whether these diverse roles all involve junction-cytoskeletal linkage or whether some are independent functions (Choi, 2013 and references therein).

    The small GTPase Rap1 plays diverse cellular roles. Mammalian Rap1 isoforms are perhaps best known for regulating integrin-based cell matrix adhesion, but Rap1 also regulates cell-cell AJs in both Drosophila and mice. In murine endothelial cells, for example, Rap1, its effector Krit1, and VE-cadherin form a complex that regulates endothelial cell junctions and stabilizes apical-basal polarity (Choi, 2013 and references therein).

    In Drosophila imaginal disc cells, Rap1 regulates the symmetric distribution of DE-cadherin (DEcad) around the apical circumference of each cell. Rap1 carries out these functions via a diverse set of effector proteins, including Krit1, TIAM, RIAM, and Cno/Afadin. Thus, Rap1 and its effectors are candidate proteins for regulating interactions between AJs, polarity proteins and the cytoskeleton during polarity establishment and maintenance (Choi, 2013).

    The early Drosophila embryo provides among the best models of establishing and maintaining apical-basal polarity. Flies start embryogenesis as a syncytium, with 13 rounds of nuclear division without cytokinesis. Membranes then simultaneously invaginate around each nucleus, forming ~6000 cells in a process known as cellularization. Prior to cellularization, the egg membrane is already polarized and serves as a polarity cue for underlying nuclei. This ultimately becomes the apical end of the new cells. Epithelial apical-basal polarity is initiated during cellularization. In the absence of cadherin-catenin complexes, cells form normally but then lose adhesion and polarity as gastrulation begins. These data and earlier work from cell culture suggested AJs are the initial apical cue. However, it was found that Bazooka (Baz)/Par3 acts upstream of AJs in this process. Strikingly, Baz and DEcad apically co-localize in spot AJs from cellularization onset. In the absence of Baz, DEcad loses its apical enrichment and redistributes all along the lateral membrane, while in the absence of AJ proteins, Baz remains apically localized, and a subset of cells retain residual apical-basal polarity, although cell shapes are highly abnormal. Cadherin-catenin and Baz complexes form independently before cellularization, and Baz then helps position DEcad in the apicolateral position where spot AJs will form. This placed Baz atop of the polarization network, raising the question of how it is positioned apically. Two cytoskeletal networks play important roles in initial Baz positioning (Choi, 2013).

    Disrupting dynein led to Baz spreading along the lateral membrane, suggesting polarized transport along microtubules (MTs) plays a role. Depolymerizing actin also destabilized apical Baz, as did significantly overexpressing Baz, suggesting an actin-based scaffold with a saturable number of binding sites anchors Baz apically. While both actin and MTs are required for initial Baz polarization, they are not the only cues. Mislocalized Baz is re-recruited or re-stabilized apically at gastrulation onset if either initial cue is disrupted, suggesting a third cue perhaps involving aPKC/Par6 or Par1. Thus, the current model for initial establishment of apical-basal polarity involves a relatively simple pathway in which Baz is positioned apically, and then positions other apical polarity players. However, once initial polarity is established, events become more complex, with a network of mutually reinforcing and inhibitory interactions between apical and basolateral polarity complexes leading to polarity elaboration and maintenance. These were significant advances, but the proteins directing apical accumulation of Baz remained unknown. Work on apical constriction in the fly mesoderm, convergent extension during gastrulation, establishment of anteriorposterior polarity in one cell C. elegans embryos, and on apically constricting Drosophila amnioserosal cells, suggested that a complex network of interactions link AJs, the apical polarity proteins Baz and aPKC, and the actomyosin cytoskeleton. Recent work on Canoe and Rap1's roles in mesoderm apical constriction and convergent elongation (Sawyer, 2011) suggested they also fit into this network. These data led to an exploration of whether Rap1 and Cno play roles in initial apical positioning of AJs and Baz and thus in the establishment and early maintenance of polarity (Choi, 2013).

    In regulating polarity establishment, Rap1 and Cno could act by several possible mechanisms. Their role in AJ positioning may be solely due to their effects on Baz localization, or alternatively Rap1 and Cno may independently affect the localization of both Baz and AJs. In the latter case, Cno may directly link AJs to the apical actin scaffold, as it was suggested to act in apical constriction. Rap1 and Cno also clearly regulate Baz positioning. Since Baz apical positioning requires an apical actin scaffold and dynein based MT transport, whether Rap1 and Cno act indirectly by regulating cytoskeletal organization was examined. However, the data suggest this is not the case: both the MT and actomyosin cytoskeletons appear normal in mutants. Thus the most likely model is that Rap1 and Cno are required for anchoring Baz apically. Consistent with this, when Cno was ectopically localized to artificial cell-cell contacts in cultured fly cells, it was able to recruit Baz to that site. This could occur directly, for example, by Cno binding Baz, or indirectly, via unknown intermediaries. Strikingly, however, when Baz was over-expressed in cellularizing embryos, presumably saturating its apical binding sites, it accumulated basolaterally and recruited DEcad but not Cno to these ectopic sites. Thus Cno and Baz do not co-localize obligatorily. It likely that each has multiple binding partners and that when pools are limiting, as Cno may be in this latter experiment, ectopic Baz cannot recruit Cno away from a preferred binding site. Of course, it remains possible that Cno and Rap1 also regulate Baz positioning through effects on MT transport or, given Cno's apical localization, unloading at an apical docking site. It will be important to test these possibilities. As is discussed in more detail below, it will also be important to define the Cno- and Rap1-independent mechanisms that partially restore apical Baz localization after gastrulation onset (Choi, 2013).

    Since Rap1 is uniformly distributed along the apical-basal axis during cellularization, the most likely hypothesis is that it is locally activated apically by a GEF. A number of Rap1GEFs exist, many of which are conserved between mammals and flies. Recent work from the Reuter lab demonstrated that, like Cno and Rap1, the Rap1 GEF Dizzy (Dzy/PDZ-GEF) plays an important role in coordinated mesodermal apical constriction, suggesting it is the GEF acting upstream of Cno and Rap1 in that process. They also suggest that Rap1 and Dzy help regulate establishment of AJs. While similar in outline, their analysis of AJs differs from this one in detail, as they see strong effects on DEcad localization without similar effects on Arm. This is surprising, since these two proteins of the cadherin-catenin complex generally localize very similarly at the cortex. However, these differences aside, their data are consistent with Dzy acting with Cno and Rap1 in AJ establishment-it will be important to examine the effects of Dzy on Baz localization. It will also be important to determine how pre-existing egg membrane polarity is translated into localized Rap1 activity (Choi, 2013).

    In addition to the parallel roles of Rap1 and Cno in regulating initial apical-basal polarization, this study identified a second role for Rap1 in establishing and maintaining columnar cell shape. The data suggest that this is partially or completely Cno-independent, and thus one of the many other Rap1 effectors may play a role in this process. It will be exciting to examine embryos mutant for other Rap1 effectors, such as Krit1/Bili, TIAM/Still life, RIAM/Pico, or RhoL to see if they are required for establishing columnar cell shape. baz and aPKC mutants also had defects in establishing columnar cell architecture. It is possible that each protein provides an independent mechanistic input into this process. This is consistent with the observed differences in the details of how columnar cell shape is disrupted, with Baz and aPKC primarily regulating apical cell area, while Rap1 affects cell shape at multiple apical-basal positions. A more speculative but perhaps less likely possibility is that Rap1 uses Baz and aPKC as effectors in establishing columnar cell shape. Fly Rap1 can form a complex with aPKC and Par6, and Rap1 acts upstream of cdc42/Par3/aPKC in regulating polarity of cultured neurons (Choi, 2013 and references therein).

    Having identified Rap1's direct effector(s) in regulating cell shape, it is necessary to move downstream. Based on analogies with other epithelial tissues in fly development, it is hypothesized establishing columnar cell shape involves regulating apical tension. Other small GTPases play key roles in this; e.g., Rho and cdc42 have striking and opposing roles in apical tension regulation during fly eye development. In that context, Rho acts via separate effectors to maintain AJs and apical tension-it regulates tension via Rok, Diaphanous, and ultimately myosin contractility. It will be interesting to determine whether the defects in apical cell shape in the absence of Rap1, Baz, or aPKC also reflect unbalanced contractility in different nascent cells, and which contractility regulators are involved. However, for now, this is speculative (Choi, 2013).

    Previous work has suggested a linear hierarchy regulating polarity establishment, with Baz at the top, positioning AJs and aPKC. The current work extends this hierarchy, positioning Rap1 and Cno upstream of Baz in this process. However, the data further suggest that viewing polarity establishment as a linear process is significantly over-simplified. It is now known that all of the relevant players -- including the AJ proteins, Baz, Cno and aPKC -- are at the cortex in syncytial embryos, prior to cellularization and the initiation of apical-basal polarity. This places them in position to cross-regulate one another. Consistent with this, the data suggest that viewing relationships with an 'upstream-downstream' point of view misses important reciprocal interactions that occur as polarity is established. Two examples point this out most clearly. First, earlier work suggested that localization of aPKC occurs 'downstream' of Baz, as apical positioning of aPKC at gastrulation onset requires Baz function. The new data reveal that Rap1 and Cno are, in turn, 'upstream' of Baz, and thus, if things work in a strictly linear fashion, Rap1 and Cno should be 'upstream' of aPKC. However, in contrast to this simple view, this study found that precise positioning of Cno during cellularization requires aPKC - in its absence, Cno is not cleared from the apical region, and the apical-basal cables of Cno at tricellular junctions are not properly assembled. In a similar fashion, Baz, which in a linear model is 'downstream' of Cno, also regulates precise positioning of Cno during cellularization. aPKC and Baz also play important roles in Cno localization during the early polarity maintenance phase beginning at gastrulation onset. Together, these data suggest that initial positioning of proteins along the apical-basal axis involves a network of protein interactions, similar to that previously suggested to regulate polarity elaboration during the extended germband phase and beyond, as cells develop the full suite of epithelial junctions. It will now be important to define mechanisms by which aPKC and Baz act to precisely position Cno: two broad possibilities are that they act on Cno directly, or that they modulate the fine scale architecture of the actin cytoskeleton, with indirect effects on Cno. It will also be exciting to determine if other polarity determinants, like the basolateral proteins Discs Large, Scribble or Lgl, or the basolateral kinase Par1 also play roles in polarity establishment, as they do in polarity maintenance. Consistent with this possibility, recent work from the Harris lab suggests Par1 is important for the gastrulation onset rescue of Baz localization in embryos in which early cues are disrupted. Finally, it will be interesting to identify the cues that come into play at gastrulation onset, which partially restore apical Baz localization, as part of the increasingly complex network of partially redundant regulatory cues that give polarity its robustness (Choi, 2013).

    A NudE/14-3-3 pathway coordinates dynein and the kinesin Khc73 to position the mitotic spindle

    Mitotic spindle position is controlled by interactions of cortical molecular motors with astral microtubules. In animal cells, Partner of Inscuteable (Pins) acts at the cortex to coordinate the activity of Dynein and Kinesin-73 (Khc73; KIF13B in mammals) to orient the spindle. Though the two motors move in opposite directions, their synergistic activity is required for robust Pins-mediated spindle orientation. This study identified a physical connection between Dynein and Khc73 that mediates cooperative spindle positioning. Khc73's motor and MBS domains link Pins to microtubule plus ends, while its stalk domain is necessary for Dynein activation and precise positioning of the spindle. A motif in the stalk domain binds, in a phospho-dependent manner, 14-3-3ζ, which dimerizes with 14-3-3ε. The 14-3-3ζ/ε heterodimer binds the Dynein adaptor NudE to complete the Dynein connection. The Khc73 stalk/14-3-3/NudE pathway defines a physical connection that coordinates the activities of multiple motor proteins to precisely position the spindle (Lu, 2013).

    Mitotic spindle orientation requires the coordination of several pathways that act on astral microtubules. These pathways may establish cortical-microtubule connections and generate the forces necessary for movement of this large cellular structure with metaphase spindle lengths varying from 2 mm in yeast to 60 mm of a Xenopus single-cell stage. The spindle-orientation protein Pins has a domain that has been thought to capture microtubules (Pinslinker), and another that generates force (PinsTPR). This study attempted to understand how these two pathways function together by taking advantage of an induced polarity system in cultured S2 cells in which the two pathways can be selectively activated. This system allowed for the identification of the Khc73 stalk domain as a critical element that links PinsTPR and Pinslinker pathways. This observation was used as a platform for establishing a complete physical connection between the two pathways. This study has also clarified the role of 14-3-3 proteins in spindle orientation, establishing that their interaction with Pins is likely to be indirect (through Dlg and Khc73) (Lu, 2013).

    Khc73 performs two functions in Pins-mediated spindle positioning. First, it functions in the Pinslinker pathway to mediate cortical microtubule capture through its MBS and motor domains, respectively. The N-terminal portion of Khc73 is sufficient for linker activity, which is likely occurring through a DlgGK/Khc73MBS interaction at the cortex and a microtubule/ Khc73motor interaction at the spindle. This suggests that Khc73's motor domain could function at the cortex by itself, however, Ed:Khc73motor did not have spindle positioning activity, indicating that other factors could be required or the motor domain is not functional in this context (e.g., as a monomer with the coiled-coil stalk). Khc73 must therefore rely on Dlg as an adaptor to target it to the cortex, which is where it can potentially function to facilitate the initial contact of astral microtubules (Lu, 2013).

    Although Khc73's MBS domain directly interacts with Dlg, Khc73 is not seen to colocalize with cortical Pins, even though Dlg robustly localizes to Pins crescents. Instead, the motor protein is seen distinctly at the ends of microtubule, suggesting that Khc73 moves to the plus ends where it may be poised for capture by the cortical Pinslinker/Dlg complex. Thus, Khc73's N-terminal domains are likely to facilitate cortical microtubule capture by linking microtubule plus ends to cortical Dlg (Lu, 2013).

    In addition to facilitating cortical microtubule capture, this study found that Khc73 also forms a physical connection to the PinsTPR/Mud/Dynein pathway with its stalk region, which is essential for the synergistic function of the two pathways. Khc73 may activate Dynein by delivering NudE to the cortex, where Dynein is presumably localized by PinsTPR/Mud. Although it is not possible to observe the localization of Dynein in S2 cells for technical reasons, there is good evidence that it is cortically localized by way of PinsTPR/Mud. In HeLa cells, Dynein localizes to the cortex with the mammalian homolog of Mud, NuMA, along with mPins, during mitosis (Lu, 2013).

    It is proposed that a 14-3-3 motif in Khc73's stalk region activates an 'idling' cortically localized Dynein by cargoing NudE. Interestingly, although the Khc73 14-3-3 motif mutant Khc73S1374A has a distribution of spindle-orientation angles that isn't random, the distribution is bimodal such that the spindle angles are either fully aligned or orthogonal to the polarity axis. The bimodal phenotype is distinct from the Khc73motor+MBS fragment, which has a canonical intermediate distribution of spindle angles, suggesting that there may be additional regions or domains in the stalk that are contributing to the bimodal phenotype. It is hypothesized that an element within Khc73's stalk region is required for the proper application of the forces generated from by two motor proteins to properly orient the mitotic spindle. Nevertheless, biochemical and genetic studies demonstrate that the 14-3-3 binding motif is, at the very least, required for proper Pins-mediated spindle positioning and required for Khc73's interaction with the 14-3-3 proteins and NudE (Lu, 2013).

    Pins mediates spindle positioning by coordinating two motor proteins that, as a pair, facilitate the cortical capture of microtubules and also provide pulling forces to robustly orient the mitotic spindle. A model is proposed in which orientation occurs through an ordered series of events, beginning with the initial polarization of Pins, followed by recruitment of Mud through its PinsTPR domain and Dlg through Pinslinker region. Cortical Mud then recruits cytoplasmic Dynein, which is not yet active and will remain inert, but poised at the cortex. Khc73 localizes to the plus ends of microtubules, where it establishes cortical-microtubule contacts through direct binding to Dlg and also delivers NudE to cortical Dynein, thereby activating it. As astral microtubules enter the proximity of the Dynein complex, Dynein can generate specifically timed cortical pulling forces necessary for robust spindle positioning. Future work will be directed at dissecting the precise timing of these synergistic events that underlie differentiation and tissue architecture (Lu, 2013).

    The conserved Discs-large binding partner Banderuola regulates asymmetric cell division in Drosophila

    Asymmetric cell division (ACD) is a key process that allows different cell types to be generated at precisely defined times and positions. In Drosophila, neural precursor cells rely heavily on ACD to generate the different cell types in the nervous system. A conserved protein machinery that regulates ACD has been identified in Drosophila, but how this machinery acts to allow the establishment of differential cell fates is not entirely understood. To identify additional proteins required for ACD, an in vivo live imaging RNAi screen was carried out for genes affecting the asymmetric segregation of Numb in Drosophila sensory organ precursor cells. Banderuola (Bnd / Wide awake) was identified an essential regulator of cell polarization, spindle orientation, and asymmetric protein localization in Drosophila neural precursor cells. Genetic and biochemical experiments show that Bnd acts together with the membrane-associated tumor suppressor Discs-large (Dlg) to establish antagonistic cortical domains during ACD. Inhibiting Bnd strongly enhances the dlg phenotype, causing massive brain tumors upon knockdown of both genes. Because the mammalian homologs of Bnd and Dlg are interacting as well, Bnd function might be conserved in vertebrates, and it might also regulate cell polarity in higher organisms. It is concluded that Bnd is a novel regulator of ACD in different types of cells. The data place Bnd at the top of the hierarchy of the factors involved in ACD, suggesting that its main function is to mediate the localization and function of the Dlg tumor suppressor. Bnd has an antioncogenic function that is redundant with Dlg, and the physical interaction between the two proteins is conserved in evolution (Mauri, 2014).

    Although most cell divisions are symmetric, some cells can divide asymmetrically into two daughter cells that assume different fates. During development, asymmetric cell division (ACD) allows specific cell types to be generated at precise locations relative to surrounding tissues. To achieve this, the axis of ACD needs to be coordinated with the architecture and polarity of the developing organism. Over the past years, a conserved protein machinery for ACD has been identified, but how this machinery connects to the organism architecture is less clear (Mauri, 2014).

    The fruit fly Drosophila melanogaster is one of the best-understood model systems for ACD. In particular, the development of the Drosophila CNS and peripheral nervous system relies heavily on ACD and has contributed much to current understanding of this process. In the peripheral nervous system, external sensory (ES) organs are formed by two outer cells (hair and socket) and two inner cells (neuron and sheath). The four cell types arise from a single sensory organ precursor (SOP) cell, which divides asymmetrically into an anterior pIIb cell and a posterior pIIa cell. In a second round of ACD, pIIa and pIIb generate the outer or inner cells of the ES organ, respectively. The difference between pIIa and pIIb cells arises from different levels of Notch signaling in the two daughter cells. This difference is established by the asymmetric segregation of the Notch inhibitor Numb into the pIIb cell. Numb is known to regulate endocytosis, but how it inhibits Notch signaling is not precisely understood (Mauri, 2014).

    In SOP cells, the polarity axis is coordinated with the anterior-posterior planar polarity axis of the overlying epithelium. Planar polarity involves the localization of mutually inhibitory components of a well-characterized machinery to the anterior or posterior plasma membrane. In SOP cells, the planar polarity protein Strabismus (Stbm) localizes to the anterior cortex and initiates the reorganization of plasma membrane domains to establish the axis of ACD. One of the most upstream events of this process is the recruitment of the membrane-associated guanylate kinase (MAGUK) Discs-large (Dlg) to the anterior cortex. This may involve a direct interaction of Dlg with the planar polarity protein Stbm. Dlg was originally identified as a tumor suppressor involved in the regulation of epithelial cell polarity and later shown to play a role in ACD and synaptogenesis. Despite its widespread functions, the biochemical pathways regulated by Dlg in those various cell types are not entirely understood (Mauri, 2014).

    In SOP cells, Dlg associates with the adaptor protein Pins to direct the protein Bazooka (Baz) to the basal-posterior side of the dividing SOP cell. Together with Par-6 and aPKC, Baz forms the so-called Par protein complex that plays a pivotal role during ACD in many different cell types. Eventually, the kinase aPKC phosphorylates Numb, mediating its release from the posterior plasma membrane and thereby causing its accumulation to the anterior side (Mauri, 2014).

    To ensure the asymmetric segregation of Numb to the anterior pIIb cell, the mitotic spindle has to be oriented along the polarity axis. This function is mediated by Pins through the binding of the microtubule binding protein Mushroom body defect (Mud), which forms a cortical attachment site for astral microtubules, aligning the spindle into the correct orientation. The binding to Pins requires the heterotrimeric G protein Gαi, which associates with Pins to mediate its recruitment to the anterior plasma membrane and switches it to an open conformation in which Pins can bind Mud (Mauri, 2014).

    The same protein machinery directs ACD in neuroblasts, the stem cell-like progenitors of the Drosophila CNS. Neuroblasts divide asymmetrically into self-renewing daughter neuroblasts and smaller ganglion mother cells (GMCs) that generate two differentiating neurons through a terminal symmetric division. The asymmetric segregation of the cell fate determinants Numb, Prospero (Pros), and Brat into the GMC is required for proper differentiation. The asymmetric partitioning of Pros and Brat is mediated by the adaptor protein Miranda, and the asymmetric localization of both Miranda and Numb depends on phosphorylation by aPKC. Mutations in any of the three segregating determinants lead to the generation of excessive numbers of neuroblasts and ultimately cause the formation of lethal, transplantable brain tumors. As in SOP cells, Pins, Dlg, and Baz are required for ACD in neuroblasts, but they act in a characteristically different manner. First, neuroblast divisions are oriented along the apical-basal axis and not the planar polarity axis. Second, Pins, Dlg, and Baz colocalize apically in neuroblasts while they occupy opposite domains in SOP cells. In part, those differences can be explained by the recruitment of the adaptor protein Inscuteable (Insc) in the apical complex. Insc is not expressed in SOP cells, but in neuroblasts, it coordinates cortical polarity and spindle alignment by connecting Pins to Baz, ensuring the correct segregation of cell fate determinants in the differentiating daughter cell. In addition, Dlg has a neuroblast-specific role in mediating spindle orientation, acting downstream of Pins to align the spindle pole through the interaction with the kinesin motor Khc-73. Pins, Dlg, and Khc-73 also regulate a pathway called 'telophase rescue' that corrects ACD defects during late mitotic stages. This pathway realigns cortical polarity along the spindle axis independently of the Par complex through a Dlg cortical clustering mechanism to ensure that determinants eventually segregate asymmetrically and daughter cell fates are correctly specified. How Dlg performs those seemingly divergent roles in SOPs and neuroblasts is currently unclear (Mauri, 2014).

    Because knowledge about ACD is evidently incomplete, several RNAi screens were performed to identify additional players required for the correct establishment of daughter cell fates. This study used the results from one of those screens to identify Banderuola (Bnd; CG45058, FlyBase name: Wide awake, Wake) Banderuola is a a weathervane in the form of a rooster. Bnd is a new key regulator of ACD that acts both in neuroblasts and in SOP cells. Baz, Pins, and Dlg are all mislocalized in bnd mutant SOP cells, placing Bnd at the top of the hierarchy for ACD. In bnd mutant neuroblasts, the asymmetric segregation of cell fate determinants is disrupted because aPKC and Dlg fail to accumulate apically. Importantly, Bnd interacts physically and genetically with Dlg, suggesting that it supports Dlg in performing its divergent functions in various cell types. Because Bnd is conserved in evolution, our data identify a new member of the universal machinery for ACD that might direct cell polarity in vertebrates as well (Mauri, 2014).

    These results establish Bnd as a new component of the machinery for asymmetric cell division. bnd RNAi or loss-of-function mutations cause defects in the establishment of polarity and the positioning of the mitotic spindle in mitotic SOP cells. bnd was shown to be required for ACD and continued self-renewal activity in Drosophila larval neuroblasts. Because Bnd interacts both biochemically and genetically with the tumor suppressor protein Dlg, it is proposed that it exerts its function during ACD by regulating the function of Dlg. Moreover, the spindle rotation phenotype that were observed in mitotic SOP cells in bnd mutants is very similar to that of dlgsw mutants, further strengthening the possibility that the two proteins are functionally connected. Because the mammalian homologs of these two proteins also interact, this function might be conserved in higher organisms as well (Mauri, 2014).

    The process of ACD involves the establishment of a polarity axis, the orientation of the mitotic spindle, the polarized distribution of cell fate determinants, and, ultimately, the establishment of different daughter cell fates. In SOP cells, the axis of polarity is established when Dlg and Pins interact with components of the planar polarity pathway to concentrate anteriorly. Because Bnd binds to Dlg and is required for Pins and Dlg localization, but not for planar polarity, the data indicate that it acts at the very top of this hierarchy. Because Dlg is also mislocalized in bnd mutant neuroblasts, the role of Bnd in this tissue appears to be similar. Nevertheless, because the defect in asymmetry establishment is not completely penetrant, it is plausible that bnd function is partially redundant. Alternatively, it might also be that the residual protein derived from maternal contribution is sufficient to maintain, at least partially, the asymmetric partitioning of determinants. Further experiments will be needed to address these issues and clarify the instructive role of Bnd in establishing cell asymmetry (Mauri, 2014).

    How could Bnd perform its function on a molecular level? Bnd::GFP localizes at the centrosomes, on the spindle, and, transiently, at the cell cortex. Because Bnd contains both Ankyrin repeats and an FN3 domain, it could mediate protein-protein interactions leading to the anterior localization of Dlg downstream of the PCP pathway. The localization of Dlg and Pins to the anterior side of dividing SOP cells is regulated by Strabismus (Stbm) and Dishevelled (Dsh). It is thought that Dsh excludes Dlg/Pins from the posterior side, whereas Stbm binds Dlg at the anterior cortex, promoting the association with Pins. This hypothesis is reinforced by the fact that Dlg interacts directly with the PDZ binding motif (PBM) of Stbm in Drosophila embryos. However, Pins is localized to the anterior cortex in stbm mutant SOP cells expressing a Stbm protein lacking the PBM domain. Hence, the localization of Dlg/Pins can be regulated independently of a direct binding to Stbm. It is tempting to speculate that Bnd could be a mediator between the PCP pathway and the establishment of the asymmetry axis in mitotic SOP cells (Mauri, 2014).

    Alternatively, however, Bnd could also affect the function of Dlg and other cortical proteins through its RA domain. RA domains mediate binding to small GTPases and regulate their activity. Small GTPases are involved in the modification of the actomyosin network, and the establishment of polarity is influenced by myosin activity and by the contractility of the actomyosin mesh. In particular, Cdc42, a small GTPase of the Rho family, plays a central role in the establishment of polarity in a wide variety of biological contexts, including the localization of Par6/aPKC to the apical cortex of neuroblasts. More recent data have also implicated small Ras-like GTPases in regulating cortical polarity and spindle orientation. The Rap1/Rgl/Ral signaling network was shown to mediate those events through the regulation of the PDZ domain protein Canoe, which is a known binding partner of Pins. It is intriguing to hypothesize that Bnd could be part of a similar signaling network impinging on Dlg. Because the RA domain of Banderuola is not conserved in higher organisms, however, the first hypothesis is favored that rests on the conserved domains of the protein (ANK domains, FN3 domain, and Bnd motif). Hence, Bnd could act as an adaptor that mediates protein-protein interactions and regulates the function of binding partners such as Dlg (Mauri, 2014).

    Why Bnd is also found at centrosomes and at the spindle is harder to explain. In fact, Bnd is the only known protein apart from Mud that localizes to both the centrosome and the cell cortex during ACD. It could help in promoting the alignment of the spindle through the interaction with the Pins/Gαi/Mud complex, but this cannot explain the entire phenotype because microtubules are not strictly required for polarity establishment during ACD. Although no biochemical interaction were detected between Bnd and Pins, Gαi, or Mud, this interaction could be transient, or it could depend on polymerized microtubules. It will be compelling to verify the localization of the endogenous protein because this would consolidate the data derived from the protein overexpression experiments. Furthermore, this could allow unraveling in detail the dynamics of Bnd cortical localization and its alignment with the SOP polarity axis, which could be concealed in overexpression conditions (Mauri, 2014).

    In neuroblasts, Bnd is required for self-renewal and asymmetric protein segregation and has an antioncogenic function that is redundant with Dlg. In bnd mutants, defects were observed leading to neuroblast loss. The remaining neuroblasts are misshapen, displaying abnormalities in the asymmetric protein segregation and reduced mitotic activity. Although the FRT site remaining in the bnd mutants prevents addressing this question through a clonal analysis, the hypothesis is favored that the phenotype is cell autonomous and is due to premature differentiation of neuroblasts. Indeed, this is consistent with the phenotype in the SOP lineage because genetic manipulations resulting in a pIIa to pIIb transformation (like Numb overexpression or Notch loss of function) often cause neuroblasts to divide symmetrically into two differentiating daughter cells (Mauri, 2014).

    The localization of both the basal determinants and Dlg itself are affected in bnd mutants. Dlg is known to mediate the basal localization of cell fate determinants in Drosophila neuroblasts. The abnormal localization of aPKC in bnd mutant neuroblasts could also be explained as an effect of dlg LOF because aPKC localization is affected in dlg mutants. Thus, the various protein mislocalization phenotypes in bnd mutant neuroblasts could be explained by a model in which Bnd exerts its function solely by localizing Dlg (Mauri, 2014).

    The tumor phenotypes, on the other hand, suggest that the two genes act in parallel. Overproliferation phenotypes are observed only upon LOF of both genes, and bnd LOF enhances the dlg RNAi phenotype. In fact, this type of genetic interaction has been described for pins and lgl before: whereas pins mutant neuroblasts underproliferate due to self-renewal failure, pins lgl double mutants have a massive overproliferation of neuroblasts due to an aberrant self-renewal program triggered by aPKC. A similar mechanism could underlie the overproliferation was observed upon double RNAi of bnd and dlg. An alternative explanation for the double knockdown phenotype is provided by the additional role that Dlg has in the telophase rescue pathway, which might be independent from bnd. This pathway is known to mediate the establishment of Pins/Gαi cortical polarity, even in the absence of the Par complex, through a Dlg-dependent mechanism. The pathway is active in wild-type neuroblasts but becomes essential only when components of the apical Par complex are missing. It is possible that the telophase rescue pathway ensures the asymmetric segregation of cell fate determinants upon bnd RNAi. When dlg is inhibited as well, however, this pathway could be compromised, resulting in overproliferation and tumor formation (Mauri, 2014).

    Dlg has four mammalian homologs. Like the Drosophila protein, they localize at the basolateral cortex in epithelia and have been shown to regulate cell polarity in various cell types. During rat astrocyte migration, for example, Dlg1 is required in association with APC for the polarization of the microtubule cytoskeleton at the leading edge of the migrating cell. Dlg-mediated polarity can be also considered a gatekeeper against tumor progression: Dlg1 is a target of oncoviral proteins and is often mislocalized or downregulated in late-stage tumors, implicating a causal connection between Dlg1 and cancer. As the interaction between Bnd and Dlg is conserved, Banderuola could be an evolutionarily conserved regulator of Dlg activity, and these studies may therefore be relevant for a variety of biological processes in higher organisms as well (Mauri, 2014).

    Aurora A triggers Lgl cortical release during symmetric division to control planar spindle orientation

    Mitotic spindle orientation is essential to control cell-fate specification and epithelial architecture. The tumor suppressor Lgl localizes to the basolateral cortex of epithelial cells, where it acts together with Dlg and Scrib to organize apicobasal polarity. Dlg and Scrib also control planar spindle orientation but how the organization of polarity complexes is adjusted to control symmetric division is largely unknown. Lgl redistribution during epithelial mitosis is reminiscent of asymmetric cell division, where it is proposed that Aurora A promotes aPKC activation to control the localization of Lgl and cell-fate determinants. This study shows that the Dlg complex is remodeled during Drosophila follicular epithelium cell division, when Lgl is released to the cytoplasm. Aurora A controlled Lgl localization directly, triggering its cortical release at early prophase in both epithelial and S2 cells. This relied on double phosphorylation within the putative aPKC phosphorylation site, which was required and sufficient for Lgl cortical release during mitosis and could be achieved by a combination of aPKC and Aurora A activities. Cortical retention of Lgl disrupted planar spindle orientation, but only when Lgl mutants that could bind Dlg were expressed. Taken together, Lgl mitotic cortical release is not specifically linked to the asymmetric segregation of fate determinants, and the study proposes that Aurora A activation breaks the Dlg/Lgl interaction to allow planar spindle orientation during symmetric division via the Pins (LGN)/Dlg pathway (Carvalho, 2015).

    Evolutionarily conserved polarity complexes establish distinct membrane domains and the polarized assembly of junctions along the apicobasal axis has been extensively characterized. One general feature is that it relies on mutual antagonism between apical atypical protein kinase C (aPKC) and Crumbs complexes and a basolateral complex formed by Scribble (Scrib), Lethal giant larvae (Lgl), and Discs large (Dlg). This study used the Drosophila follicular epithelium as an epithelial polarity model to address how polarity is coordinated during symmetric division. Dlg and Scrib have been shown to provide a lateral cue for planar spindle orientation. Accordingly, Scrib and Dlg remain at the cortex during follicle cell division. In contrast, Lgl is released from the lateral cortex to the cytoplasm during mitosis. This subcellular reallocation begins during early prophase, since Lgl starts to be excluded from the cortex prior to cell rounding, one of the earliest mitotic events, and is completely cytoplasmic before nuclear envelope breakdown (NEB). Thus, the Dlg complex is remodeled at mitosis onset in epithelia (Carvalho, 2015).

    The subcellular localization of Lgl is controlled by aPKC-mediated phosphorylation of a conserved motif, which blocks Lgl interaction with the apical cortex. To address the mechanism of cortical release during mitosis, nonphosphorytable form Lgl3A-GFP was expressed in the follicular epithelium. Lgl3A-GFP remains at the cortex throughout mitosis indicating that Lgl dynamics during epithelial mitosis also rely on the aPKC phosphorylation motif. Although the apical aPKC complex depolarizes during follicle cell division, Lgl cortical release precedes aPKC depolarization. Using Par-6-GFP as a marker for the aPKC complex and the Lgl cytoplasmic accumulation as readout of its cortical release, it was found that maximum cytoplasmic accumulation of Lgl occurs when most Par-6 is still apically localized (~70% relative to interphase levels). Thus, Lgl cortical release is the first event of the depolarization that characterizes follicle cell division, indicating that Lgl reallocation does not require extension of aPKC along the lateral cortex (Carvalho, 2015).

    Although the major pools of Lgl and aPKC are segregated during interphase, Lgl has a dynamic cytoplasmic pool that rapidly exchanges with the cortex. Thus, further activation of aPKC at mitosis onset would be expected to shift the equilibrium toward cytoplasmic localization. Lgl dynamic redistribution in epithelia is similar to the neuroblast, where activation of Aurora A (AurA) leads to Par-6 phosphorylation and subsequent aPKC activation. To test whether a similar mechanism induced Lgl cortical release during epithelial mitosis, Lgl subcellular localization was analyzed in aPKC mutants and in par-6 mutants unphosphorylatable by AurA. Lgl cytoplasmic accumulation is unaffected in par-6; par-6S34A mutant cells. Temperature-sensitive aPKCts/aPKCk06403 mutants display strong cytoplasmic accumulation of Lgl during prophase, with a minor delay relatively to the wild-type). Moreover, homozygous mutant clones for null (aPKCk06403) and kinase-defective (aPKCpsu141) alleles also display Lgl cortical release during mitosis. These results implicate that although aPKC activity may contribute for Lgl mitotic dynamics, the putative aPKC phosphorylation motif is under the control of a different kinase, which triggers Lgl cortical release in the absence of aPKC (Carvalho, 2015).

    AurA is a good candidate to induce Lgl cortical release as it controls polarity during asymmetric division. Furthermore, Drosophila AurA is activated at the beginning of prophase, which coincides with the timing of Lgl cytoplasmic reallocation. To examine whether AurA controls Lgl dynamics in the follicular epithelium, homozygous mutant clones were generated for the kinase-defective allele aurA37. In contrast to wild-type cells, only low amounts of cytoplasmic Lgl were detected during prophase in aurA37 mutants, which display a pronounced delay in the cytoplasmic reallocation of Lgl during mitosis. This delayed cortical release of Lgl has been previously reported during asymmetric cell division in aurA37 mutants, possibly resulting from residual kinase activity. Thus, AurA is essential to trigger Lgl cortical exclusion at epithelial mitosis onset (Carvalho, 2015).

    The idea that Lgl mitotic reallocation is directly controlled by a mitotic kinase implies that Lgl should display similar dynamics regardless of the polarized status of the cell. Consistently, Lgl-GFP is also released from the cortex before NEB in nonpolarized Drosophila S2 cells. Furthermore, Lgl3A-GFP is retained in the cortex during mitosis, revealing that Lgl cortical release is also phosphorylation dependent in S2 cells. Treatment with a specific AurA inhibitor (MLN8237), or with aurA RNAi, strongly impairs Lgl cortical release during prophase, as Lgl is present in the cortex at NEB. However, inhibition of AurA still allows later cortical exclusion, which could result from the activity of another kinase. Despite their distinct roles, AurA and Aurora B (AurB) phosphorylate common substrates in vitro. Therefore, whether AurB could act redundantly with AurA was analyzed. Inactivation of AurB with a specific inhibitor, Binucleine 2, enables normal Lgl cytoplasmic accumulation before NEB and still allows later cortical exclusion in cells treated simultaneously with the AurA inhibitor As AurB does not seem to participate on Lgl mitotic dynamics, RNAi directed against aPKC was used to examine whether it could act redundantly with AurA. aPKC depletion did not block Lgl cortical exclusion, but it was slightly delayed. However, simultaneous AurA inhibition and aPKC RNAi produced almost complete cortical retention of Lgl during mitosis. Thus, AurA induces Lgl release during early prophase, but aPKC retains its ability to phosphorylate Lgl during mitosis (Carvalho, 2015).

    To address which serine(s) within the phosphorylation motif of Lgl control its dynamics during mitosis, individual and double mutants were enerated. As complete cortical release occurs before NEB, the ratio of cytoplasmic to cortical mean intensity of Lgl-GFP at NEB was quantified to compare each different mutant. All the single mutants displayed similar dynamics to LglWT, exiting to the cytoplasm prior to NEB. In contrast, all double mutants were cortically retained during mitosis, indicating that double phosphorylation is both sufficient and required to efficiently block Lgl cortical localization (Carvalho, 2015).

    The ability to doubly phosphorylate Lgl would explain how AurA drives Lgl cortical release. Accordingly, the sequence surrounding S656 perfectly matches AurA phosphorylation consensus, whereas the S664 surrounding sequence shows an exception in the -3 position. In contrast, the sequence surrounding S660 does not resemble AurA phosphorylation consensus, and AurA does not directly phosphorylate S660 in vitro as detected by phosphospecific antibodies against S660. That S656 is directly phosphorylated by recombinant AurA was confirmed in vitro using a phosphospecific antibody for S656. Moreover, AurA inhibition or aurA RNAi results in a similar cortical retention at NEB to LglS656A,S664A, suggesting that AurA also controls S664 phosphorylation during mitosis, whereas aPKC would be the only kinase active on S660. Consistent with this, aPKC RNAi increases the cortical retention of LglS656A,S664A, mimicking the localization of Lgl3A. Furthermore, whereas S660A mutation does not significantly affect the cytoplasmic accumulation of Lgl in aPKC RNAi, S656A and S664A mutations disrupt Lgl cortical release in aPKC-depleted cells, leading to the degree of cortical retention of LglS656A,S660A and LglS660A,S664A, respectively. Altogether, these results support that AurA controls S656 and S664 and that these phosphorylations are partially redundant with aPKC phosphorylation to produce doubly phosphorylated Lgl, which is released from the cortex (Carvalho, 2015).

    RNAi-mediated knockdown of Lgl in vertebrate HEK293 cells results in defective chromosome segregation. Furthermore, overexpressed Lgl-GFP shows a slight enrichment on the mitotic spindle suggesting that relocalization of Lgl could be important to control chromosome segregation. However, lgl mutant follicle cells assemble normal bipolar spindles, and although it was possible to detect minor defects on chromosome segregation, the mitotic timing (time between NEB and anaphase) is indistinguishable between lgl and wild-type cells. Additionally, loss of Lgl activity allows proper chromosome segregation in both Drosophila S2 cells and syncytial embryos. Thus, Lgl does not seem to have a general role in the control of faithful chromosome segregation in Drosophila (Carvalho, 2015).

    Nevertheless, Lgl cortical release could per se play a mitotic function, as key mitotic events are controlled at the cortex. In fact, the orientation of cell division requires the precise connection between cortical attachment sites and astral microtubules, which relies on the plasma membrane associated protein Pins (vertebrate LGN). Pins uses its TPR repeat domain to bind Mud (vertebrate NUMA), which recruits the dynein complex to pull on astral microtubules, and its linker domain to interact with Dlg, which participates on the capture of microtubule plus ends. Notably, Pins/LGN localizes apically during interphase in Drosophila and vertebrate epithelia, being reallocated to the lateral cortex to orient cell division. Pins relocalization relies on aPKC in some epithelial tissues, but not in chick neuroepithelium and in the Drosophila follicular epithelium, where Dlg provides a polarity cue to restrict Pins to the lateral cortex. Dlg controls Pins localization during both asymmetric and symmetric division, and a recent study has shown that vertebrate Dlg1 recruits LGN to cortex via a direct interaction. However, Dlg uses the same phosphoserine binding region within its guanylate kinase (GUK) domain to interact with Pins/LGN and Lgl. Thus, maintenance of a cortical Dlg/Lgl complex during mitosis is expected to impair the ability of Dlg to bind Pins and control spindle orientation (Carvalho, 2015).

    Interaction between the Dlg's GUK domain and Lgl requires phosphorylation of at least one serine within the aPKC phosphorylation site. Although the phosphorylation-dependent binding of Lgl to Dlg remains to be shown in Drosophila, crystallographic studies revealed that all residues directly involved in the interaction with p-Lgl are evolutionarily conserved from C. elegans to humans. Thus, whereas Lgl3A does not form a fully functional Dlg/Lgl polarity complex, double mutants should bind Dlg's GUK domain and are significantly retained at the cortex during mitosis due to the inability to be double phosphorylated. This led to an examination of their ability to support epithelial polarization during interphase and to interfere with mitotic spindle orientation. Rescue experiments were performed in mosaic egg chambers containing lgl27S3 null follicle cell clones. lgl mutant clones display multilayered cells with delocalization of aPKC. This phenotype is rescued by Lgl-GFP, but not by Lgl3A-GFP. More importantly, in contrast to LglS660A,S664A, which extends to the apical domain in wild-type cells and fails to rescue epithelial polarity in lgl mutant cells, LglS656A,S660A and LglS656A,S664A can rescue epithelial polarity, localizing with Dlg at the lateral cortex and below aPKC. Hence, aPKC-mediated phosphorylation of S660 or S664 is sufficient on its own to control epithelial polarity and to confine Lgl to the lateral cortex (Carvalho, 2015).

    Whether exclusion of Lgl from the cortex and the consequent release from Dlg would be functionally relevant for oriented cell division was examined. Expression of Lgl-GFP or Lgl3A-GFP does not affect planar spindle orientation during follicle cell division. In contrast, Lgl double mutants display metaphasic cells in which the spindle axis, determined by centrosome position, is nearly perpendicular to the epithelial layer. Live imaging revealed that these spindle orientation defects were maintained throughout division as it was possible to follow daughter cells separating along oblique and perpendicular angles to the epithelia. Moreover, equivalent defects on planar spindle orientation were detected upon expression of LglS656A,S664A in the lgl or wild-type background, indicating that cortical retention of Lgl exerts a dominant effect. Interestingly, LglS656A,S660A and LglS656A,S664A induce higher randomization of angles, whereas LglS660A,S664A, which is less efficiently restricted to the lateral cortex, produces a milder phenotype. Altogether, these results indicate that retention of Lgl at the lateral cortex disrupts planar spindle orientation only if Lgl can interact with Dlg (Carvalho, 2015).

    Despite the ability of LglS656A,S660A-GFP to rescue epithelial polarity in lgl mutants, strong overexpression of LglS656A,S660A-GFP, but not of other Lgl double mutants, can dominantly disrupt epithelial polarity during the proliferative stages of oogenesis. One interpretation is that LglS656A,S660A forms the most active lateral complex of the mutant transgenes, disrupting the balance between apical and lateral domains. Therefore whether the dominant effect of Lgl cortical retention on spindle orientation could solely result from Dlg mislocalization was assessed. Dlg is properly localized at the lateral cortex in LglS656A,S660A-expressing cells presenting misoriented spindles, but this position does not correlate with the orientation of the centrosomes. Thus, cortical retention of Lgl interferes with Dlg's ability to transmit its lateral cue to instruct spindle orientation, which may result from an impairment of the Dlg/Pins interaction (Carvalho, 2015).

    In conclusion, these findings outline a mechanism that explains how the lateral domain is remodeled to accomplish oriented epithelial cell division, unveiling that AurA has a central role in controlling the subcellular distribution of Lgl. AurA regulates the activity of aPKC at mitotic entry during asymmetric division, and these results are consistent with the ability of aPKC to phosphorylate and collaborate in Lgl cortical release. However, in epithelia, aPKC accumulates in the apical side during interphase, where it induces apical exclusion of Lgl, in part by generating a phosphorylated form that binds Dlg. Consequently, aPKC has a reduced access to the cortical pool of Lgl at mitotic entry and would be unable to rapidly induce Lgl cortical exclusion. These data show that cell-cycle-dependent activation of AurA removes Lgl from the lateral cortex through AurA's ability to control Lgl phosphorylation on S656 and S664 independently of aPKC. Thus, AurA and aPKC exert the spatiotemporal control of Lgl distribution to achieve unique cell polarity roles in distinct cell types (Carvalho, 2015).

    It is proposed that release of Lgl from the cortex allows Dlg interaction with Pins to promote planar cell division in Drosophila epithelia. Lgl cortical release requires double phosphorylation, indicating that whereas Lgl-Dlg association involves aPKC phosphorylation, multiple phosphorylations break this interaction, acting as an off switch on Lgl-Dlg binding. Triple phosphomimetic Lgl mutants display weak interactions with Dlg, suggesting that multiple phosphorylations could directly block Lgl-Dlg interaction. Alternatively, the negative charge of two phosphate groups may suffice to induce association between the N- and C-terminal domains of Lgl, impairing its ability to interact with the cytoskeleton and plasma membrane as previously proposed. This would reduce the local concentration of Lgl available to interact with Dlg, enabling the interaction of Dlg's GUK domain with the pool of Pins phosphorylated by AurA. Therefore, AurA converts the Lgl/Dlg polarity complex generated upon aPKC phosphorylation into the Pins/Dlg spindle orientation complex. This study, underlines the critical requirement of synchronizing the cell cycle with the reorganization of polarity complexes to achieve precise control of spindle orientation in epithelia (Carvalho, 2015).

    Uninflatable and Notch control the targeting of Sara endosomes during asymmetric division

    Cell fate decision during asymmetric division is mediated by the biased partition of cell fate determinants during mitosis. In the case of the asymmetric division of the fly sensory organ precursor cells, directed Notch signaling from pIIb to the pIIa daughter endows pIIa with its distinct fate. Previous studies have shown that Notch/Delta molecules internalized in the mother cell traffic through Sara endosomes and are directed to the pIIa daughter. This study shows that the receptor Notch itself is required during the asymmetric targeting of the Sara endosomes to pIIa. Notch binds Uninflatable, and both traffic together through Sara endosomes, which is essential to direct asymmetric endosomes motility and Notch-dependent cell fate assignation. The data uncover a part of the core machinery required for the asymmetric motility of a vesicular structure that is essential for the directed dispatch of Notch signaling molecules during asymmetric mitosis (Loubery, 2014).

    Dlg5 maintains apical polarity by promoting membrane localization of Crumbs during Drosophila oogenesis

    Apical-basal polarity plays critical roles in the functions of epithelial tissues. However, the mechanisms of epithelial polarity establishment and maintenance remain to be fully elucidated. This study shows that the membrane-associated guanylate kinase (MAGUK) family protein Dlg5 is required for the maintenance of apical polarity of follicle epithelium during Drosophila oogenesis. Dlg5 localizes at the apical membrane and adherens junction (AJ) of follicle epithelium in early stage egg chambers. Specifically, the major function of Dlg5 is to promote apical membrane localization of Crumbs, since overexpression of Crumbs but not other major apical or AJ components can rescue epithelial polarity defects resulting from loss of Dlg5. Furthermore, by performing a structure-function analysis of Dlg5, it was found that the C-terminal PDZ3 and PDZ4 domains are required for all Dlg5's functions as well as its ability to localize to apical membrane. The N-terminal coiled-coil motif can be individually targeted to the apical membrane, while the central linker region can be targeted to AJ. Lastly, the MAGUK core domains of PDZ4-SH3-GUK can be individually targeted to apical, AJ and basolateral membranes (Luo, 2016).

    How cell polarity is established and maintained is an important question in the fields of cell and developmental biology. During animal development, polarized cells such as epithelial cells maintain their apical-basal polarity despite undergoing dramatic shape changes and tissue remodeling during morphogenesis. Genetic screens done in developing Drosophila and C. elegans have uncovered a number of highly conserved apical and basolateral regulators essential for the establishment and maintenance of apical-basal polarity. Specifically, the Par3/Par6/aPKC complex and the Crumbs (Crb)/Stardust (Sdt)/Patj complex are thought to define and maintain the identity of apical membrane, and the Discs-large (Dlg)/Lethal giant larvae (Lgl)/Scribble (Scrib) complex delineates the basolateral membrane. Finally, the junctional complex of E-cadherin/β-catenin/α-catenin initiates and maintains the adherens junction (AJ), which divides the cortex into apical and basolateral regions. Decades of research have formed a consensus model, in which the apical-basal polarity is generated and maintained by a mutually antagonistic interaction between the apical regulators and the basolateral regulators. Recently, a mathematic modeling study done in Drosophila follicle epithelia suggested that in addition to the negative feedback between apical regulators and basolateral regulators, a positive feedback loop among apical polarity regulators is required to maintain the apical-basal polarity in epithelia. A central component of this positive feedback loop is the transmembrane protein Crb, and its apical membrane localization was thought to be the key to maintenance of apical-basal polarity (Luo, 2016).

    Discs-large 5 (Dlg5) belongs to the MAGUK family, and it is highly conserved across species including human, mouse, chicken, zebra fish and Drosophila. MAGUK members also include Dlg and Sdt, which act as molecular scaffolds and are core components of the basolateral Dlg/Lgl/Scrib complex and the apical Crumb complex respectively. Dlg5 was first identified in human and was found to be expressed in placenta and in prostate gland epithelia. Since then, Dlg5 was found to interact with a variety of junctional, cytoskeletal, trafficking, and receptor molecules, including β-catenin, P55, vinexin, Girdin, Citron kinase, Syntaxin, Smoothened and TGF-β receptors. And its functions vary from inhibiting cancer cell migration to mediating receptor signaling, but most of these functions were obtained from cell culture studies. Detailed genetic analysis of Dlg5 was first done using Dlg5 knockout mice, which displayed failure of epithelial tube maintenance resulting in brain hydrocephalus and kidney cysts. These defects were likely due to disruption of apical polarity and AJ. This study focused on Dlg5's requirement of AJ function and found that Dlg5 physically interacted with the β-catenin/cadherin complex and was found together with β-catenin/cadherin complex in both the Rab11-labeled recycling vesicles and the AJ. A more recent work using the same Dlg5-/- mice found that Dlg5 was also required for lung morphogenesis. Specifically, deletion of Dlg5 resulted in loss of apical polarity markers such as aPKC, and Dlg5 was partially colocalized with aPKC in the apical membrane of the wild type lung epithelia. But how Dlg5 exerts its functions in the apical membrane and regulate apical polarity is unknown (Luo, 2016 and references therein).

    In Drosophila, RNAi knockdown of dlg5 affected the cohesion and morphology of border cell clusters as well as delaying their migration. Recently, genetic analysis of Drosophila Dlg5 revealed that its mutation caused embryonic lethality and loss of germ cells in the embryonic gonad. Moreover, reduction of Dlg5 in the follicle cells in the adult ovary leads to defects in egg chamber budding, stalk cell overgrowth, ectopic polar cell induction and abnormal distribution of E-cadherin. However, detailed analysis of whether and how Drosophila Dlg5 regulates epithelial or apical polarity has not been done. This study reports that a genetic screen for follicle epithelial morphogenesis has identified the Drosophila Dlg5 as an essential player for maintenance of apical polarity by promoting Crb's apical membrane localization (Luo, 2016).

    Dlg5 is specifically required for the maintenance of apical polarity and AJ of follicle epithelia during early stages of oogenesis, since both loss-of-function mutation and RNAi knockdown of dlg5 affected only apical polarity regulators and the sub-apical AJ components but not the basolateral regulators. Furthermore, the apical markers (Crb, Sdt, Patj, aPKC and Par6) were more severely reduced than the sub-apical AJ markers (Arm, E-cad and Baz) with the exception of N-cad, suggesting that the loss of apical polarity is the main cause of severe morphological defects in Dlg5-deficient follicle cells. A previous study found that loss of Crb, aPKC and Par6 did not affect the lateral localization of Dlg, whereas loss of Arm caused Dlg spreading to the apical membrane of follicle cells. This is consistent with the result that no apical spreading of Dlg was observed in dlg5 mutant clones, further confirming that loss of Dlg5 affected apical polarity more severely than the AJ function. Indeed, rescue of apical polarity defects by Crb but not Arm expression further validated this notion (Luo, 2016).

    Importantly, this study demonstrates that Dlg5 positively regulates apical polarity by specifically promoting Crb's apical membrane localization, based on the following results. First, double staining revealed that Crb reduction was sometimes more severe than reduction of other apical markers (aPKC and Par6) in dlg5 mutant clones. Second, overexpression of Crb but not other apical or AJ regulators (aPKC, Par6, Arm) could completely rescue dlg5's apical polarity defects. Third, the increased membrane localization and membrane spreading of Crb as caused by blocking the Rab5-mediated endocytosis could be dramatically suppressed by dlg5 mutation. Moreover, the apical enrichment of Dlg5 in the early and mid-stage follicle epithelia (stage 1-stage 9) further suggests that Dlg5 could function at the apical region to promote Crb's membrane localization. On the other hand, Crb might conversely enhance Dlg5's localization to the apical membrane, since overexpression of Crb and hence its membrane spreading toward the basolateral region led to stronger localization of Dlg5 in the basolateral membrane. A previous study has reported that deletion of Dlg5 in mouse resulted in loss of aPKC but not Par3 (homologous with Drosophila Baz) in the developing lung epithelia and that Dlg5 was partially colocalized with aPKC, which are similar to the current findings. But how Dlg5 regulated the apical polarity during mouse lung morphogenesis was not understood. Based on the current results, it would be worthwhile to check whether murine Dlg5 promotes apical polarity by primarily regulating one of the three mammalian CRB paralogs (CRB1, CRB2, CRB3) (Luo, 2016).

    As a MAGUK family member, Dlg5 is thought to function as a scaffold protein. Previous works have focused on which domains of Dlg5 physically interact with junctional and membrane-bound proteins like β-catenin, vinexin and smoothened, and trafficking regulators like syntaxin 4 in vitro or in cultured cells. But whether such domains are essential for its function and localization in vivo and which domains possess apical or AJ membrane targeting ability have not yet been addressed. Structure-function analysis demonstrates that the C-terminal fragment including MAGUK core (GUK, SH3, PDZ4) and PDZ3 is necessary but not entirely sufficient for Dlg5's functions. Furthermore, deletion of this C-terminal fragment (Δ4) caused most of Dlg5 to re-distribute to the cytoplasm, losing its membrane localization in the apical, AJ, and lateral regions. Interestingly, PDZ3 and PDZ4 (a subset of C terminal fragment) were also required for Dlg5's functions, and their deletion (Δ5) likewise resulted in the loss of apical and lateral (but not AJ) membrane localization. One interesting difference between the localization patterns of the other deletion mutants (that still possessed rescue abilities; Δ1-3, Δ6-8) and the patterns of Δ4 and Δ5 is that other deletions all retained some degree of apical localization, in contrast to the lack of localization in the apical membrane for Δ4 and Δ5. Together, these results suggest that MAGUK core and PDZ3's requirement for Dlg5's membrane localization in general and PDZ3-PDZ4's requirement for Dlg5's apical membrane localization may be critical for Dlg5's functions in the follicle cells. This is consistent with Dlg5's role in promoting Crb's apical localization. Lastly, this study found that the N-terminal coiled coil domain, the middle linker region and the MAGUK core could be individually membrane-targeted to apical, AJ and all (apical, AJ and basolateral) regions respectively (Luo, 2016).

    Role of Tau, a microtubule associated protein, in Drosophila photoreceptor morphogenesis

    Cell polarity genes have important functions in photoreceptor morphogenesis. Based on recent discovery of stabilized microtubule cytoskeleton in developing photoreceptors and its role in photoreceptor cell polarity, microtubule associated proteins might have important roles in controlling cell polarity proteins' localizations in developing photoreceptors. Tau, a microtubule associated protein, was analyzed in this study to find its potential role in photoreceptor cell polarity. Tau colocalizes with acetylated/stabilized microtubules in developing pupal photoreceptors. Although it is known that tau mutant photoreceptor has no defects in early eye differentiation and development, it shows dramatic disruptions of cell polarity proteins, adherens junctions, and the stable microtubules in developing pupal photoreceptors. This role of Tau in cell polarity proteins localization in photoreceptor cells during the photoreceptor morphogenesis was further supported by Tau's overexpression studies. Tau overexpression caused dramatic expansions of apical membrane domains where the polarity proteins localize in the developing pupal photoreceptors. It is also found that Tau's role in photoreceptor cell polarity depends on Par-1 kinase. Furthermore, a strong genetic interaction between tau and crumbs was found. Tau was found to have a crucial role in cell polarity protein localization during pupal photoreceptor morphogenesis stage, but not in early eye development including eye cell differentiation (Nam, 2016).


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    Zygotically transcribed genes

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