Interactive Fly, Drosophila

org Interactive Fly, Drosophila

Three snail family genes -- snail, escargot and worniu -- encode related zinc finger transcription factors that mediate Drosophila central nervous system (CNS) development. Simultaneous removal of all three genes causes defective neuroblast asymmetric divisions; inscuteable transcription/translation is delayed/suppressed in the segmented CNS. Furthermore, defects in localization of cell fate determinants and orientation of the mitotic spindle in dividing neuroblasts are much stronger than those associated with inscuteable loss of function. In inscuteable neuroblasts, cell fate determinants are mislocalized during prophase and metaphase, yet during anaphase and telophase the great majority of mutant neuroblasts localize these determinants as cortical crescents overlying one of the spindle poles. This phenomenon, known as 'telophase rescue', does not occur in the absence of the snail family genes; moreover, in contrast to inscuteable mutants, mitotic spindle orientation is completely randomized. These data provide further evidence for the existence of two distinct asymmetry-controlling mechanisms in neuroblasts both of which require snail family gene function: an inscuteable-dependent mechanism that functions throughout mitosis and an inscuteable-independent mechanism that acts during anaphase/telophase (Cai, 2001).

CNS development is abnormal in Df(2L)osp29 embryos due to deletion of Sna family proteins. Both Sna and Wor are expressed strongly in all NBs, including those in the procephalic region, during early neurogenesis. The expression of Esg is also seen in NBs and other tissues, as visualized with anti-Esg immunostaining. Expression of Esg can be detected in the midline cells as well as GMCs during embryonic development. The functions of these three genes are overlapping; the early CNS defects are detected only when all three genes are removed simultaneously. In order to test whether the defects of localization of Mir/Pros and Pon/Numb seen in Df(2L)TE35BC-3 embryos are due to the absence of the three sna family genes, the localization of Mir/Pros and Pon/Numb was examined in embryos single mutant for sna, esg or wor, a double mutant for sna/esg and deletions that removed sna/wor or esg/wor, as well as embryos double mutant for sna/esg and further subjected to wor double-stranded RNA (RNAi) treatment. In single and double mutant embryos, both Mir/Pros and Pon/Numb form normal basal crescents in mitotic NBs. Only the sna/esg double mutant embryos that have been injected with wor RNAi reproduce the phenotype found in Df(2L)TE35BC-3 embryos (Cai, 2001).

In wild-type embryos, NBs are located between the ectoderm and mesoderm. The Df(2L)TE35BC-3 embryos lack mesoderm. Therefore, it is possible that correct NB asymmetry requires signal(s) from the mesoderm, and the asymmetry defects seen in Df(2L)TE35BC-3 could be due simply to the absence of mesoderm in these embryos. This is unlikely since NB asymmetry is intact in sna embryos, which lack mesoderm and share the abnormal morphology of Df(2L)TE35BC-3 embryos. Furthermore, the partial rescue of mesoderm in Df(2L)TE35BC-3 embryos by ectopic expression of the Sna protein driven by twist-gal4 does not reverse the asymmetry defects. Thus, it is concluded that mislocalization of Mir/Pros and Pon/Numb in Df(2L)TE35BC-3 embryos is due to the absence of all three sna family genes. Based on this conclusion, Df(2L)TE35BC-3 is referred to as sna/esg/wor deficient and was used in subsequent studies (Cai, 2001).

In wild-type embryos, Baz, Insc and Pins form a complex that is localized to the apical cortex of the dividing NBs. The apical complex is required for the asymmetric distribution of cell fate determinants such as Pros and Numb to the basal cortex of NBs and coordinates the orientation of the mitotic spindle along the apical-basal axis of the NB. In embryos deficient for the sna family genes, Mir/Pros and Pon/Numb are no longer concentrated to the basal cortex of mitotic NBs, indicating defects in NB asymmetry. It is possible that the asymmetry defects seen in sna/esg/wor-deficient NBs are due to the alteration of Insc expression. Anti-Insc staining indicates that Insc protein is indeed undetectable in the segmented CNS of sna/esg/wor-deficient embryos. Although the signal intensity in the procephalic region is comparable to that in the wild-type controls, the number of cells with anti-Insc staining appears to be decreased. This altered expression of Insc in the mutant embryos suggests that the mislocalization of Mir/Pros and Pon/Numb in sna/esg/wor-deficient embryos is, at least in part, due to a lack of Insc protein expression in dividing NBs. As expected, Baz protein levels are low and undetectable in the great majority of mutant NBs. The lack of easily detectable Baz in NBs is probably due to the instability of the protein when Insc is absent since the baz mRNA levels remain unchanged in sna/esg/wor NBs. Pins protein localization is also affected in sna/esg/wor-deficient embryos (Cai, 2001).

The down-regulation of Insc protein in NBs is also dependent on the simultaneous loss of sna, esg and wor functions. Insc expression in double mutant embryos of sna/esg was similar to that of wild-type embryos. In sna/esg double mutant embryos, further removal of the third member of sna gene family, wor, with RNAi leads to the total loss of Insc protein expression. Moreover, ectopic expression of any one of the sna family genes under the control of an early neural driver sca-gal4 in sna family gene mutant embryos largely restores the Insc expression in NBs (sna 79%; esg 64% and wor 44%), further indicating that Insc expression is indeed regulated by the Sna family proteins (Cai, 2001).

insc transcript levels were examined in the sna/esg/wor-deficient embryos. In wild-type stage 9-10 embryos, insc RNA is expressed prominently in NBs of the segmented CNS and in the procephalic region. The transcript level is maintained in the segmented CNS and procephalic NBs throughout embryogenesis. In sna/esg/wor-deficient embryos, RNA in situ hybridization data indicate that the insc RNA is absent in the segmented CNS at stages 9-10 but is detectable in the procephalic NBs. This suppression of insc RNA transcription in the segmented CNS of sna/esg/wor-deficient embryos provides evidence that the Sna family proteins are essential for insc mRNA transcription during early neurogenesis. The suppression of insc transcription in the segmented CNS is transient and insc RNA can be detected, at a lower level, in late stage 11 embryos. However, Insc protein in the segmented CNS of sna/esg/wor-deficient embryos remains undetectable at late stage 11 when the insc RNA levels partially recover by an unknown mechanism. It is obvious that translation of insc RNA in late stage 11 embryos is inhibited in the segmented CNS of embryos deficient for sna/esg/wor. Although the inhibition mechanism is unknown, it is believed that the insc 5'- and/or 3'-untranslated regions (UTRs) are involved since Insc protein can be ectopically expressed in sna/esg/wor-deficient embryos from a uas-insc transgene in which the 5'- and 3'-UTRs have been partially removed. Considering that the Sna family proteins are localized to nuclei, it is unlikely that they interact directly with 5'- and/or 3'-UTRs of insc RNA. Presumably other genes regulated by the Sna family proteins mediate the observed translational effect (Cai, 2001).

The observation of delayed and decreased insc mRNA transcription and the inhibition of Insc protein synthesis in the segmented CNS of sna/esg/wor-deficient embryos suggests the dual regulation of insc expression by the Sna family proteins at both transcriptional (stage 9-10) and translational (stage 11 onwards) levels. This dual regulation mechanism is prominent in the segmented CNS but insc RNA and protein expression in the procephalic region is only partially affected in sna/esg/wor-deficient embryos. The mechanism that enables the partial restoration of insc transcription in NBs of the segmented CNS at late stage 11 in the absence of sna family gene function remains to be identified (Cai, 2001).

In insc²² mutant NBs, in which the apical complex required for correct asymmetric division is abolished, basal components such as Mir/Pros and Pon/Numb often form random crescents, sometimes broad and loose, from prophase to metaphase; however, Pros/Mir and Pon/Numb can eventually be redistributed to the 'budding site' of the future GMCs, although sometimes not as exclusively as seen in wild-type embryos, at anaphase and telophase even when the spindle is misorientated. Consequently, the great majority of all GMCs inherit, at least in part, cell fate determinants such as Pros and adopt correct GMC fate. This phenomenon, referred to as 'telophase rescue', does not occur in NBs lacking the three sna family genes. For example, in sna/esg/wor-deficient NBs, basal proteins Mir/Pros and Pon/Numb form a randomly localized crescent in dividing NBs but, unlike in insc embryos, these proteins are not redistributed at anaphase/telophase to the region of the cortex that gives rise to the GMC. Consequently, the great majority of the GMCs do not inherit the basal proteins Mir/Pros and Pon/Numb and thus lose their GMC identities. This finding explains why GMCs are not specified correctly in Df(2L)osp29 embryos (Cai, 2001).

Furthermore, it is known that the mitotic spindle in NBs rotates 90° during metaphase so that it is realigned along the apical-basal (A/B) axis of the embryos; in insc mutants, this spindle rotation during metaphase occurs only in a small proportion (~20%) of NBs; nevertheless, even some of these NBs are able to reorient spindles late in mitosis. The NB spindle orientation during anaphase or telophase was measured in wild-type and mutant embryos and they were catagorized into four equal quadrants depending on the angle that the spindle forms with the A/B axis. Based on the spindle orientation in wild-type embryos, all spindles with an angle >45° relative to the A/B axis during late mitosis are considered to be misoriented. The misoriented spindles in insc²²mutant embryos are limited; the great majority of NBs (90%) have their spindles oriented within 45° of the A/B axis, compared with 100% in wild-type NBs. In contrast to wild-type and insc NBs, in sna/esg/wor-deficient NBs, spindle orientation is completely randomized with almost equal distribution for each of the four quadrants. Moreover, a small number of NBs (10%) completely reverse their polarity, giving rise to a small apical GMC, which has never been reported in any known asymmetry mutant (Cai, 2001).

These observations indicate that removal of Insc alone has only a limited effect on NB asymmetric divisions in terms of basal protein localization and spindle orientation late in mitosis, suggesting that the Insc-dependent mechanism is not the only apparatus that controls the asymmetric divisions in NBs. It appears that an Insc-independent mechanism exists that functions in parallel to coordinate the asymmetry events at later stages (anaphase onwards) of mitosis. This Insc-independent asymmetry-controlling mechanism, which is responsible for the 'telophase rescue' phenomenon and for prevention of random spindle orientation in insc²² embryos, is destroyed upon removal of the three sna family genes. However, one might argue that the severe asymmetry defects seen in the absence of the sna family genes might be artifactual, caused by the combination of loss of insc expression and the absence of the mesoderm. This possibility is suggested because in insc/sna double mutant embryos, which lack both insc and the mesoderm, NBs exhibit phenotypes that are indistinguishable from those seen in the insc single mutant. It has therefore been concluded that in the absence of the sna family genes, both the Insc-dependent and -independent asymmetry-controlling mechanisms are destroyed, leading to asymmetry defects that are more severe than those seen in insc single mutants (Cai, 2001).

The existence of two distinct asymmetry-controlling mechanisms in wild-type NBs raises an interesting issue: how do these two mechanisms work in concert to mediate asymmetric divisions? Since embryos deficient for the sna family genes lack both mechanisms, it was reasoned that by restoring the Insc-dependent mechanism in these embryos the consequences of missing just the insc-independent mechanism could be assessed. Ectopic expression of full-length Insc protein with an early neural driver sca-gal4 in NBs of sna family gene mutant embryos shows complete rescue of the protein localization defects. The apical complex forms normally, as indicated by the formation of apical Insc as well as Pins and Baz crescents. The defects in basal protein localization are also completely rescued; Mir/Pros and Pon/Numb form tight basal crescents in mitotic NBs. These results suggest that, with respect to protein localization, Insc protein is the only component missing in the Insc-dependent asymmetry machinery, and replacement of Insc through ectopic expression is sufficient to restore wild-type localization of the apical and basal components. Furthermore, it indicates that the Insc-independent mechanism is cryptic with respect to protein localization since it is dispensable when the Insc-dependent mechanism is in place. Either mechanism alone is able to distribute basal proteins to the cortex of the future GMC 'budding site' with clear temporal and efficiency differences: the Insc-dependent mechanism localizes basal proteins starting in late prophase in the form of tight crescents, while the Insc-independent mechanism is only able to redistribute, sometimes partially, mislocalized basal proteins late in mitosis (telophase rescue) (Cai, 2001).

The spindle misorientation phenotype in sna family gene mutant embryos is also largely corrected by ectopic Insc expression. However, unlike protein localization, the rescue of mitotic spindle orientation is incomplete; the population of NBs with misoriented spindles drops from 45% to only 12%. These data suggest that both the Insc-dependent and -independent mechanisms are required for correct spindle orientation in wild-type embryos since ~10% of the mitotic spindles are misoriented in anaphase/telophase NBs defective for either mechanism. However, a complete randomization of spindle orientation is seen when both mechanisms are absent (Cai, 2001).

Thus, the underlying cause for the asymmetry defects associated with some deficiencies uncovering the 35B-D region of the genome, e.g. Df(2L)TE35BC-3, is the simultaneous loss of three members of the sna gene family: sna, esg and wor. All available lethal complementation groups uncovered by Df(2L)TE35BC-3, all deficiencies that remove only two out of the three sna family members and a sna/esg double mutant generated from recombination do not show any defects in any aspect of NB asymmetric division; only embryos double mutant for sna/esg, and further subjected to wor RNAi, reproduce the asymmetry defects seen in the deficiencies. These data indicate that the defects in sna/esg/wor-deficient embryos are caused by the simultaneous functional loss of all three sna family genes. The observation that the ectopic expression of sna, esg or wor reverses the asymmetry phenotypes in the segmented CNS of sna/esg/wor-deficient embryos further supports this conclusion. These conclusions are in agreement with an earlier study reporting that the sna family genes are required for CNS development (Cai, 2001).

It has been observed that in insc embryos, cell fate determinants such as Pros and Numb are mislocalized early during mitosis; however, in anaphase and telophase, the effect termed 'telophase rescue' causes the misplaced crescents to redistribute and overlie one spindle pole, enabling the basal cell fate determinants to segregate, exclusively or partially, to the GMCs. The insc loss-of-function alleles insc²², insc^P49 and insc^P72 all show telophase rescue. It has been found that essentially all NBs in insc embryos can redistribute Pros and Numb, at least partially, into GMCs. These observations suggest the existence of a second asymmetry-controlling mechanism that does not require insc functions, which operates late in mitosis to coordinate protein localization with spindle orientation. These observations explain why insc mutants have minimal effect on GMC cell fate. The Insc-independent mechanism corrects the earlier errors caused by absence of Insc during anaphase/telophase, thereby enabling cell fate determinants to be inherited by the GMC. This mechanism is apparently less efficient, as shown by the fact that in some insc NBs, normally basal components form a broad and loose crescent and are only partially sequestered into GMCs. Furthermore, the observation that mitotic spindle orientation is only mildly affected in insc NBs is also consistent with an Insc-independent compensatory mechanism (Cai, 2001).

Analysis of NB divisions in embryos deficient for the three sna family genes provides further support for the existence of an Insc-independent mechanism. In these embryos, the Insc-dependent mechanism is clearly abolished; both the transcription and the translation of insc are suppressed in the mutant NBs. In addition, telophase rescue no longer occurs; the normally basally localized components are misplaced in mitotic NBs and not redistributed to the future GMCs even at anaphase/telophase. Moreover, the spindle orientation in embryos deficient for the sna family genes becomes randomized; ~45% of NBs exhibit misoriented spindles with an angle >45° with respect to the A/B axis at anaphase/telophase, which is not seen in wild-type NBs and is at a much higher frequency than that seen in insc²² NBs. Thus, NBs deficient for the sna family genes show two defects that are not seen in insc NB: (1) the absence of telophase rescue, and (2) randomization of the spindle orientation late in mitosis. These observations indicate that both the Insc-dependent and -independent mechanisms require the sna family genes (Cai, 2001).

These two mechanisms can apparently function independently. In insc NBs, the Insc-independent mechanism functions in the absence of the Insc-dependent mechanism to correct the earlier (prophase to metaphase) asymmetry defects during anaphase/telophase. In sna/esg/wor-deficient NBs that have been forced to express Insc, the Insc-dependent mechanism can act in the absence of the Insc-independent mechanism to mediate the localization of the basal components from prophase to telophase, obviating the requirement for telophase rescue; however, although the Insc-dependent mechanism can reduce the extent of the mitotic spindle orientation defects seen in the sna/esg/wor NBs, it does not restore wild-type spindle orientation. Therefore, it appears that both mechanisms are required and act in concert to mediate mitotic spindle orientation. However, with respect to localization of the basal components, the effects of the Insc-independent mechanism are only visible when the Insc-dependent mechanism is absent (Cai, 2001).

For the Insc-dependent mechanism, three components have been identified: Baz, Insc and Pins are known to form an apically localized functional complex. The function of this complex requires the participation of all members. Insc appears to be the only component of the Insc-dependent mechanism missing in sna/esg/wor-deficient embryos since ectopic expression of Insc restores its function. Little information is available on the components of the Insc-independent mechanism. Other members of asymmetry machinery identified so far in NBs are the basal components such as Mir/Pros, Pon/Numb, Stau and pros RNA. These downstream components are controlled and coordinated by both Insc-dependent and -independent mechanisms (Cai, 2001).

In embryos deficient for the sna family genes, one of the major defects is the absence of Insc protein expression in the segmented CNS. RNA in situ hybridization indicates that the insc RNA transcripts are not detected in NBs of stage 9-10 embryos. Even in late stage 11 embryos when the insc RNA levels partially recover, Insc protein is never seen in the segmented CNS, indicating that the down-regulation of insc occurs at both the transcriptional and translational levels. In the procephalic region of these sna/esg/wor-deficient embryos, Insc expression is only partially affected. The 5'- and/or 3'-UTRs of the insc transcript appear to play an important role in the translational regulation of Insc expression. This is supported by two observations: (1) Insc protein can be detected in sna/esg/wor embryos following ectopic expression of a cDNA construct containing the complete insc coding region but with the 5'- and 3'-UTRs partially removed; (2) transcripts derived from lacZ driven by a 1.2 kb insc 5' CNS promoter sequence are not subjected to this translational repression in sna/esg/wor embryos, although their expression pattern is identical to that of Insc in the CNS. Given that the Sna family proteins are localized to nuclei, it is unlikely that they play a direct role in translational regulation. Other unknown intermediates must be involved (Cai, 2001).

Snail, a zinc-finger transcriptional repressor, is a pan-neural protein, based on its extensive expression in neuroblasts. Previous results have demonstrated that Snail and related proteins, Worniu and Escargot, have redundant and essential functions in the nervous system. The Snail family of proteins control central nervous system development by regulating genes involved in asymmetry and cell division of neuroblasts. In mutant embryos that have the three genes deleted, the expression of inscuteable is significantly lowered, while the expression of other genes that participate in asymmetric division, including miranda, staufen and prospero, appears normal. The deletion mutants also have much reduced expression of string, suggesting that a key component that drives neuroblast cell division is abnormal. Consistent with the gene expression defects, the mutant embryos lose the asymmetric localization of Prospero RNA in neuroblasts and lose the staining of Prospero protein that is normally present in ganglion mother cells. Simultaneous expression of inscuteable and string in the snail family deletion mutant efficiently restores Prospero expression in ganglion mother cells, demonstrating that the two genes are key targets of Snail in neuroblasts. Mutation of the dCtBP co-repressor interaction motifs in the Snail protein leads to reduction of the Snail function in central nervous system. These results suggest that the members of the Snail family of proteins control both asymmetry and cell division of neuroblasts by activating, probably indirectly, the expression of inscuteable and string (Ashraf, 2001).

Both snail and worniu have extensive expression in neuroblasts, while that of escargot is transient and sparse. Furthermore, based on genetic analysis, snail and worniu have more important role than escargot in the regulation of CNS development. The expression of snail and worniu in GMCs was carefully examined. In situ hybridization has revealed that worniu RNA, in contrast to its extensive expression in neuroblasts, is present in only a small number of GMCs. Even in later staged embryos, when there should be multiple GMCs surrounding each neuroblast, the staining in no more than one small cell next to each neuroblast could be detected. The limited staining in the GMCs is probably due to the segregation of some RNA from the parental neuroblast. Once the GMC is formed, the active transcription of worniu probably ceases. The protein and RNA expression of snail was also examined. The results showed that there is also very limited expression of snail in GMCs. snail RNA-containing GMCs were rarely detected next to neuroblast. Consistent with RNA expression, antibody staining revealed that the protein is predominantly in the neuroblasts (Ashraf, 2001).

In mutants containing deletions that uncover escargot, worniu and snail, many early neuroblast markers are normal, but ftz expression in GMCs is abnormal. The regulation of ftz depends on Prospero, a homeodomain protein that controls GMC fate. Prospero protein and mRNA are preferentially segregated to GMCs from the neuroblast through the process of asymmetric division. Genes that are involved in asymmetric segregation of Prospero include inscuteable, miranda and staufen. The expression of these possible Snail family target genes was examined in neuroblasts (Ashraf, 2001).

Mutant embryos collected from deficiency strains that uncover the 35D1 chromosomal region, including the snail family genes were examined. In wild-type embryos, the expression of inscuteable can be detected in delaminating neuroblasts. After delamination, many neuroblasts show localization of the Inscuteable RNA. Embryos homozygous for the region 35D1 osp29 deletion, however, had significantly lower levels of the RNA and the staining was detected in a much smaller number of neuroblasts. Transgenic copies of snail, worniu or escargot efficiently rescues the expression of inscuteable RNA, demonstrating that it is the uncovering of the snail family of genes in the deletion that causes the phenotype. The rescue transgenes are under the control of the 2.8 kb snail promoter, which contains the neuroblast expression element. A 1.6 kb snail promoter construct that contains the mesoderm element but lacks the CNS element could not rescue the defect, demonstrating that expression of the transgenes within neuroblasts is essential for the function (Ashraf, 2001).

The segregation of Prospero protein into GMCs from neuroblasts is a critical event during asymmetric cell division. Since inscuteable plays a role in the segregation of prospero gene products into GMCs, whether there is Prospero protein in GMCs of mutant embryos was examined. Prospero protein staining can be easily detected in many wild type GMC nuclei. The staining is largely absent in the deletion that uncovers the snail family locus; only a few cells with the size of normal GMCs had clear nuclear staining. A band of cells along the midline also had Prospero staining, but these cells probably represent an expansion of the midline. It has been well documented that in all snail mutants there is derepression of the mid-line determinant single-minded in the blastoderm stage embryo (Ashraf, 2001).

To determine whether there are defects within GMCs in addition to the loss of Prospero, the expression of Hunchback, which is present transiently in early neuroblasts and later in many GMCs was examined. In the deletion mutant, the Hunchback protein in GMCs is also absent, while staining in cells surrounding the amnioserosa appeared normal. Transgenes of snail, worniu and escargot rescue the staining of Prospero and Hunchback, indicating that these GMC determinants are downstream of the Snail family. The results also suggest that the regulation of ftz by the Snail family is indirect, probably through an earlier event such as segregation of Prospero from neuroblast to GMC (Ashraf, 2001).

If the misregulation of inscuteable in the deletion mutant is the cause of the loss of Prospero and ftz expression in GMCs, the expression of inscuteable should correct the defects even in the absence of Snail family of proteins. A line carrying an inscuteable transgenic construct driven by the 2.8 kb snail promoter was crossed into the osp29 deletion genetic background. However, the rescue of Prospero expression in GMCs was variable and not nearly as strong as those embryos expressing the snail family transgenes. This suggests that inscuteable may not be the only important target gene of Snail. Another line of evidence supporting the idea of an additional target gene comes from the comparison of the phenotypes in osp29 and inscuteable mutant embryos. In inscuteable mutants, the Prospero crescent is formed but the mitotic spindle rotation is randomized. As a result, the Prospero protein frequently is present both in neuroblasts and GMCs. This phenotype is less severe than the almost total loss of Prospero GMC staining in osp29 deletion mutant. Therefore, it is surmised that in addition to the misregulation of inscuteable, there may be other defects that lead to the more severe phenotype in the deletion mutants (Ashraf, 2001).

The severe CNS defects are likely due to a combination of loss of inscuteable and string expression. Similar to the results obtained for inscuteable, transgenic expression of string alone has a weak and variable effect in the rescue of Prospero expression in GMCs. When both inscuteable and string are simultaneously expressed in neuroblasts of osp29 mutants using the UAS-Gal4 system, clear staining of Prospero in many cells resembling GMCs is observed. The staining is particularly apparent alongside the expanded midline, characteristic of mutant embryos with no Snail function in early mesoderm. The results support the idea that both inscuteable and string are relevant targets of the Snail family (Ashraf, 2001).

A clearly demonstrated in vivo function of Snail is transcriptional repression. The repression function is mediated through the recruitment of dCtBP (Drosophila C-terminal binding protein), which acts as a co-repressor for Snail to regulate target genes such as rhomboid, lethal of scute and single-minded. There are two conserved P-DLS-R/K motifs in Snail, as well as in Worniu and Escargot, and they have been shown to be critical for recruiting dCtBP. Mutations of these motifs abolish the repressor function of Snail in the blastoderm. To gain insight into the molecular mechanism of how Snail regulates CNS development, transgenic copies of snail, which had the dCtBP interaction motifs mutated were introduced into the osp29 deletion background. M1 contains the N-terminal motif mutation and M2 contains the C-terminal motif mutation. The expression of inscuteable and ftz was examined. The assay shows that the double mutant (M12) lost most of the ability to rescue, and M1 has lost some ability to rescue. However, M2 functioned quite efficiently, closer to that of the wild-type protein, to rescue inscuteable and ftz expression. These results demonstrate that the dCtBP interaction motifs are essential for the Snail function in the CNS, consistent with the idea that Snail acts as a repressor in neuroblasts to regulate gene expression. Thus, the activation of inscuteable and string by the Snail family may be indirect (Ashraf, 2001).

Abstrakt regulates Insc levels and asymmetric division of neural and mesodermal progenitors

In Drosophila, both neural and muscle progenitors divide asymmetrically. In these cells the Inscuteable (Insc) protein complex coordinates cell polarity and spindle orientation. Abstrakt (Abs) is a DEAD-box protein that regulates aspects of cell polarity in oocytes and embryos. A conditional allele of abs was used to investigate its role in neural and muscle progenitor cell polarity. In neuroblasts loss of apical Insc crescents, failure in basal protein targeting, and defects in spindle orientation were observed. In the GMC4-2a cell loss of apical Insc crescents, defects in basal protein targeting, and equalization of sibling neuron fates are observed; muscle precursors show a similar equalization of sibling cell fates. These phenotypes resemble those of insc mutants; indeed, abs mutants show a striking loss of Insc protein levels but no change of insc RNA levels. Furthermore, the Abs protein physically interacts with insc RNA. These results demonstrate a novel role for Abs in the posttranscriptional regulation of insc expression, which is essential for proper cell polarity, spindle orientation, and the establishment of distinct sibling cell fates within embryonic neural and muscle progenitors (Irion, 2003).

Mitotic neuroblasts form an apical cortical protein complex containing Bazooka (the Drosophila homolog of nematode and mammalian Par-3), Par-6, atypical Protein Kinase C, Inscuteable (Insc), Partner-of-Inscuteable, and Gαi proteins . These apical proteins have three functions: to promote basal cell fate determinant localization, to orient the mitotic spindle along the apical/basal axis, and to promote the formation of an asymmetric spindle leading to the generation of daughters of unequal size. The basally localized determinants include Miranda (Mir) and Numb (Nb), which were used as markers in this study. Their basal localization ensures their preferential segregation into the basal daughter cell, called ganglion mother cell (GMC), during neuroblast division and ensures proper GMC fate specification (Irion, 2003).

To assay abs function, a temperature-sensitive allele was used in combination with a small deficiency uncovering the abs locus (abs^14B/Df(3R)231-5, hereafter referred to as abs^14B embryos) in which the maternally contributed Abs protein can be inactivated by a shift to the restrictive temperature. Wild-type embryos at the restrictive temperature and abs^14B embryos at the permissive temperature show normal apical (Insc) and basal (Mir) cortical protein crescents in mitotic neuroblasts, as well as normal apicobasal orientation of the mitotic spindle. In contrast, abs^14B embryos that are shifted to the restrictive temperature display severe defects in neuroblast polarity: Mir frequently shows uniform cortical distribution or occasionally accumulates as mispositioned lateral crescents. Furthermore, mitotic spindles occasionally fail to orient along the apical-basal axis. The similarity of these phenotypes and those that were previously reported for mutations affecting components of the Insc complex prompted the assaying of Insc protein localization in abs mutants. Interestingly, Insc protein is not detectable above background levels at the restrictive temperature in abs^14B mutant neuroblasts, although apical Insc localization is not affected in abs^14B embryos at the permissive temperature or in wild-type embryos at the restrictive temperature. It is concluded that loss of abs function leads to the loss of detectable Insc protein in neuroblasts and generates the phenotype previously seen in insc mutants. The simplest interpretation is that Insc expression and/or Insc protein stability is impaired in abs mutants, which leads to the observed defects in neuroblast asymmetric cell division (Irion, 2003).

To determine if abs has a more general role in regulating Insc levels and asymmetric cell division, asymmetric division of the ganglion mother cell GMC4-2a, which produces a pair of identified sibling neurons, RP2 and RP2sib, was examined. During wild-type GMC4-2a divisions, the mitotic spindle is apicobasally oriented; Insc is localized to the apical cortex, whereas Numb is localized as a basal cortical crescent and segregates preferentially into the more basal daughter cell, where it acts to downregulate Notch (N) signaling and induce the RP2 cell fate. The RP2 sibling cell does not inherit Numb, cannot downregulate N signaling, and adopts the secondary RP2sib fate. GMC4-2a and RP2 express the Even-skipped (Eve) transcription factor, but RP2sib does not; thus, there is only one Eve+ cell at the RP2 position in wild-type embryos. However, in abs^14B embryos shifted to the restrictive temperature prior to GMC4-2a division, approximately 32% of the hemisegments had a duplicated Eve+ cell at the RP2 position. This phenotype was rarely seen either in control embryos (from a stock homozygous for abs^14B along with two copies of a functional abs⁺ transgene, henceforth referred to as abs^24:14B, that rescues the abs lethality subjected to the same temperature-shift regime or in abs^14B embryos at the permissive temperature. The duplicated Eve+ cells are likely to be duplicated RP2 neurons because they express two additional markers (22C10 and Zfh1) for mature RP2 neurons (Irion, 2003).

To elucidate the origin of the duplicated RP2 neurons, anti-Eve staining was used to follow the development of the GMC4-2a lineage in wild-type and abs^14B embryos. The results indicate that the extra RP2 neuron arises as the result of a transformation of the RP2sib to the RP2 cell fate (Irion, 2003).

Pon directly binds Numb protein and reflects the localization of Numb in all cells analyzed so far. In control abs^24:14B and in wild-type embryos shifted to the restrictive temperature (33°C), Pon localizes as a basal crescent in mitotic GMC4-2a. In abs^14B embryos subjected to the same temperature shift regime, approximately 50% (18/34) of metaphase GMC4-2a cells show cortical distribution, misplaced crescents, or weak basal crescents of Pon, and approximately 25% of the cells show no obvious Pon crescents. Hence, the symmetric segregation of Numb to both daughter cells in a proportion of the dividing GMC4-2a cells could account for the RP2 duplication phenotype seen in the abs^14B embryos (Irion, 2003).

Because the abs phenotype is similar to the insc phenotype in both neuroblasts and GMC4-2a, Insc localization during the GMC4-2a cell division was investigated. In control embryos, Insc always forms an apical crescent in metaphase GMC4-2a cells. In contrast, at the restrictive temperature, the majority of the abs^14B mutant GMC4-2a cells showed no clear apical crescents of Insc. Consistent with the finding that Insc localization is affected in abs^14B, the duplicated RP2 cells seen at the restrictive temperature exhibit equal nuclear size, as is also seen in insc embryos but not in mutants that disrupt sibling cell fate choice at the postmitotic level (Irion, 2003).

The role of abs during embryonic muscle progenitor divisions was investigated. The muscle progenitor P15 divides asymmetrically to produce two daughter cells with distinct identities. Numb is asymmetrically localized in the dividing P15 and preferentially segregates to the daughter cell that will become the founder for the single Eve-positive muscle DA1; the sibling cell is Eve-negative. abs^14B embryos subjected to a 45 min pulse at the restrictive temperature showed duplications of the Eve-positive DA1 in 34% (23/68) of the hemisegments. In the control abs^24:14B embryos, 135/136 of the hemisegments showed a single Eve-positive DA1. Thus, abs is also required for the asymmetric division of some muscle progenitors (Irion, 2003).

The abs and insc mutant phenotypes in asymmetrically dividing cells are very similar, and abs mutants show a loss of Insc protein crescents in neuroblasts, in GMCs, and throughout the embryo. Thus, the abs phenotype can be most simply modeled as a defect in establishing or maintaining normal levels of apical Insc protein in all of these cell types. The loss of Insc crescents could be caused either by an overall decrease in the levels of Insc or by a failure to localize Insc correctly in these cells. In situ hybridization experiments revealed no reduction in insc RNA expression, so abs does not appear to regulate insc at the transcriptional level. Western blots were used to test whether the total amount of Insc protein was affected in abs mutant embryos. The Insc protein migrates as an approximately 100 kDa band. Wild-type and abs^14B embryos were shifted to the restrictive temperature and analyzed after 0, 30, and 60 min. The levels of Insc protein decreased progressively in abs^14B embryos until they were nearly undetectable at 60 min, whereas they remained constant or even increased (depending on the age distribution of embryos at the beginning of the experiment) in wild-type embryos. Other proteins remain constant, and several proteins can be translated de novo at the restrictive temperature, indicating that abs function is not generally required for protein synthesis. Together, these data indicate that the most upstream defect associated with a reduction in abs function is a reduction in the levels of the Insc protein (Irion, 2003).

If Abs indeed acts on asymmetric cell divisions by maintaining high levels of Insc, it should be possible to circumvent the requirement for Abs at least in part by raising Insc levels experimentally. To test this, the GAL4-UAS system was used to express high levels of insc within neuroblasts in embryos lacking functional Abs protein. This led to a marked rescue of the RP2 phenotype (Irion, 2003).

Because Abs is a DEAD-box protein, it seemed conceivable that it might exert its effect on Insc protein levels by a direct interaction with insc RNA. A yeast-three hybrid assay was used to test this. The assay is based on the interaction of the HIV-1 RNA binding protein Rev with RNA molecules containing a Rev responsive element (RRE). Rev is fused to the GAL4 DNA binding domain, whereas the putative RNA binding protein, in this case Abs, is fused to the activation domain. The two fusion proteins are then bridged by a hybrid RNA consisting of an RRE-containing sequence fused to the RNA to be tested, in this case insc RNA. insc RNA is clearly able to interact with Abs in this system. Both the full-length RNA and a construct lacking the 5' third of the RNA show an interaction. However, no single fragment of the 3' part of the RNA was found to be able to interact with Abs (Irion, 2003).

Thus Abs directly binds Insc mRNA in vitro; loss of Abs leads to lowered Insc protein levels but not lowered mRNA levels, and loss of Abs leads to a failure to properly localized cell fate determinants in at least three asymmetrically dividing cell types: neuroblasts, GMCs and muscle progenitors. It is concluded that Abs has a role in controlling cell polarity and asymmetric cell division in multiple cell types, in part through the posttranscriptional regulation of Insc levels (Irion, 2003).

Protein Interactions

Several proteins, including Numb and Miranda, segregate into the basal daughter cell and are needed for the determination of its correct cell fate. Both the apical-basal orientation of the mitotic spindle and the localization of Numb and Miranda to the basal cell cortex are directed by Inscuteable, a protein that localizes to the apical cell cortex before and during neuroblast mitosis. The apical localizaton of Inscuteable requires Bazooka, a protein containing a PDZ domain that is essential for apical-basal polarity in epithelial cells. Bazooka localizes with Inscuteable in neuroblasts and binds to the Inscuteable localization domain in vitro and in vivo. In embryos lacking both maternal and zygotic bazooka function, Inscuteable no longer localizes asymmetrically in neuroblasts and is instead uniformly distributed in the cytoplasm. Mitotic spindles in neuroblasts are misoriented in these embryos, and the proteins Numb and Miranda fail to localize asymmetrically in metaphase. These results suggest that direct binding to Bazooka mediates the asymmetric localization of Inscuteable and connects the asymmetric division of neuroblasts to the axis of epithelial apical-basal polarity (Schober, 1999).

In Drosophila neuroblasts, inscuteable controls both spindle orientation and the asymmetric localization of the cell-fate determinants Prospero and Numb. Inscuteable itself is localized in an apical cortical crescent and thus reflects the intrinsic asymmetry of the neuroblast. Localization of Inscuteable depends on Bazooka, a protein containing three PDZ domains with overall sequence similarity to Par-3 of Caenorhabditis elegans. Bazooka and Inscuteable form a complex that also contains Staufen, a protein responsible for the asymmetric localization of Prospero messenger RNA. It is proposed that, after delamination of the neuroblast from the neuroepithelium, Bazooka provides an asymmetric cue in the apical cytocortex that is required to anchor Inscuteable. Since Bazooka is also responsible for the maintenance of apical-basal polarity in epithelial tissues, it may be the missing link between epithelial polarity and neuroblast polarity (Wodarz, 1999).

Inscutable and Prospero interact physically. The C-terminal 108 amino acids of Insc are sufficient to confer an interaction with Staufen, while other residues of Insc appear to inhibit the interaction mediated by the C-terminal 108 amino acids. The C-terminal region (residues 769-1026) of Stau confers this specific interaction. Both Staufen and Inscuteable proteins are cortically localized in the apex of neuroblasts; the apical localization of Staufen protein requires the presence of Inscuteable (Li, 1997).

Paradoxically, Staufen is required for the basal localization of Prospero mRNA during mitosis. Prospero mRNA is localized to the apical cortex during interphase, however the change in PROS mRNA localization from the apical cortex at interpahse to the basal cortex at prophase fails to occur in animals that lack zygotic staufen. In staufen and inscuteable mutant neuroblasts, the PROS mRNA remains primarily on the apical cortex during mitosis, indicating that the apical cortical localization of PROS mRNA during interphase requires neither insc nor stau function. However, the basal cortical relocalization that takes place at prophase requires both insc and stau function. Since staufen mutation fails to affect either Inscuteable protein localization or mitotic spindle orientation in neuroblasts, it is concluded that stau acts downstream of inscuteable (Li, 1997).

Staufen binds to the 3' untranslated regions of Prospero mRNA, suggesting that Staufen's role in Prospero mRNA redistribution is mediated through this interaction. How then does Prospero mRNA get to the basal cortex during mitosis? It is assumed that the Inscuteable/Staufen-independent mechanism that operates to effect localization of PROS mRNA to the apical cortex during interphase is normally overridden by the Insc/Stau-mediated process during mitosis. There is strong evidence that suggests that during early development specific signals localized to the 3' UTR of Bicoid mRNA can recruit Staufen to form ribonucleoprotein particles that are subsequently transported in a process that requires intact microtubules (Ferrandon, 1994). It is therefore appealing to suggest that Stau might play a similar role in the neuroblast to transport PROS mRNA from the apical to basal cortex in the transition between interphase and mitosis. With respect to PROS mRNA localization, the role of Insc may be to facilitate Stau protein/PROS mRNA interaction with perhaps other components necessary for the transport of PROS mRNA (Li, 1997).

Cellular diversity in the Drosophila central nervous system is generated through a series of asymmetric cell divisions in which one progenitor produces two daughter cells with distinct fates. Asymmetric basal cortical localization and segregation of the determinant Prospero during neuroblast cell divisions play a crucial role in effecting distinct cell fates for the progeny sibling neuroblast and ganglion mother cell. Similarly asymmetric localization and segregation of the determinant Numb during ganglion mother cell divisions ensures that the progeny sibling neurons attain distinct fates. The most upstream component identified so far which acts to organize both neuroblast and ganglion mother cell asymmetric divisions, is encoded by inscuteable. The Inscuteable protein is itself asymmetrically localized to the apical cell cortex and is required both for the basal localization of the cell fate determinants during mitosis and for the orientation of the mitotic spindle along the apical/basal axis. The functional domains of Inscuteable have been defined. Amino acids 252-578 appear sufficient to effect all aspects of its function, however, the precise requirements for its various functions differ. The region aa288-497 is necessary and sufficient for apical cortical localization and for mitotic spindle (re)orientation along the apical/basal axis. A larger region (aa288-540) is necessary and sufficient for asymmetric Numb localization and segregation; however, correct localization of Miranda and Prospero requires additional sequences from aa540-578. The requirement for the resolution of distinct sibling neuronal fates appears to coincide with the region necessary and sufficient for Numb localization (aa288-540). These data suggest that apical localization of the Inscuteable protein is a necessary prerequisite for all other aspects of its function. Although Inscuteable RNA is normally apically localized, RNA localization is not required for protein localization or any aspects of inscuteable function (Tio, 1999).

The Drosophila central nervous system develops from stem cell like precursors called neuroblasts, which divide unequally to bud off a series of smaller daughter cells, called ganglion mother cells. Neuroblasts show cell-cycle-specific asymmetric localization of both RNA and proteins: at late interphase, Prospero mRNA and Inscuteable, Prospero and Staufen proteins are all apically localized; at mitosis, Inscuteable protein remains apical, whereas Prospero mRNA, Prospero protein and Staufen protein form basal cortical crescents. In vitro culture of neuroblasts was used to investigate the role of intrinsic and extrinsic cues and the cytoskeleton in asymmetric localization of Inscuteable, Prospero and Staufen proteins. Neuroblast cytokinesis is normal in vitro, producing a larger neuroblast and a smaller ganglion mother cell. Apical localization of Inscuteable, Prospero and Staufen in interphase neuroblasts is reduced or eliminated in vitro, but all three proteins are localized normally during mitosis (apical Inscuteable, basal Prospero and Staufen). Microfilament inhibitors result in delocalization of all three proteins. Inscuteable becomes uniform at the cortex, whereas Prospero and Staufen become cytoplasmic; inhibitor washout leads to recovery of microfilaments and asymmetric localization of all three proteins. Microtubule disruption has no effect on protein localization, but disruption of both microtubules and microfilaments results in cytoplasmic localization of Inscuteable. It is concluded that both extrinsic and intrinsic cues regulate protein localization in neuroblasts. Microfilaments, but not microtubules, are essential for asymmetric protein anchoring (and possibly localization) in mitotic neuroblasts. These results highlight the similarity between Drosophila, Caenorhabditis elegans, vertebrates, plants and yeast: in all organisms, asymmetric protein or RNA localization and/or anchoring requires microfilaments (Broadus, 1997).

Neuroblasts in the developing Drosophila CNS asymmetrically localize the cell fate determinants Numb and Prospero as well as Prospero RNA to the basal cortex during mitosis. The localization of Miranda to the apical cortex, its interaction with Inscuteable in vitro and its role in localizing several downstream factors suggests that Miranda occupies a central link between Inscuteable at the apical cortex and the localization of Prospero, Staufen, and Prospero RNA to the basal cortex. How, early in mitosis, the apically localized Inscuteable dictates basal localization of intrinsic factors for asymmetric cell division may be elucidated by further studies on the genetic and cell biological mechanisms of the asymmetric localization of Miranda. The localization of Prospero requires the function of inscuteable and miranda, whereas Prospero RNA localization requires inscuteable and staufen function (Shen, 1998).

Miranda forms a crescent on the apical cortex of neuroblasts in late interphase. Later in mitosis, Miranda forms a crescent on the basal neuroblast cortex. Asymmetric localization of both Numb and Prospero has been shown to be dependent on the actin cytoskeleton. The actin dependence of Miranda localization was tested using the actin depolymerizing drug latrunculin A. After treatment of Drosophila embryos with 200 µM latrunculin A for 20 min, asymmetric localization of Miranda is completely disrupted, while membrane association is unperturbed. It is concluded that the asymmetric localization of Miranda during mitosis is an actin-dependent process. All Miranda fragments that contain the amino-terminal 298 amino acids exhibit the same asymmetric localization pattern as wild type Miranda. In contrast, a fragment containing amino acids 114-298 localizes to the cytoplasm and fails to segregate preferentially into the basal daughter cell, as does a fragment containing all residues carboxy-terminal to amino acid 300 (Shen, 1998).

The observation that Miranda protein fragments form an apical crescent that may coincide with the apical Inscuteable crescent led to a test of the possibility that Miranda interacts physically with Inscuteable. In an in vitro binding assay, Inscuteable coprecipitates with Miranda. An Inscuteable fragment from amino acids 252 to 615 also interacts with Miranda (Shen, 1998).

Miranda contains multiple functional domains: an amino-terminal asymmetric localization domain, which interacts with Inscuteable; a central Numb interaction domain, and a more carboxy-terminal Prospero interaction domain. Miranda and Staufen have similar subcellular localization patterns and interact in vitro. miranda function is required for the asymmetric localization of Staufen. Miranda localization is disrupted by the microfilament disrupting agent latrunculin A. These results suggest that Miranda directs the basal cortical localization of multiple molecules, including Staufen and Prospero mRNA, in mitotic neuroblasts in an actin-dependent manner (Shen, 1998).

Protein Interactions: Partner of Inscuteable

Asymmetric localization is a prerequisite for inscuteable to function in coordinating and mediating asymmetric cell divisions in Drosophila. Partner of Inscuteable (Pins), a new component of asymmetric divisions, is required for Inscuteable to asymmetrically localize. In the absence of pins, Inscuteable becomes cytoplasmic and asymmetric divisions of neuroblasts and mitotic domain 9 cells show defects reminiscent of insc mutants. Pins colocalizes with Insc and interacts with the region of the Insc protein necessary and sufficient for directing its asymmetric localization. Analyses of pins function in neuroblasts reveal two distinct steps for Insc apical cortical localization: a pins-independent, bazooka-dependent initiation step during delamination (interphase) and a later maintenance step during which Baz, Pins, and Insc localization are interdependent (Yu, 2000a).

Pins was identified using a yeast two hybrid assay to identify proteins that exhibit interaction with the Insc asymmetric localization domain. Northern blots reveal a major embryonic transcript of 3.3 kb. Sequence analyses of full-length cDNAs predict a Pins coding region of 658 amino acids. Analyses of the predicted protein reveal the presence of seven TPR repeats, which serve as a general protein-protein interaction motif in the N-terminal half of Pins. Moreover, in database searches, a putative human homolog of unknown function, LGN, with 46% identity and 63% similarity over the entire length of the coding region was identified. Pins is able to bind to all Insc-GST fusion proteins containing the asymmetric localization domain but not to GST alone nor to Insc-6, which lacks the asymmetric localization domain. The TPR repeat-containing the N-terminal region of Pins, is necessary and sufficient for direct interaction with the Insc asymmetric localization domain in vitro (Yu, 2000a).

To determine the subcellular localization of Pins, antibodies were generated against a Pins fusion protein. In NBs, Pins is localized as a crescent to the apical cortex. Apical crescents can be detected in interphase NB following delamination. More intensely labeled Pins apical crescents can be seen during mitosis from prophase to anaphase. In telophase, Pins shows a weak cortical distribution that disappears only after telophase. Double labelings with anti-Insc indicate that, with the exception of delaminating NBs, the two proteins are largely colocalized during the NB cell cycle. In delaminating NBs, high levels of Insc staining can be seen on the apical stalk, which extends from the NB toward the surface of the neuroectoderm. In comparison, high levels of apical Pins are detected only following NB delamination. These observations suggest that the initial localization of Insc to the apical stalk of NBs during delamination (interphase) may precede that of the Pins apical localization; however, during mitosis the two proteins are colocalized as apical crescents. Apical cortical crescents of Pins can also be found in the dividing cells of the procephalic mitotic domain 9 (Yu, 2000a).

As a first step toward understanding the role pins might play with respect to the genes that are known to be involved in asymmetric cell divisions, Pins distribution was examined in embryos homozygous for loss-of-function alleles of mir, pros, pon, numb, and insc. With the exception of insc mutants, Pins expression is wild type (WT) in these mutants. In insc null embryos, Pins distribution is no longer asymmetric in mitotic NBs as well as dividing cells of mitotic domain 9 (with complete penetrance); Pins distribution is primarily cortical and the intensity of anti-Pins staining is also strongly reduced. Hence asymmetric localization of Pins requires insc (Yu, 2000a).

To assess the effects of removing both maternal and zygotic pins on Insc localization, genotypically mutant embryos derived from mutant mothers either homozygous or transheterozygous for the mutants pins^P62 and pins^P89 were obtained. These embryos do not produce detectable amounts of Pins as judged by immunofluorescence and Westerns with the anti-Pins antibody and are referred to as Pins^- embryos. Insc localization is dramatically affected in these embryos. In mitotic NBs and interphase NBs that have completed delamination, as well as in dividing cells of mitotic domain 9, Insc is localized to the cytoplasm. In NBs this failure to asymmetrically localize appears to be a defect in maintenance, since the initial apical localization of Insc occurs normally. This is most convincingly seen in delaminating NBs that are known to have completed S phase and are at the G2 stage of the cell cycle. Delaminating NBs possess a membrane stalk that emanates from their apical surface; this stalk retains contact with the epithelial surface, and this is where apical cortical localization of Insc is initially seen. This initial localization of Insc to the apical stalk occurs normally in Pins^- embryos; however, apical Insc localization cannot be maintained and later in interphase and during mitosis, Insc no longer associates with the cortex and adopts a cytoplasmic localization. Hence while the initial apical localization of Insc during delamination does not require pins, the maintenance of this asymmetric localization later in interphase and throughout mitosis is pins dependent (Yu, 2000a).

In the absence of baz function, Insc does not localize apically even in delaminating NBs and is cytoplasmic later in the cell cycle. Not surprisingly, in embryos lacking both maternal and zygotic baz, Pins distribution in mitotic NBs is mostly cortical, similar to its distribution in insc mutant NBs. Interestingly, Baz localization to the apical cortex of NBs is itself affected by pins and insc loss of function. In Pins^- NBs, the apical cortical Baz crescents normally present in WT mitotic NBs cannot be detected from metaphase onward. However, occasional weak crescents can be found in mutant interphase/prophase NBs and these are always localized to the apical cortex. The Baz distribution in insc mutant NBs is similar to that seen in Pins^- embryos. These observations suggest that the maintenance and/or stability of apical Baz in NBs requires both insc and pins. Taken together these results indicate that the initial localization of Insc (e.g., to the apical stalk) requires baz but not pins; however, the maintenance of apical Baz/Pins/Insc later in the cell cycle (e.g., at metaphase) are mutually dependent, requiring all three components (Yu, 2000a).

To further explore the relationship between pins and insc, attention was focused to the epithelial cells that normally express but do not apically localize Pins. insc is necessary for the apical localization of Pins in NBs and cells of mitotic domain 9. Would the ectopic expression of Insc in epithelial cells be sufficient to recruit Pins to the apical cortex? Ectopically expressed Insc, driven from a hsp70-insc transgene, localizes to the apical cortex in WT epithelial cells and, interestingly, causes Pins, which is normally localized to the lateral cortex, to also localize to the apical cortex. Conversely, apical localization of ectopically expressed Insc is dependent on pins. Insc ectopically expressed in Pins^- epithelial cells does not localize as an apical crescent; rather it adopts a cytoplasmic distribution (primarily toward the apical side of the cell) during interphase and is undetectable during mitosis, presumably due to rapid degradation. This apparent instability of ectopically expressed Insc may be the reason why the 90° rotation in the mitotic spindles (that occurs as a consequence of Insc ectopic expression in the WT epithelial cells) no longer occurs when Insc is expressed in Pins^- embryos. These results indicate that the ectopic expression of Insc is sufficient for Pins to be recruited to the apical cortex of WT epithelial cells; moreover, similar to NBs, there is also a mutual dependence of Pins and ectopically expressed Insc for the apical localization of both proteins in these cells (Yu, 2000a).

The role of insc in orienting mitotic spindle, localizing Pros/Mir and Pon/Numb in neural progenitors, mediating alternative cell fate, and effecting nuclear size asymmetry of specific sibling neurons has been previously demonstrated. In order to ascertain the role of pins in mediating these processes, the phenotype of Pins^- embryos was analyzed with anti-beta-tubulin to assess spindle orientation in cells of mitotic domain 9. Anti-Mir, anti-Pros, anti-Pon, anti-Numb, and DNA stainings were used to examine protein localization in NBs. Anti-Eve staining was used to assess whether distinct cell fates and distinct nuclear cell sizes are specified for RP2/RP2sib, a pair of sibling neurons. Pins^- embryos display phenotypes similar to those seen in insc mutants. Mitotic spindle orientation is defective. In the cells of mitotic domain 9, the phenotype is similar to that seen in insc mutants where the 90° reorientation (which normally occurs in WT resulting in the orientation of the spindle along the apical/basal axis) fails to occur in the mutant. Mitotic spindle orientation of NBs in the segmented CNS, deduced from DNA staining, also often fails to adopt an apical/basal orientation. Mir/Pros and Pon/Numb normally localize as basal crescents in WT metaphase NBs. However, in Pins^- metaphase NBs these proteins often show defective localization, in the form of mislocalized crescents and cortical localization, similar to that seen in insc mutants. The quantitation of these phenotypes has been shown in both Pins^- and insc mutant metaphase NBs. Where misplaced Mir/Pon crescents (>45° deviation from basal) form, they can either overlie one of the mitotic spindle poles (coupled) or not (uncoupled). An interesting difference between the pins and insc phenotype is that the frequency of coupled protein crescents is higher in Pins^- NBs than in insc NBs. These observations suggest that the coordination of mitotic spindle orientation with protein localization may be less disrupted in Pins^- than in insc metaphase NBs (Yu, 2000a).

Resolution of distinct fates for the sibling neurons RP2 and RP2sib also frequently fail to occur. In ~60% of the mutant hemisegments, duplicated RP2 neurons (Eve-expressing neurons at the RP2 position) are found at the expense of the RP2sib. Moreover, the two RP2 neurons appear to have indistinguishable nuclear sizes, a phenotype also seen in insc mutants. In ~15% of the hemisegments, no Eve-expressing RP2/RP2sib neurons are produced due to a failure to correctly specify the GMC that is the progenitor for RP2/RP2sib. This similarity in the pins and insc loss of function across a range of phenotypes indicates that the pins-mediated maintenance of Insc asymmetric localization is necessary for the correct execution of neural progenitor asymmetric cell divisions. By driving the expression of a uas-pins transgene in neural tissue with a sca-Gal4 driver in Pins^- embryos, the above phenotypes can be rescued and Insc crescents can be restored in mitotic NBs, confirming that it is the loss of pins that is responsible for these phenotypes (Yu, 2000a).

A protein complex containing inscuteable and the galpha-binding protein pins orients asymmetric cell divisions in Drosophila

Where does Pins fit in the pathway that establishes and maintains cell asymmetry? Two proteins of approximately 70 kDa and 40 kDa are reproducibly coimmunoprecipitated with Inscuteable. The 70 kDa protein has been identified as Inscuteable. The sequences of two short peptide fragments of the 40 kDa protein could be determined. The sequences occur in both the Drosophila Gαi protein and Galphao protein (Galpha47A, Swissprot accession number P16377), but not in any other Drosophila protein or EST. It cannot currently be determine whether the 40 kDa band is Drosophila Galphao or Galphai. To test for a direct interaction between Inscuteable, Pins and Galphai/Galphao, in vitro binding assays were performed. In vitro translated Pins protein binds strongly to Inscuteable. Very weak binding is also detected between Insc and both Galphai and Galphao. In contrast, both Galphai and Galphao bind strongly to a Pins. These results suggest that the complex containing Inscuteable, Pins and Galphai/Galphao forms as a result of a direct protein interaction between Inscuteable and Pins, and between Pins and Galphai/Galphao, even though the weak interaction between Galphai/Galphao and Inscuteable may also contribute (Schaefer, 2000).

The fact that Pins contains three GoLoco domains, which are thought to be modulators of Galpha signaling, and that Pins exists in a complex with Galpha in vivo, offers the intriguing possibility that a heterotrimeric G-protein signaling cascade is involved in directing asymmetric cell divisions in Drosophila (Schaefer, 2000). No evidence exists that would suggest the involvement of extracellular signals (through G-protein coupled receptors) in orienting neuroblast divisions. Furthermore, asymmetric localization of Inscuteable during metaphase and asymmetric cell division can occur in cultured neuroblasts in the absence of any extracellular signal. Therefore, knowing whether and how G-proteins in asymmetric cell division awaits identification of further pathway elements (Schaefer, 2000).

In Drosophila, distinct mechanisms orient asymmetric cell division along the apical-basal axis in neuroblasts and along the anterior-posterior axis in sensory organ precursor (SOP) cells. Heterotrimeric G proteins are essential for asymmetric cell division in both cell types. The G protein subunit Galphai (FlyBase designation G-oalpha65A) localizes apically in neuroblasts and anteriorly in SOP cells before and during mitosis. Interfering with G protein function by Galphai overexpression or depletion of heterotrimeric G protein complexes causes defects in spindle orientation and asymmetric localization of determinants. Galphai is colocalized and associated with Pins, a protein that induces the release of the ßgamma subunit and might act as a receptor-independent G protein activator. Thus, asymmetric activation of heterotrimeric G proteins by a receptor-independent mechanism may orient asymmetric cell divisions in different cell types (Schaefer, 2001).

While a significant amount of Galphai coimmunoprecipitates with Insc and Pins, no Galphao can be detected in the immunoprecipitate. It is concluded that Galphai but not Galphao is part of the Insc/Pins complex in vivo. To determine the subcellular localization of Galphai, Drosophila embryos were stained for Galphai, DNA, and Insc or Bazooka. Before stage 12 of embryogenesis, Galphai is expressed in all cells and localizes to the cell cortex. Costaining for the apical marker Bazooka reveals that Galphai is concentrated basolaterally in epithelial cells. Upon neuroblast delamination, when the expression of Insc starts, Galphai concentrates in an apical stalk that extends into the epithelial cell layer and then colocalizes with Insc in a crescent along the apical cell cortex during interphase, prophase, and metaphase until anaphase, when Insc disappears and Galphai becomes delocalized. Galphai but not the associated ß subunit is asymmetrically localized in neuroblasts, suggesting that Gß13F is also bound to other Galpha subunits, possibly Galphao (Schaefer, 2001).

To test whether Insc is required for asymmetric Galphai localization in neuroblasts, insc^P72 mutant embryos were stained for Galphai and DNA. During neuroblast delamination, Galphai fails to localize apically in insc mutants and in 87% of insc mutant metaphase neuroblasts, the protein is distributed around the whole cell cortex. To test whether ectopic expression of Insc is sufficient for the apical localization of Galphai, insc was ubiquitously expressed from a heat-inducible transgene. While Galphai is localized basolaterally in epidermal cells of heat-shocked control embryos, heat-shock-induced ectopic expression of insc in these cells results in apical concentration of Galphai. Thus, expression of insc is both required and sufficient for apical recruitment of Galphai (Schaefer, 2001).

Since Galphai directly binds to Pins, the subcellular localization of Galphai was tested in pins mutants. No apical localization of Galphai was observed in 100% of the pins mutant metaphase neuroblasts. This might be an indirect consequence of the defect in Insc localization in pins mutant metaphase neuroblasts. However, initiation of Galphai localization also fails in 88% of pins mutant delaminating neuroblasts. Insc is normally localized in pins mutants at this stage and so it is concluded that both Insc and Pins are required for the apical localization of Galphai in neuroblasts (Schaefer, 2001).

Staining for the neuronal marker Asense has shown that neuroblasts are correctly specified, delaminate, and enter mitosis shortly after delamination both in conc and Gß13F mutants. Furthermore, staining for DmPar-6 reveals no defects in epithelial polarity. However, while 86% of the asymmetric cell divisions in conc mutant neuroblasts are oriented along the apical-basal axis, only 26% of the divisions in Gß13F mutant neuroblasts have this orientation, whereas the others are misoriented by more than 30 degrees. Miranda localizes into a basal cortical crescent in 100% of the conc mutant metaphase neuroblasts, but only in 6% of the Gß13F mutant neuroblasts. In 29% of the Gß13F mutant neuroblasts, crescents are misoriented, whereas in 65%, Miranda is largely cytoplasmic. Defects in asymmetric localization are also observed for Numb. Thus, Gß13F mutants have defects in asymmetric cell division similar to or stronger than those observed in insc mutants, and therefore Insc distribution was analyzed in these mutants. When neuroblasts delaminate from the neuroectoderm, Insc begins to accumulate in a stalk that extends into the epithelium, and this initial localization is unchanged in Gß13F mutants. In Gß13F mutants, cortical localization of the protein is progressively lost after delamination. Weak cortical Insc crescents were found in 11% of the metaphase neuroblasts, but in 25%, the protein was partially, and in 64% completely, localized into the cytoplasm. Thus, heterotrimeric G proteins are required for maintaining Insc localization and for directing spindle orientation and asymmetric protein localization during neuroblast division (Schaefer, 2001).

The GDP-bound form of Galpha is thought to be inactive and tightly associated with its ßgamma subunit. To test whether Galphai in the Insc/Pins complex is bound to the ß subunit, the Insc/Pins/Galphai complex was immunoprecipitated using a ß-Gal-tagged version of the functional domain of Insc. No Gß13F can be found in the complex, even though a significant amount of Gß13F can be detected in a control experiment where equal amounts of Galphai are precipitated by anti-Galphai. Thus, Galphai is bound to Gß13F in vivo but is free of the ß subunit in the complex with Insc and Pins. To test whether Pins is responsible for the release of the ß subunit, Galphai was immunoprecipitated in the presence of recombinant Pins protein. A significant amount of Gß13F is bound to Galphai in control experiments, but addition of an MBP (maltose binding protein)-fusion of full-length Pins (MBP-Pins) or the Pins GoLoco domains (MBP-GoLoco) during the immunoprecipitation causes the release of the ß subunit. The same effect can be achieved by addition of a 38 aa peptide corresponding to the last GoLoco domain of the Pins protein, but not with a peptide in which a conserved phenylalanine had been mutated to arginine. Thus, the Pins GoLoco domains cause the dissociation of Gß13F from Galphai (Schaefer, 2001).

These results suggest that Galphai exists in an unusual form in Drosophila neuroblasts that is bound to GDP but free of the ß subunit. Furthermore, the observation that recombinant Pins triggers the release of the ß subunit from Galphai is consistent with the hypothesis that Pins activates heterotrimeric G proteins without nucleotide exchange on the alpha subunit in the absence of an extracellular ligand (Schaefer, 2001).

To test whether G proteins also function in Insc-independent asymmetric cell division, the distribution of Galphai was analyzed in SOP cells during pupal development. In interphase, when Numb is homogeneously distributed around the cell cortex, Galphai is asymmetrically localized to the anterior cell cortex in SOP cells. During metaphase, both Numb and Galphai are found at the anterior cell cortex and in telophase, they segregate into the same daughter cell. Similar results were obtained for Pins. Thus, Pins and Galphai localize asymmetrically in SOP cells but in contrast to neuroblasts, they are at the same side as Numb (Schaefer, 2001).

In neuroblasts, the Insc protein is critical for the asymmetric localization of Galphai and its binding partner Pins. Neuroblasts arise from epithelial cells in which Insc is not expressed and Galphai is localized basolaterally. When neuroblasts delaminate, Insc expression starts and the protein functions as an adaptor that links the Pins/Galphai complex to the Bazooka/DmPar-6/DaPKC complex that is inherited from the apical cortex of the epithelial cells. Neither Pins nor Galphai are required for Insc localization during this stage. In delaminated neuroblasts, however, Insc, Pins, and Galphai become codependent for their apical localization. At this point, their subcellular localization in various mutants can no longer be explained simply by protein-protein interactions of the known components. When Galphai is overexpressed, for example, Pins is recruited to the cell cortex whereas Insc relocalizes into the cytoplasm, suggesting that the two proteins no longer interact. Thus, events that happen downstream of Galphai seem to be involved in maintaining the colocalization of the more upstream components. The simplest model is that G proteins establish a positional cue at the apical cell cortex during neuroblast delamination -- this cue is needed for maintaining apical protein localization in delaminated neuroblasts and ultimately, for orienting asymmetric cell division. In Drosophila, this downstream activity remains to be identified, but a similar feedback loop for asymmetric protein localization is found in yeast and here its molecular components are well understood. Local activation of a heterotrimeric G protein in response to the pheromone alpha-factor recruits Cdc24 to the site of G protein activation. Cdc24 is an exchange factor that locally activates the small G protein Cdc42 and activated Cdc42, in turn, is needed to maintain Cdc24 localization. Thus, the initiation of an autoregulatory feedback loop at a particular position may be a common theme in cell polarity (Schaefer, 2001).

LGN/mInsc and LGN/NuMA complex structures suggest distinct functions in asymmetric cell division for the Par3/mInsc/LGN and Galphai/LGN/NuMA pathways

Coupling of spindle orientation to cellular polarity is a prerequisite for epithelial asymmetric cell divisions. The current view posits that the adaptor Inscuteable (Insc) bridges between Par3 and the spindle tethering machinery assembled on NuMA-LGNGαi^GDP, thus triggering apico-basal spindle orientation. The crystal structure of the Drosophila ortholog of LGN (known as Pins) in complex with Insc reveals a modular interface contributed by evolutionary conserved residues. The structure also identifies a positively charged patch of LGN binding to an invariant EPE-motif present on both Insc and NuMA (Mushroom body defect or Mud). In vitro competition assays indicate that Insc competes with NuMA for LGN binding, displaying a higher affinity, and that it is capable of opening the LGN conformational switch. The finding that Insc and NuMA are mutually exclusive interactors of LGN challenges the established model of force generators assembly, which this study revises on the basis of the newly discovered biochemical properties of the intervening components (Culurgioni, 2011).

This study reports the characterization of the Pins^TPR dInsc^PEPT complex and provides a molecular explanation for the mutual exclusive interaction of Insc and NuMA to LGN. While this manuscript was in preparation, Zhu and coworkers arrived to similar conclusions analyzing the structure of the LGN-NuMA complex (Zhu, 2011).

A 38-residue fragment of Drosophila Insc encompasses the Pins^TPR binding region. This fragment of Insc shares a high sequence similarity to functional homologues recently discovered in mammals, fully supporting the notion that the basic mechanism responsible for the recruitment of force generators at polarity sites is evolutionary conserved. With the exception of a short N-terminal α-helix, the Insc^PEPT is intrinsically unstructured, and lines on the scaffold provided by the superhelical TPR arrangement of Pins with an extended conformation. The interaction surface is organized around a core module involving the EPE motif of Insc^PEPT and the central TPR5-6 of Pins, whose specificity is primarily dictated by charge complementarity. The binding is further stabilized by polar and hydrophobic interactions contributed by the αA helix of Insc^PEPT. Not surprisingly, the large interaction surface characterizing the topology of the Pins^TPR;Insc^PEPT heterotypic dimer accounts for an elevate;d binding affinity (of about 5 nM for the fly proteins and 13 nM for the human counterparts). The structure of mouse LGN^191–350, corresponding to what is named TPR5-8, with Insc^19–40 suggests that vertebrate proteins assemble with organizational principles similar to the fly ones. However, the short mouse constructs only depict the interaction of LGN^TPR with the αA helix of Insc^PEPT, up to the first Glu of the EPE motif. Intriguingly, the mouse LGN;Insc interaction seems to be characterized by lower affinity compared to human and fly ones (with K_D of 63 nM for LGN^TPR5–8;Insc^19–40, and of 47 nM for LGN^TPR1–8;Insc^20–57) (Zhu, 2011).

The evidence that NuMA forms a complex with the same LGN^TPR domain associating to Insc raised the question of whether it binds in a similar manner. Indeed, comparison of the primary sequence of Insc^PEPT with the known LGN-binding portion of NuMA revealed the presence of an EPE triplet that turned out to be essential for the LGN recognition, with a similar molecular signature of the EPE motif of the Insc^PEPT. Notably, the NuMA ortholog in fly (Mud) codes for two consecutive EDE-EGE motifs in the Pins-binding region, whose interplay remains to be clarified. The structure of LGN in complex with NuMA^PEPT fully supports the notion that the EPE-interaction module represents a common region required for docking unstructured ligands on the LGN^TPR scaffold. In the case of NuMA, the interface is further contributed by a helical fragment forming a bundle with helices αA2 and αA3 of LGN^TPR. The consequence of the partial overlap between the Insc and NuMA binding sites is that their concomitant loading on LGN is excluded (Zhu, 2011).

A key step during the assembly of the force generators is the opening of the LGN conformational switch that keeps the molecule in an inactive state. Binding of NuMA to LGN^TPR induces the release of the intramolecular interactions holding the molecule in a closed form. In agreement with the similarity in the binding modes, it was demonstrated that also Insc disengages the LGN GoLoco motifs from the TPR domain. Together these findings imply that the GoLoco motifs contact the TPR repeats in the same region occupied by Insc and NuMA. Primary sequence inspection revealed that the GoLocos of both Pins and LGN do not contain EPE triplets, suggesting that either the head-to-tail interaction involves alternative TPR patches sterically occluded by the presence of Insc and NuMA, or that less conserved negatively charged triplets are accommodated on the same TPR5-6 of LGN (Zhu, 2011).

The well established model for force generators recruitment at polarity sites rests on the assumption that Insc and NuMA can be part of the same apically localized multisubunit complexes containing Par proteins. This model stems from colocalization experiments showing that in asymmetric mitoses Par3, Insc, LGN, and NuMA cluster together in apical crescents, complemented by coimmunoprecipitation assays in which LGN;Gαi were found in association with Par3;Insc and NuMA. The finding that Insc and NuMA are mutually exclusive partners of LGN is both unexpected and puzzling. In particular, the higher affinity characterizing the Insc binding to LGN shifts the balance of the unmodified proteins towards the Insc;LGN complex formation, which is instrumental in recruiting LGN with Par proteins at the onset of mitosis but cannot account for microtubule-pulling forces. What is the possible mechanism for transferring LGN from Insc to NuMA? The architecture of the Insc^PEPT;Pins^TPR structure whereby an extended ligand is accommodated on a large domain allows a high degree of regulation of the interaction strength. Posttranslational modifications on either side of the dimer might locally alter the contacts without affecting the rest of the interface, as it has been demonstrated for the similarly organized complex between the cytoplasmic domain of E-cadherin and β-catenin. Such modulating modifications can in principle occur on Insc, NuMA, or on LGN. To date, no experimental information is available regarding putative Insc or NuMA modifications. More controversial is the literature relative to LGN phosphorylations. In mitotic Drosophila neuroblasts, Pins has been found phosphorylated by Aurora-A on Ser436 at about half of the linker connecting the TPR domain with the GoLoco motifs. Using an “induced polarity” assay in S2 cells, phospho-Ser436^Pins was shown to trigger a redundant NuMA-independent spindle orientation pathway engaging the membrane associated Dlg protein. It is to date unclear if such pathway is conserved in vertebrates. Notably, during oriented symmetric cell divisions of MDCK cells, phosphorylation on a similarly positioned Ser401 of LGN functions in excluding force generators from the apical cortex in order to prevent apico-basal spindle orientation. In this context, phospho-Ser401^LGN would selectively prevent binding of LGN to apical Gαi. Based on structural and biochemical results, it is difficult to provide a molecular explanation as to whether these LGN phosphorylations could also impact on the Insc and NuMA binding. Recent observations support the notion that the pool of NuMA;LGN;Gαi colocalizing with Par3;Insc in embryonic mouse skin progenitors is tightly regulated to set the balance between symmetric and asymmetric divisions, though no mechanism for this has been put forward. In summary, more has to be learned to understand what brings LGN from Insc to NuMA (Zhu, 2011).

An additional question relates to the mechanism maintaining effective NuMA;LGN;Gαi^GDP species at the correct cortical sites in the absence of Insc. Based on the knowledge acquired in this study, a step-wise model is proposed that can be schematized as follows (see Both NuMA and Insc open the LGN conformational switch): (1) in the early phases of mitosis LGN is brought to the apical membrane in conjunction with Par proteins by the high-affinity interaction with the preformed Par3;Insc complex. Binding of LGN to Insc triggers the conformational switch transition enabling the relocation of Gαi^GDP moieties previously distributed all around the plasma-membrane with Gβγ; (2) upon mitotic progression, when LGN is already at the correct sites, a yet unidentified molecular event alters the relative affinities of Insc and NuMA for LGN to shift the balance between the Insc-bound and the NuMA-bound LGN pools. It is hypothesized that the four Gαi subunits present on LGN at this stage are sufficient to transiently hold cortical NuMA;LGN;Gαi^GDP in proximity of Par proteins to allow directional microtubule pulling. It is speculated that NuMA;LGN;Gαi^GDP is a short-lived complex and disassemble, possibly assisted by a specialized GEF for Gαi such as as Ric-8A (see Drosophila Ric-8), releasing apo-LGN in the cytoplasm to start a new cycle. Such a dynamical interaction network would allow for a continuous regulation of the force exerted on astral microtubules throughout mitosis. Future attempts to validate the model in vivo will greatly benefit from the biochemical tools presented in this study (Zhu, 2011).

Protein Interactions: Cornetto

Drosophila neuroblasts divide asymmetrically along the apical-basal axis. The Inscuteable protein localizes to the apical cell cortex in neuroblasts from interphase to metaphase, but disappears in anaphase. Inscuteable is required for correct spindle orientation and for asymmetric localization of cell fate determinants to the opposite (basal) cell cortex. Inscuteable also directs asymmetric protein localization to the apical cell cortex during later stages of mitosis. In a two-hybrid screen for Inscuteable-binding proteins, coiled-coil protein Cornetto, which shows a highly unusual subcellular distribution in neuroblasts, has been identified. Computer searches did not identify any known protein domains, but both the central region of the protein (amino acids 370-420 and 470-590) and the C terminus (amino acids 890-960) are predicted by the COILS program to form a coiled coil at very high probability. All two-hybrid clones contain the coiled coil domains, suggesting that they are the Inscuteable-binding part of the protein. The coiled-coil regions of Cornetto show high homology to other coiled-coil proteins, but sequence similarity searches failed to identify clear orthologs of cornetto in other species. Although the protein is uniformly distributed in the cytoplasm during metaphase, it concentrates apically in anaphase and forms an apical crescent during telophase in an inscuteable-dependent manner. Upon overexpression, Cornetto localizes to astral microtubules and microtubule spin-down experiments demonstrate that Cornetto is a microtubule-binding protein. After disruption of the actin cytoskeleton, Cornetto localizes with microtubules throughout the cell cycle and decorates the mitotic spindle during metaphase. These results reveal a novel pattern of asymmetric protein localization in Drosophila neuroblasts and are consistent with a function of Cornetto in anchoring the mitotic spindle during late phases of mitosis, even though cornetto mutant analysis suggests that this function might be obscured by genetic redundancy (Bulgheresi, 2001).

inscuteable: Biological Overview | Evolutionary Homologs | Developmental Biology | Effects of Mutation | References

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