To analyze Mud localization in asymmetric cell division, Mud in embryonic neuroblasts were stained by using an anti-Mud antibody (Yu, 2006). At neuroblast delamination, Mud colocalizes with Pins on the apical cell cortex. This cortical localization is maintained through interphase, when alternative methods of fixation also reveal a pool of Mud on the nuclear rim (see also Yu, 2006). At metaphase, when the spindle aligns with the apical crescents of Mud and Pins, Mud can also be observed on spindle poles. At telophase, Mud preferentially segregates into the neuroblast. This localization is consistent with recent work showing that Mud decorates mitotic and meiotic spindle poles and is required for positioning spindles in meiosis II (Yu, 2006). Although Mud is expressed in larval brains, fixation conditions could not be found for analyzing Mud localization in larval tissue. It is concluded that Mud colocalizes with Pins on the cortex of asymmetrically dividing neuroblasts (Bowman, 2006).
To test if the localization of Mud depends on its binding partner Pins, pins Δ50 maternal and zygotic mutant embryos were analyzed. The apical enrichment of Mud is lost in pins mutant neuroblasts in metaphase, and the cortical association is weaker, but Mud remains associated with spindle poles. To test whether Pins is sufficient for directing apical localization of Mud, transgenic inscuteable under the control of the hsp70 promoter was used to express Inscuteable in epithelial cells. Epithelial cells normally divide parallel to the plane of the epithelium. Introduction of ectopic Inscuteable recruits Pins and Gαi from the basolateral to the apical cortex, inducing a spindle reorientation. Mud is also recruited apically and colocalizes with Pins. This suggests that Pins recruits Mud to the apical cortex of epithelial cells in the presence of Inscuteable, and, by extension, that Pins recruits Mud apically in neuroblasts. It is concluded from these experiments that Pins is required and sufficient for the apical recruitment of Mud, but that the spindle pole localization of Mud is independent of Pins. Both the apical localization of Mud and its association with microtubules are consistent with a role in spindle orientation (Bowman, 2006).
In the developing central nervous system of Drosophila, proliferation follows a reproducible and well-described spatial and temporal pattern. This pattern involves a defined number and distribution of neural stem cells (neuroblasts), as well as a precisely regulated time course of division of these neuroblasts. Mutations in the mushroom body defect (mud) gene interfere with the regulation of this pattern in a rather specific manner. In the abdominal neuromeres a subset of neuroblasts prolongs the period of proliferation. Additional daughter cells persist into the imago. Similar defects are expressed in the anterior ventral nerve cord and in the lateral central brain region. In the mushroom body cortex, however, mutations in mud affect the proliferation pattern by increasing the number of neuroblasts. These additional neuroblasts behave like normal mushroom body neuroblasts according to their time course of proliferation and the specification of their progeny (Prokop, 1994).
A Pins-Gαi interacting protein that also binds microtubules is a good candidate for a regulator of spindle orientation in asymmetric cell division. To find out if Mud controls spindle orientation, larval neuroblast divisions weRE analyzed in animals homozygous for mud4, a presumptive null allele affecting all Mud isoforms (Guan, 2000). For this, third instar larval brains were immunostained for Miranda and Centrosomin. Neuroblasts were defined as Miranda-expressing cells greater than 10 μm in diameter. In wild-type neuroblasts, Miranda forms a crescent in metaphase, and it segregates into a single daughter cell at telophase. In mud zygotic mutants, the Miranda crescent can be bisected by the cleavage plane and inherited by both daughter cells. Missegregation of Miranda could be due to defective spindle orientation, or it could be a secondary consequence of a general loss of polarity. Alternatively, Mud could regulate mitotic spindle morphology or formation. All mud mutant neuroblasts form crescents of aPKC and opposing crescents of Insc and Miranda. Furthermore, spindles in mud mutant neuroblasts appear bipolar with no gross morphological differences from wild-type (see also Yu, 2006). From these data, it is concluded that mud mutants form functional spindles and the neuroblasts are correctly polarized. Consistent with this, Brat and Numb form crescents in mud mutant neuroblasts, but the spindle is not aligned with them. Therefore, the spindle orientation defect is a direct consequence of Mud loss of function. To quantify this defect, the angle between a line connecting the two centrosomes and a line bisecting the crescent of Miranda was measured in metaphase neuroblasts. A small angle indicates tight coupling of the mitotic spindle with the polarity axis. In wild-type, the measured angle is almost always less than 10°. In mud mutants, the majority of spindles show more oblique orientations, and only a minority of spindles have measured angles of 10° or less. It is concluded that Mud is required for coordinating the mitotic spindle with the axis of polarity. Together, these observations demonstrate that mud mutant neuroblasts polarize correctly, but, in the absence of Mud, the polarized cortical domains cannot direct the orientation of the mitotic spindle. As a result, cell fate determinants can fail to segregate asymmetrically (Bowman, 2006).
Mud gets its name from defective formation of the mushroom body, an adult brain structure required for olfactory learning and memory. The neurons forming the mushroom body, called Kenyon cells, are generated by four mushroom body neuroblasts that divide repeatedly throughout embryonic, larval, and pupal development. Mushroom body neuroblasts, like all neuroblasts in Drosophila, divide asymmetrically to yield a GMC that produces two neurons and a self-renewing neuroblast. Notably, wild-type mushroom body neuroblasts form crescents of Miranda, which segregate into a single small cell. This shows that neuroblasts of this lineage segregate cell fate determinants asymmetrically. In mud mutants, Miranda also forms a crescent, but spindle misorientation leads to missegregation of Miranda in ~4% of mushroom body neuroblasts. It is proposed that the remaining neuroblasts divide asymmetrically by repositioning either the spindle or the cell polarity axis during telophase. A similar rescue of defects during asymmetric cell division at late stages of mitosis has been described for other mutants, where it is called telophase rescue. Although the vast majority of mud mutant neuroblasts still divide asymmetrically, in those cells that inherit equal amounts of Miranda, and presumably equal amounts of the apical complex members known to regulate cell size asymmetry, the daughter cell size is equal. It is concluded that mushroom body neuroblasts segregate Miranda and therefore its binding partners Brat and Prospero asymmetrically, but faulty spindle orientation leads to occasional missegregation in mud mutants (Bowman, 2006).
Missegregation of cell fate determinants can result in the transformation of GMCs to neuroblasts. Therefore whether symmetric segregation of Miranda resulted in increased numbers of mushroom body neuroblasts in mud mutants was examined. For this, the mushroom body-specific GAL4 line OK107 and UAS CD8-GFP were used to label mushroom body neuroblasts and their progeny, and Miranda, which is present in neuroblasts but rapidly degraded in GMCs, was immunostained. While the number of mushroom body neuroblasts in wild-type late third instar brains is always 4, the average number of mushroom body neuroblasts in mud mutants is nearly 14. Neuroblast number increases over time; an average of around eight mushroom body neuroblasts were observed in the early third instar brains of mud mutants. From these experiments, it is concluded that mud mutants generate excess mushroom body neuroblasts, a conclusion consistent with an earlier study (Prokop, 1994). Increased numbers of neuroblasts were observed in the posterior half of the larval brain hemisphere, ventral nerve cord, and the anterior brain regions of mud mutants. Ectopic neuroblasts in mud mutants express the neuroblast marker Deadpan and incorporate BrdU, showing that they are correctly specified and mitotically active (Bowman, 2006).
In brat mutants, transformation of GMCs to neuroblasts leads to a decrease in the number of neurons. To see if this is true for mud mutants, Kenyon cells were analyzed in late third instar larval brains by using OK107-GAL4-driven CD8-GFP to mark the progeny of the mushroom body neuroblasts. Wild-type larval brains contain around 500 Kenyon cells, but, in mud mutants, the average number of Kenyon cells increases. This suggests that unlike in brat mutants, the ectopic neuroblasts in mud mutants produce ectopic progeny cells. It is proposed that the penetrant symmetric division phenotype in brat mutants makes neuroblasts nearly incapable of producing GMCs, while the frequent asymmetric divisions in mud mutants can still give rise to differentiated progeny (Bowman, 2006).
To test whether the ectopic neuroblasts and progeny cells develop normally, the morphology of the mushroom body was investigated in mud mutant adults. During development, repeated divisions of mushroom body neuroblasts sequentially generate three types of morphologically distinct Kenyon cells. These neurons project axons that form the characteristic lobed structure of the adult mushroom body. Kenyon cells born from late embryogenesis to the early third instar project their axons into the γ lobe, Kenyon cells born between early third instar and puparium formation project branched axons into the α′ and β′ lobes, and, finally, Kenyon cells born after puparium formation project their branched axons into the α and β lobes. Like larval brains, mud mutant adult brains contain an increased number of Kenyon cells. The number of wild-type Kenyon cells is lower than expected, but OK107 may not detectably label every Kenyon cell. Interestingly, while a small γ lobe is present in mud mutants, the ectopic Kenyon cells are unable to project axons into the α′β′ or αβ lobes. The absence of these lobes could indicate a role for Mud in axon guidance. Alternatively, the presence of the γ lobe suggests that Kenyon cells born early in development, when the maternal supply of Mud is sufficient, are correctly specified. Consistent with this, the mud mutant γ lobe expresses low levels of Fasciclin-II, as in wild-type γ lobes. At later stages, the reduced levels of Mud may result in misspecification of the α′β′ and αβ neurons, resulting in an absence of projections. Taken together, these observations suggest that in mud mutants, the occasional transformation of a GMC into a neuroblast causes overproliferation and cell fate misspecification (Bowman, 2006).
In addition to their well-known effects on the development of the mushroom body, mud mutants are also female sterile. This study shows that, although the early steps of ovary development are grossly normal, a defect becomes apparent in meiosis II when the two component spindles fail to cohere and align properly. The products of meiosis are consequently mispositioned within the egg and, with or without fertilization, soon undergo asynchronous and spatially disorganized replication. In wild-type eggs, Mud is found associated with the central spindle pole body that lies between the two spindles of meiosis II. The mutant defect thus implies that Mud should be added to the short list of components that are required for the formation and/or stability of this structure. Mud protein is also normally found in association with other structures during egg development: at the spindle poles of meiosis I, at the spindle poles of early cleavage and syncytial embryos, in the rosettes formed from the unfertilized products of meiosis, with the fusomes and spectrosomes that anchor the spindles of dividing cystoblasts, and at the nuclear rim of the developing oocyte. In contrast to its important role at the central spindle pole body, in none of these cases is it clear that Mud plays an essential role. But the commonalities in its location suggest potential roles for the protein in development of other tissues (Yu, 2006).
This work describes a fully penetrant phenotype that is associated with strong mutations in the mud gene. Specifically, mud mutants have a striking maternal-effect lethal phenotype: although eggs produced by mutant females look normal and accept sperm, they fail to undergo normal cleavage divisions. Instead of forming a well-ordered syncytial blastoderm, these eggs accumulate disordered arrays of replicated material and then necrose. A number of mutants in other genes have been reported to have abnormal early embryonic mitoses. One class of these, comprising strong alleles of the genes pan gu (png), plutonium (plu), and giant nuclei (gnu), reflects defects in a complex with protein kinase activity. It seems unlikely that mud is a member of this class. While png, plu, and gnu mutants typically form one to five giant nuclei, strong alleles of mud characteristically generate dozens of smaller chromatin masses that are scattered throughout the egg. Moreover, in contrast to mutations in genes of the giant nuclei class, mud mutations do not interfere with degradation of maternal mRNA. A closer resemblance to the mud phenotype is shown by mutants that have defects in spindle-associated proteins. In such mutants, as in mud, the meiotic products undergo inappropriate and disorganized mitoses that produce eggs with scattered chromatin masses. The possibility is considered that Mud and the products of one or more of these genes (ncd, alphaTub67C, or gammaTub37C) serve mutually interdependent roles. If so, double heterozygotes might display a maternal-effect phenotype lacking in any of the single heterozygotes. However, when either the mud3 or the mud4 mutation was combined with the ncdD, alphaTub67C1, or gammaTub37C3 mutation, no decrease in fertility was seen over the robust levels seen in the single heterozygotes. Although this outcome is not decisive, it argues against possibilities such as Mud being involved in the localization of these spindle components. In any case, it should be emphasized that the observation that unfertilized mutant eggs undergo the same rounds of disorganized mitoses as do early embryos strongly implies that the mud maternal lethal phenotype is due to a defect in meiosis and not in a subsequent stage of development (Yu, 2006).
What is the basis for the meiotic defect? A central spindle pole body that incorporates several centrosomal proteins together with a diffuse aster of alpha-tubulin is normally found between the two spindles that make up the meiosis II apparatus. As judged by alpha-tubulin staining, this structure is defective or absent in mud mutant eggs. In wild-type eggs, Mud is found associated with the spindle pole body, implying that the protein is needed for the formation or stability of this structure. Because Mud staining appears to surround the alpha-tubulin framework, an attractive possibility is that the former is needed for the latter to be recruited to or be maintained at the central spindle pole body. In any case, just as with weak alleles of polo, which also show defects in the formation of the central spindle pole body, disjoined meiosis II spindles, and disorganized early mitoses, the observations of this study provide a plausible scenario for the female sterility of mud mutants. To wit, without a functional spindle pole body the two meiosis II spindles are not properly held together and the products of meiosis do not get correctly positioned with respect to the egg cortex. It is presumed that positioning is important for condensation of the meiotic products into inactive polar bodies. These structures may normally serve to shield the dead-end meiotic products from the replication machinery, which according to this scenario is competent to operate in unfertilized Drosophila eggs. If so, when proper condensation fails, inappropriate replication ensues. Regardless of the correctness of this model, the similar phenotypes of mud and polo mutations suggest that corresponding proteins might serve interdependent roles in activated eggs. But no decrease in fertility was observed in double heterozygotes of polo1 and either mud3 or mud4. It remains to be seen whether Polo and Mud are similarly distributed in activated oocytes (Yu, 2006).
In addition to its association with the central spindle pole body, Mud protein is readily detected at other spindle poles. However, in those cases where it can be tested, it appears that Mud is not essential for the formation or function of these structures. In this regard, Mud is reminiscent of the fly orthologs of CP190 and pericentrin, which, despite being consistently found at the centrosome, are dispensable for mitosis. It may be that the structures built at the spindle poles are designed to withstand the undersupply of a few ingredients. If so, it might be useful to look for synthetic phenotypes when mud mutations are combined with those of other genes whose products are concentrated at spindle poles. Of course, because eggs that lack a maternal contribution of functional Mud develop so anomalously, it is undecided whether it plays an essential role at the spindle poles of embryonic mitoses or in polar bodies. Insight into these cases will have to await the availability of a conditional mutant that can be shifted to nonpermissive conditions after meiosis II is completed. Despite the doubts about its role at places other than the central spindle pole body, the frequent associations of Mud with microtubular structures in the oocyte and egg suggest that the protein might be regarded as a MAP; direct tests of this hypothesis are underway. Another attractive, albeit speculative, idea is that Mud is functionally related to the vertebrate NuMA protein. Like Mud, NuMA is a large coiled-coil protein that is found at spindle poles. The two proteins are apparently not orthologous in that they cannot be globally aligned outside of their coiled-coil regions; they also differ in subcellular localization during interphase. However, the carboxy terminus of one particular isoform of Mud shares a short region of similarity with NuMA, hinting at a conserved interaction (Yu, 2006).
Two other sites of Mud localization that suggest a connection with microtubules are fusomes and spectrosomes. These are membrane-rich structures that form during the earliest stages of ovary development and are surrounded by microtubules. Mud is present within these structures at a time when they serve to anchor the mitotic spindles of the dividing cystoblasts but not later, when they serve to focus the microtubule network in postmitotic cells. Although this distribution hints at a role for Mud in spindle anchoring, inactivation of the gene causes no obvious defect in fusomes or spectrosomes. These structures not only form normally in mud mutants but the processes they govern during early oogenesis proceed without defect. It is concluded that, if Mud plays a role in building fusomes and spectrosomes or connecting them to spindles, it is a role for which there is adequate redundancy (Yu, 2006).
In cells that are not in metaphase or anaphase, Mud can also be found at the nuclear envelope. It is not clear how Mud gets from the nuclear rim of the oocyte and the early embryo, respectively, to the meiotic and mitotic spindle apparatus. In contrast, Mud might lose contact with the nuclear envelope, where it could have been held by its transmembrane domain, and be directed to the spindle by a distinct targeting signal. But, since the spindles of the Drosophila germ line and pre-syncytial egg are typically enclosed by membranous structures, the movement of Mud might be part of a concerted redistribution of elements of the nuclear envelope. Although the mechanism is thus unclear, it should be pointed out that shuffling between the nuclear envelope and the spindle is not unique to Mud but has been reported for several other proteins. Another open question is whether Mud plays a microtubule-related role at the nuclear rim. However, even though the oocyte nucleus is surrounded by a cage of microtubules and the dynactin complex is concentrated at its rim, the structure and positioning of the oocyte nucleus are not grossly affected by loss of Mud function. Accordingly, the idea is favored that Mud is simply stored at the nuclear rim to ensure a local supply for subsequent delivery to the spindle apparatus but the possibility that a parallel system renders obscure a more active function for Mud at the nuclear envelope cannot be ruled out (Yu, 2006).
To what extent do the observations on the female sterility shed light on the other phenotypes of mud mutants, particularly the role of Mud in the development of the adult nervous system? It is easy to imagine that a protein that can associate with the nuclear envelope might be part of the mechanism that prepares the way for exit from the cell cycle. And a protein that is commonly found around spindle poles might be involved in the regulation of spindle orientation that governs the transition from symmetric to asymmetric division of neuroblasts and thus the switch from neuroblast proliferation to stem cell behavior. Similarly, a protein that associates with microtubules might play a role in the precision of growth cone movement that is needed for proper axon pathfinding. Thus, although speculative, concrete suggestions for places to look for Mud action in the nervous system can be gleaned from the insights gained from a description of this protein in the oocyte and early embryo (Yu, 2006).
Asymmetric cell division generates cell diversity during development1, 2 and regulates stem-cell self-renewal in Drosophila and mammals. In Drosophila, neuroblasts align their spindle with a cortical Partner of Inscuteable (Pins)-Galphai crescent to divide asymmetrically, but the link between cortical polarity and the mitotic spindle is poorly understood. This study shows that Pins directly binds, and coimmunoprecipitates with, the NuMA-related Mushroom body defect (Mud) protein. Pins recruits Mud to the neuroblast apical cortex, and Mud is also strongly localized to centrosome/spindle poles, in a similar way to NuMA. In mud mutants, cortical polarity is normal, but the metaphase spindle frequently fails to align with the cortical polarity axis. When spindle orientation is orthogonal to cell polarity, symmetric division occurs. It is proposed that Mud is a functional orthologue of mammalian NuMA and Caenorhabditis elegans Lin-5, and that Mud coordinates spindle orientation with cortical polarity to promote asymmetric cell division (Siller, 2006).
The Mud–Pins interaction was confirmed by showing that a short C-terminal portion of Mud containing the NLM domain and 142 amino acids of the amino-terminal sequence (amino acids 1825–1997) directly interacts with Pins in vitro. Further analysis revealed that Mud binds the amino-terminal Pins tetratricopeptide (TPR)1–7 domain, but not the C-terminal GoLoco domain. Although Insc binds TPR1–4, no Mud binding was observed to any region of Pins that was smaller than TPR1–7, indicating that all seven TPRs are required for proper presentation of the Mud-binding epitope. Consistent with the Mud–Pins direct interaction, Mud and Pins can be coimmunoprecipitated from embryonic lysates. The Mud–Pins interaction is likely to be evolutionarily conserved, since homologous domains in NuMA and Mud mediate their interaction with LGN and Pins, respectively. It is concluded that the Mud C-terminus can bind the Pins TPRs, and both proteins are part of a common protein complex in vivo (Siller, 2006).
In embryonic neuroblasts, Mud and Pins were both enriched at the cortex over the apical centrosome/spindle pole from late interphase and up to the end of metaphase. By late anaphase–telophase, Mud showed bipolar apical and basal cortical crescents over both spindle poles; this can be seen most clearly in neuroblasts that are cultured in vitro, where there are fewer surrounding cells. In addition, Mud shows strong spindle-pole/centrosome localization and weaker spindle and astral microtubule localization in all neuroblasts. In larval neuroblasts, Mud is always present at the apical cortex with Pins: it either forms cortical crescents over both spindle poles or is uniformly cortical. In mud null mutants, larval neuroblasts have virtually no detectable Mud protein, confirming the antibody specificity. It is concluded that Mud and Pins form apical cortical crescents during late interphase and prophase (this is the time when spindle orientation is established in larval neuroblasts), and that Mud is also detected at the basal cortex later in mitosis, as well as on spindle poles and microtubules. The minor differences in Mud localization between embryonic and larval neuroblasts may be due to differences in fixation/visualization or in cell types. Thus, Drosophila Mud, C. elegans Lin-5 and mammalian NuMA all share a common localization profile of cell cortex, spindle poles and spindle microtubules (Siller, 2006).
Whether Mud and Pins rely on each other for apical cortical localization was tested. In mud mutant larval neuroblasts, normal apical–basal localization of Pins–Galphai and all other tested cortical polarity proteins was found. By contrast, pins or Galphai maternal-zygotic null-mutant neuroblasts always lacked apical enrichment of Mud: it was either cytoplasmic or cytoplasmic with residual uniform cortical localization, although centrosome/spindle-pole localization was unaffected. In addition, the C-terminal truncated Mud protein that is encoded by the mud allele fails to localize to the cortex or spindle poles in larval neuroblasts. It is concluded that Pins recruits Mud to the neuroblast apical cortex, probably via interaction with the Mud C-terminal domain (Siller, 2006).
The function of Mud in spindle orientation was investigated. Because Mud is maternally provided and required during meiosis, spindle orientation was analyzed in larval neuroblasts. Wild-type larval neuroblasts invariably aligned their metaphase spindle within 15° of the centre of the Pins apical crescent or the Mira basal crescent. By contrast, mud mutant neuroblasts showed significant defects in metaphase spindle alignment with the apical Pins crescent. Also, formation of bent spindles were observed in 29%-40% of all mud mutant neuroblasts, but these are not correlated with spindle-orientation defects and arise after spindle orientation is fixed. It is concluded that Mud is required for metaphase spindle orientation. Despite severe defects in metaphase spindle orientation, it was found that the mitotic spindle and cortical polarity markers were nearly always re-aligned by telophase in mudmutant neuroblasts. In the rare neuroblasts in which 'telophase rescue' of spindle-cortex alignment failed to occur, and the spindle axis remained nearly perpendicular to the cell polarity axis, it was found that the neuroblast division was invariably symmetric with regards to cortical polarity and sibling cell size. Thus, Mud specifically regulates spindle orientation, but spindle orientation defects can affect the asymmetry of cell division. It is concluded that: (1) Mud is required to align the mitotic spindle with Pins cortical polarity at metaphase; (2) a Mud-independent mechanism can rescue spindle–cortex alignment at telophase, and (3) proper spindle–cortex alignment is necessary to promote asymmetric cell division of larval neuroblasts (Siller, 2006).
Time-lapse imaging of larval neuroblasts was used to address two important questions: when do the spindle orientation defects arise in mud mutants, and how are the spindle orientation defects 'rescued' at telophase? Mitotic larval neuroblasts were imaged in whole brain explants expressing a spindle marker labelled with green fluorescent protein (transgenic line G147) and/or an enhanced yellow fluorescent protein (EYFP)–Baz apical cortical marker. In wild-type neuroblasts, it was found that the two fully separated centrosomes were always aligned along the future apical–basal axis by the end of prophase. Thus, in contrast to embryonic neuroblasts in which spindle rotation is reported to occur at metaphase, larval neuroblasts fix spindle-pole/centrosome alignment at prophase and maintain spindle orientation up to the end of telophase. Analysis of the EYFP–Baz apical cortical marker revealed that cortical polarity was always established prior to fixation of centrosome position and accurately predicted the final axis of spindle orientation. This is consistent with the tight alignment of centrosomes and cortical polarity axes that were observed from the end of prophase to telophase in fixed preparations. It is concluded that wild-type neuroblasts establish cortical polarity by prophase, establish centrosome position by the end of prophase and maintain tight spindle–cortex alignment during telophase (Siller, 2006).
In mud mutant neuroblasts, it was found that spindle orientation was also established at prophase, with little or no spindle movement through telophase. However, significant defects were found in the alignment of the mitotic spindle with the EYFP–Baz cortical crescent, including neuroblasts in which the spindle and cortical polarity axes were nearly perpendicular. When examined mud mutant neuroblasts were examined during anaphase/telophase, movement of the mitotic spindle to bring it into alignment with the EYFP–Baz cortical polarity axis was never observed, despite data from fixed preparations showing that the majority of metaphase spindle orientation defects are corrected by telophase. Finally, it was observed that mud mutants could divide asymmetrically or symmetrically. Symmetric divisions occurred only when the spindle was nearly orthogonal with the cortical polarity axis and it was inferred that these neuroblasts correspond to the equally dividing neuroblasts that were seen in fixed preparations. Three conclusions were drawn from the live imaging experiments. First, the mud spindle-orientation defects are due to a failure in centrosome/spindle-pole positioning at prophase, prior to the formation of the metaphase spindle. This further supports the conclusion that metaphase spindle morphology defects are not the source of the spindle-orientation defects. Second, mud mutants do not rotate their spindle towards the cortical polarity axis at anaphase–telophase, indicating that the observed 'telophase rescue' of spindle-cortical polarity occurs by modification of cortical protein distribution to match the spindle axis. Third, Mud does not directly promote asymmetric cell division, but it does regulate spindle orientation relative to cortical polarity, and only when the spindle is orthogonal to the cortical polarity axis does the cell division become symmetric. Thus, spindle orientation dictates whether the cell division is symmetric or asymmetric (Siller, 2006).
This study has shown that Mud has the properties of a functional orthologue of the vertebrate NuMA and C. elegans Lin-5 proteins. All three proteins contain coiled-coil regions and an adjacent NLM domain (found only in NuMA-related proteins), and all three proteins directly interact with similar Galpha-binding proteins (Pins, LGN, GPR1/2). In addition, all three proteins are localized to the cell cortex, spindle poles and spindle microtubules, and at least Mud and Lin-5 have some role in spindle orientation and generating unequal daughter cell size. However, there are differences. NuMA and Lin-5 cortical association depends on LGN and GPR1/2, respectively, whereas Mud can localize to the cortex (albeit uniformly) in the absence of cortical Galphai and Pins. Pins-independent Mud cortical localization may be mediated by the Mud C-terminal putative transmembrane domains, which are absent in NuMA and Lin-5 proteins. Conversely, NuMA and Lin-5 facilitate cortical localization of LGN and GPR1/2, respectively, whereas Mud is not required for Pins localization. Finally, it is unknown how Mud interacts with the mitotic spindle. NuMA directly binds tubulin through a domain containing the NLM motif, raising the possibility that the Mud NLM domain mediates microtubule association. Alternatively, Mud may associate with the spindle via dynein/dynactin, as has been shown for NuMA (Siller, 2006).
Pins and Galphai regulate cortical polarity, spindle orientation, spindle asymmetry and the establishment of sibling cell size differences. Previously, all Drosophila mutants in cortical polarity proteins either severely disrupted cortical polarity, thereby precluding analysis of cortical-spindle alignment, or had no effect on spindle orientation. Reduction in Mud or Khc-73 levels affects spindle orientation without altering cortical polarity; each has a partially penetrant phenotype, so they may function redundantly. mud mutants affect only spindle orientation without directly regulating any other known Pins–Galphai-dependent functions, such as regulation of cortical polarity or sibling cell size. Only when the spindle is aligned orthogonally to the Pins–Galphai crescent are there defects in sibling cell size, presumably due to the equalized activity of Pins–Galphai in both siblings. Whether each of the many essential Pins–Galphai functions has a unique effector protein, similar to the role of Mud in regulation of intrinsic spindle orientation, will be an interesting question for the future (Siller, 2006).
Reference names in red indicate recommended papers.
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date revised: 15 March 2008
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