BIOLOGICAL OVERVIEW

There are two gamma-tubulin genes in Drosophila. The gammaTubulin23C isoform is essentially ubiquitous and is required for viability and microtubule organization during mitosis and male meiosis. In contrast, the expression of gammaTubulin37C (gammaTub37C) is restricted to ovaries and early embryos (Tavosanis, 1997). Drosophila females homozygous for mutations in the gammaTub37C gene display abnormal meiotic spindles and the embryos derived from them have nuclear proliferation defects. The meiotic figures observed in females homozygous for lack-of-function alleles of gammaTub37C lack the bilateral symmetry and linear arrangement of the chromatin masses that characterize wild-type female meiotic figures during metaphase-I. The meiotic spindle is also severely disrupted in these mutants (Tavosanis, 1997; Wilson, 1998). gammaTub37C is also essential for nuclear proliferation in the early Drosophila embryo. The eggs produced by mutant mothers show an arrest of nuclear divisions during early embryogenesis because both microtubule polymerization and (as a consequence) spindle formation are blocked (Wilson, 1998; Llamazares, 1999).

gammaTub37C and gamma-tubulin ring complex protein 75 are essential for bicoid RNA localization during Drosophila oogenesis. bicoid (bcd) mRNA localization requires the activity of exuperantia and swallow at sequential steps of oogenesis and is microtubule dependent. In a genetic screen, two novel genes essential for bcd RNA localization were identified. They encode maternal gamma-Tubulin37C and gamma-tubulin ring complex protein 75 (Grip75), both of which are gamma-tubulin ring complex components. Mutations in these cytoskeletal genes specifically affect bcd RNA localization, whereas other microtubule-dependent processes during oogenesis are not impaired. This provides direct evidence that a subset of microtubules organized by the gamma-tubulin ring complex is essential for localization of bcd RNA. At stage 10b, gammaTub37C and Grip75 are found anteriorly concentrated; the formation of a microtubule-organizing center at the anterior pole of the oocyte is proposed (Schnorrer, 2002).

Asymmetry along the anterior-posterior axis of early Drosophila embryos originates from localized mRNAs at opposite poles of the freshly laid egg. bicoid (bcd) and nanos (nos) mRNAs are concentrated at the anterior and posterior poles, respectively. Bcd and Nos proteins spread from their localized sources and generate the primary morphogen gradients patterning the anterior-posterior axis of the Drosophila embryo (Schnorrer, 2002).

Previous genetic screens have identified exuperantia (exu) and swallow (swa) as essential factors for bcd mRNA localization. The process of bcd RNA localization is initiated early during oogenesis, when bcd RNA is produced in the nurse cells and assembled into large particles in the cytoplasm. Exu protein is concentrated in electron-dense structures, termed sponge bodies, in the nurse cell cytoplasm, in which the assembly of Exu- and bcd RNA-containing particles is likely to occur. These particles move into the oocyte in a microtubule-dependent manner, and bcd RNA localizes, initially together with Exu protein, at the anterior cortex of the oocyte. This anterior localization of bcd RNA is disrupted in exu mutants (Schnorrer, 2002 and references therein).

While Exu appears to be dispensable as soon as bcd RNA reaches the oocyte, swallow (swa) is required for bcd RNA localization within the oocyte. In swa mutants the initial localization is normal, but bcd RNA fails to stay anteriorly from stage 10b of oogenesis onward. In a wild-type oocyte, Swa protein colocalizes with bcd RNA at the anterior cortex at stage 10b in a microtubule-dependent manner. Swa interacts with dynein light chain and might be transported to the microtubule minus ends at the anterior pole by the dynein motor complex. Swa localization is independent of exu and bcd RNA, demonstrating that this localization does not require the early bcd RNA localization machinery acting in the nurse cells (Schnorrer, 2002 and references therein).

How the localization of bcd RNA is achieved in the oocyte is not well understood. Depolymerization of microtubules abolishes the anterior bcd RNA concentration at stage 9 and also at stage 10 of oogenesis, indicating a continuous requirement for the microtubule skeleton in order to localize and maintain bcd RNA at the anterior pole. However, drug treatment affects all microtubules at the same time, and, hence, these experiments do not allow different steps of the localization process to be distinguished or its regulation understood. Microtubules are found in an anterior to posterior gradient at stage 9. The current model of microtubule polarity is mainly based on the observation that a kinesin heavy chain ß-galactosidase (Kin:ß-gal) and an unconventional kinesin ß-galactosidase fusion (Nod:ß-gal) are concentrated at the posterior and anterior pole of a stage 9 oocyte, respectively. Therefore, the stable microtubule minus ends are thought to localize at the anterior, whereas plus ends seem to spread to the posterior. However, this model is complicated by the finding that endogenous dynein heavy chain, a motor protein transporting cargo to the minus ends, is also concentrated at the posterior pole of stage 9 oocytes. At later stages of oogenesis, at which swa has an essential function for bcd RNA localization, microtubules are mainly assembled subcortically, with an anterior concentration. Their polarity and dynamics are not well described. Importantly, both Swa and bcd RNA localize to both poles in gurken (grk) mutant oocytes, which contain a microtubule cytoskeleton with duplicated polarity, resulting in the presumptive minus ends at the anterior and posterior poles at stage 9. The ectopic bcd RNA localization at the posterior of grk oocytes argues for an active transport of bcd RNA in order to reach the posterior pole and suggests that the observed anterior localization of bcd RNA in wild-type oocytes is not established by simply trapping the imported RNA. However, there is no genetic evidence for a connection between bcd RNA localization and the microtubule cytoskeleton (Schnorrer, 2002).

During Drosophila oogenesis many different processes depend on the integrity of the microtubule cytoskeleton. It is necessary for oocyte specification in the germarium, for movement of the oocyte nucleus from posterior pole to the future dorsal-anterior corner, and for both bcd and osk RNA transport to the anterior and posterior pole, respectively. To fulfill these multiple functions properly, the cytoskeleton must be tightly controlled in a temporal and spatial manner. However, there is no centrosome or apparent microtubule-organizing center (MTOC) known in the oocyte at midoogenesis, when transport of the different RNAs to the anterior and posterior pole occurs. But, under normal cellular environments, free microtubules are extremely unstable. Microtubule organization has been studied in more detail during early Drosophila embryogenesis. There, the gamma-tubulin ring complex (gammaTuRC), which is composed of gamma-Tubulin37C and several different gamma-tubulin ring complex proteins, is located at the centrosomes, specifically at the base of a microtubule. Its ring-shaped structure serves as a template for a microtubule and allows the controlled polymerization of tubulin dimers (Moritz, 2000; Oegema, 1999). Furthermore, it prevents microtubule shrinkage at the minus ends (Schnorrer, 2002 and references therein).

In a genetic mosaic screen, using an in situ hybridization assay, mutations were isolated in two novel genes required for bcd RNA localization. These genes code for gamma-Tubulin37C (gammaTub37C) and gamma-tubulin ring complex protein 75 (Grip75), which are both components of the gamma-tubulin ring complex. The anterior concentration of bcd RNA is lost after stage 10b in both mutants, thus providing direct genetic evidence that an intact microtubule skeleton is indeed essential for correct bcd RNA localization. It is proposed that the gamma-tubulin ring complex establishes an MTOC at the anterior pole of the oocyte at stage 10b, which is essential to maintain bcd RNA localization. gammaTub37C and Grip75 mutants specifically affect the function of this MTOC, whereas other microtubule-dependent processes during oogenesis remain intact (Schnorrer, 2002).

swa is essential for bcd RNA localization at midoogenesis, when large amounts of bcd RNA are imported from the nurse cells through ring canals into the oocyte. The bcd RNA localization pattern changes from a ring shape at stage 10a to a disc shape at stage 10b. This coincides with the colocalization of bcd RNA and Swa protein at the anterior cortex. Swa localization is required for the transition of the bcd RNA localization pattern, since bcd RNA remains at the lateral cortex of stage 10b swa mutant oocytes and is never found in the middle at the anterior pole. swa likely has a specific function with respect to bcd RNA because, in swa mutants, the localization of other anteriorly concentrated components, like Nod:ß-gal or the gammaTuRC components gammaTub37C and Grip75, is not as affected as bcd RNA. Thus, swa is not required for microtubule function in general. However, Swa might be involved in regulating the dynamics of certain microtubules, since the regular centrosome and spindle distribution during embryogenesis is affected in swa mutants (Schnorrer, 2002).

It has been proposed that transport of Swa to the anterior pole of the oocyte occurs along microtubules via the minus end-directed dynein motor complex (Schnorrer, 2000). In support of this model, it has been found that SwaGFP localizes together with gammaTub37C and Cnn to centrosomes during early embryogenesis. At the end of mitosis the SwaGFP concentration at the centrosomes decreases and the localization is reestablished within the next interphase. This suggests that Swa is not an integral component of centrosomes, but localizes to microtubule minus ends in two different biological contexts. The distribution of the microtubule minus end directed motor Nod:ß-gal at stage 10b of oogenesis suggests that transport to the microtubule minus ends persists at this stage. This supports the idea that Swa also achieves its localization by active transport. Nod:ß-gal recapitulates the change in the localization pattern of bcd RNA. Therefore, this change is likely to be an active process, which, with respect to bcd RNA, requires localized Swa to function. But how is this change organized (Schnorrer, 2002)?

The screening procedure for novel genes essential for bcd RNA localization has identified mutations in gammaTub37C and Grip75, which affect bcd RNA localization at late stage 10b of oogenesis. These mutants are cytoskeletal factors that are essential for bcd RNA localization. They are both components of the same molecular complex, the gamma-tubulin ring complex, and show the same bcd RNA mislocalization and early embryonic arrest phenotype. In the early embryo, the gamma-tubulin ring complex is concentrated at the centrosomes, which organize the spindle microtubules. Other mutants in maternally expressed centrosomal proteins, which cause nuclear division problems during early embryogenesis, do not result in an aberrant bcd RNA distribution. centrosomin (cnn) or abnormal spindle (asp) mutants were tested and no difference was detected in bcd RNA localization. This demonstrates that the screening procedure is stringent enough to identify specific factors, and, since it covered only the left arm of the second chromosome, it is likely that more genes with a function in bcd RNA localization exist in the genome (Schnorrer, 2002).

gammaTub37C and Grip75 mutants are required for microtubule assembly during embryogenesis and, therefore, are necessary for starting the nuclear divisions (Llamazares, 1999; Tavosanis, 1997). However, both mutants are viable and, hence, not essential for the microtubule-dependent processes during later embryonic and larval development. For gammaTub37C this is less surprising because it is only expressed maternally and a second gamma-tubulin, gammaTub23C, fulfils zygotic functions. In contrast, Drosophila has no second paralog of Grip75 in the genome. Consequently, Grip75 plays an essential role only in certain microtubule-dependent processes, which appear to be those that also require gammaTub37C. However, both Grip75 alleles are sterile not only in females, but also in males, a phenotype described for mutants in centrosomin, but not in gammaTub37C or swa (Schnorrer, 2002).

gammaTub37C and Grip75 mutants display a specific loss of microtubule function during oogenesis. Several microtubule-dependent processes, such as oocyte specification, nuclear migration, and osk and bcd RNA transport at stage 9, are functional. bcd RNA mislocalization starts at late stage 10b. This phenotype can be explained in two ways. Either gammaTub37C and Grip75 affect all microtubules of the oocyte at a specific time point or they eliminate the function of only a subset of microtubules, while others are unaffected. The latter explanation, which proposes specialized microtubules, is supported by the fact that a maternal gamma-tubulin exists that has a relatively divergent primary sequence and comprises about 20% of the gamma-tubulin pool in oocytes. Furthermore, tubulin modifications such as acetylation or polyglycylation may distinguish certain microtubules from others. Attempts were made to address this problem by analyzing microtubule-dependent transport to the posterior pole. Posterior transport is normal in gammaTub37C mutants; however, it is possible that this transport is already completed at stage 10b, since Kin:ß-gal is no longer concentrated at the posterior at late stage 10b. Hence, posterior transport might not take place anymore and does not allow the two possibilities proposed above to be distinguished. Therefore, ooplasmic streaming, a microtubule-dependent process that occurs at the same time as the bcd RNA mislocalization in gammaTub37C, Grip75, and swa mutants, was analyzed. The fact that ooplasmic streaming is unaffected in all the mutants suggests that only a subset of microtubules, which are required for bcd RNA localization from stage 10b onward, but not for ooplasmic streaming, are affected. This conclusion is directly supported by the gamma-tubulin analysis in gammaTub37C and Grip75 mutants, which shows the presence of microtubules in these mutant oocytes at stage 10b (Schnorrer, 2002).

So far, no microtubule-organizing center (MTOC) has been described at the anterior pole at any stage of oogenesis that would allow directed microtubule-dependent transport to the anterior pole and demonstrate the polarity of the microtubule network. The gamma-tubulin ring complex is capable of nucleating microtubules in a controlled manner and, in addition, can act as a cap that stabilizes their minus ends. gammaTub37C and Grip75 are essential for these functions of the ring complex during embryogenesis and, most likely, also during oogenesis. Since Grip75GFP is a functional component of the gammaTuRC in the embryo and stably binds to gammaTub37C during oogenesis, the colocalization of Grip75GFP and gammaTub37C at the anterior pole of a stage 10b wild-type oocyte suggests that Grip75 and gammaTub37C form a distinct microtubule organizer at the anterior pole at stage 10b. Importantly, grk mutants show an ectopic posterior gammaTub37C focus, consistent with the model of microtubule minus ends at both poles in grk oocytes. Furthermore, this posterior gammaTub37C focus supports the idea that the gammaTub37C focus at the anterior pole in wild-type oocytes is not simply a consequence of dumping the nurse cell cytoplasm into the oocyte, but a distinct MTOC. This is further supported by the anterior enrichment of gammaTub37C in dumpless mutants. The proposed anterior MTOC might have a similar molecular composition as the gammaTuRC during embryogenesis and interacts with Swa protein. The disrupted bcd RNA and Nod:ß-gal localization in Grip75 and gammaTub37C mutants shows the functional significance of the proposed MTOC (Schnorrer, 2002).

The formation of a gamma-tubulin ring complex-based MTOC in the middle of the anterior pole at stage 10b may explain the 'ring to disc' transition of the localization pattern of bcd RNA and Nod:ß-gal and is consistent with the anterior colocalization of gammaTub37C and gamma-tubulin. However, gamma-tubulin is not restricted to the anterior cortex at stages 10b–11 but extends along the whole oocyte cortex. It is likely that the gamma-TuRC functions as a template to nucleate certain microtubules at the anterior pole in a controlled direction (Schnorrer, 2002).

An interesting aspect of the gammaTub37C and Grip75 mutant phenotypes is that they uncouple Swa and bcd RNA localization. Since the defect in bcd RNA localization starts slightly later in gammaTub37C and Grip75 than in swa mutants, it was expected that Swa would be localized, which is the case. But the colocalization of Swa and bcd RNA is lost at late stage 10b, arguing against the hypothesis that Swa binds directly to bcd RNA in order to transport it to, or anchor it at, the anterior pole. Furthermore, it is unlikely that Swa itself acts as molecular anchor to trap bcd RNA, which is imported from the nurse cells. The situation at the anterior pole appears to be more complicated, and the following model is proposed (Schnorrer, 2002).

In a first phase lasting until stage 10a, bcd RNA is localized in an exu- and microtubule-dependent process to the anterior pole into a ring-shaped pattern. This requires neither gammaTub37C and Grip75 nor swa to function. In a second phase, a change in microtubule organization occurs, microtubules are mainly assembled subcortically, and transport to the posterior pole stops. Swa protein localization starts at the entire anterior cortex of the oocyte, not only in its corners, possibly in a dynein-dependent manner. The bcd RNA localization pattern changes into a disc or a cap-like pattern at the anterior pole. This is likely to be an active process, which initially does not depend on gammaTub37C and Grip75 but does depend on swa. Swa might use gammaTub23C and other Grips in order to partially reorganize the microtubule cytoskeleton. In a third phase, starting at late stage 10b, gammaTub37C and Grip75 are essential to keep bcd RNA anteriorly and to complete the ring- to disc-shape transition. During this time the localized amount of bcd RNA increases continuously. Since Grip75GFP and gammaTub37C are enriched at the anterior pole from stage 10b onward, it was proposed that an MTOC, which might organize a subset of microtubules with distinct polarity, is established there at this time. In gammaTub37C and Grip75 mutants, this MTOC is disrupted and bcd RNA diffuses into the oocyte. This is presumably promoted by large amounts of nurse cell cytoplasm entering the oocyte through the ring canals at the anterior pole. Therefore, bcd RNA either requires a stable anchor or continuous transport back to the anterior pole during phases two and three. The hypothesis of continuous transport along microtubules is favored, considering the molecular nature of gammaTub37C and Grip75 (Schnorrer, 2002).

In contrast to bcd RNA, Swa stays localized at the anterior cortex in gammaTub37C and Grip75 mutants. This argues that Swa itself is less sensitive to microtubule disruption than bcd RNA and might be anchored at the anterior cortex after its initial localization. The localized amount of Swa does not increase further after stage 10b. Swa binds to the gamma-TuRC and might regulate microtubule turnover or dynein motor recycling. This binding is lost in swa mutants, demonstrating that the C terminus of Swa is required for the interaction with the gamma-TuRC. The possibility that Swa itself has the capacity to change properties or location of a MTOC remains to be tested. In the future, proteins like Exu and Swa, which seem to have specialized functions for only a few processes and are poorly conserved during evolution, might be possible to integrate into the cellular machinery required for asymmetric localization of determinants. This machinery requires a variety of conserved cytoskeletal components, which can be used for mRNA transport at different developmental stages, such as Drosophila oogenesis and embryogenesis (Schnorrer, 2002).

Endosomal Wnt signaling proteins control microtubule nucleation in dendrites

Dendrite microtubules are polarized with minus-end-out orientation in Drosophila neurons. Nucleation sites concentrate at dendrite branch points, but how they localize is not known. Using Drosophila, this study found that canonical Wnt signaling proteins regulate localization of the core nucleation protein gammaTubulin (gammaTub). Reduction of frizzleds (fz), arrow (low-density lipoprotein receptor-related protein [LRP] 5/6), dishevelled (dsh), casein kinase Igamma, G proteins, and Axin reduced gammaTub-green fluorescent protein (GFP) at branch points, and two functional readouts of dendritic nucleation confirmed a role for Wnt signaling proteins. Both dsh and Axin localized to branch points, with dsh upstream of Axin. Moreover, tethering Axin to mitochondria was sufficient to recruit ectopic gammaTub-GFP and increase microtubule dynamics in dendrites. At dendrite branch points, Axin and Dsh colocalized with early endosomal marker Rab5, and new microtubule growth initiated at puncta marked with Fz, Dsh, Axin, and Rab5. It is proposed that in dendrites, canonical Wnt signaling proteins are housed on early endosomes and recruit nucleation sites to branch points (Weiner, 2020).

Neurons extend long branched processes from a central cell body. This shape is incompatible with a centrosomal microtubule organizing center (MTOC). Mature neurons are therefore among the ranks of differentiated cells that have noncentrosomal microtubule arrays. It is particularly important to understand how neuronal microtubules are organized because the distance from the primary site of synthesis in the cell body to functional sites in axons and dendrites can be large and, therefore, place heavy demands on microtubule-based transport. In humans, slight disruptions in microtubule regulators or motors can manifest as neurodegenerative disease, underscoring neuronal reliance on perfectly orchestrated microtubule-based transport (Weiner, 2020).

If neuronal microtubules are not anchored to the centrosome, how are they organized? In all neurons so far examined, axonal microtubules have their dynamic plus ends oriented away from the cell body (plus-end-out). In dendrites of vertebrate neurons, microtubules are mixed polarity. In invertebrate neurons (Drosophila and Caenorhabditis elegans), axons have the same plus-end-out microtubule organization as vertebrates, but mature dendrites have almost all minus-end-out microtubules. In immature Drosophila dendrites, microtubules are mixed polarity and only gradually resolve to the minus-end-out mature arrangement. Thus, although the final arrangement of microtubules in vertebrate and invertebrate dendrites is somewhat different, they are the same during dendrite development (Weiner, 2020).

Although the arrangement of neuronal microtubules is clearly noncentrosomal, the source of axonal and dendritic microtubules has been controversial. Two major models for generating axonal and dendritic microtubules have been proposed. The first is that neuronal microtubules are nucleated at the centrosome, or perhaps elsewhere in the cell body, and then released for transport/sliding into axons and dendrites. This model has substantial support, including recent analyses with newer techniques. For example, live imaging of microtubules with plus-tip (+TIP) tracking proteins and photoconvertible αTubulin has provided evidence for directional transport of microtubules into and out of developing axons in mammalian and Drosophila neurons (Weiner, 2020).

The second model is that nucleation sites are found outside the cell body and that microtubules are generated locally in axons and dendrites. Evidence for this model came from the observation that centrosomal γTubulin (γTub), the core microtubule nucleation protein, decreases gradually over time and that centrosome ablation does not disrupt axon formation. Similarly, the centriole is not surrounded by γTub in Drosophila neurons in vivo, and it is dispensable for neuronal microtubule organization. One way to reconcile these two models is to assume that both are important and that, very early in neuronal development, microtubule sliding can dominate, whereas later in development and in mature neurons, microtubules are primarily locally nucleated (Weiner, 2020).

In some cell types, the Golgi complex recruits nucleation sites, and small Golgi outposts can be found in both mammalian and Drosophila dendrites. Thus, it was proposed that the Golgi might act as a noncentrosomal MTOC in dendrites. However, subsequent analysis of γTub and Golgi outposts, including a strategy to deplete Golgi from dendrites, called this proposal into question (Weiner, 2020).

Within Drosophila dendrites, γTub is concentrated at branch points. A previous study identified proteins that localize a different microtubule regulator, adenomatous polyposis coli (Apc) 2, to branch points. It was reasoned that some or all of this machinery might be used to position γTub to the same region. Whether any of the Apc2 localization proteins act upstream of γTub-green fluorescent protein (GFP) in dendrites was tested. Surprisingly, a subset of Wnt signaling proteins was required to localize γTub-GFP to dendrite branch points, regulate dendritic microtubule polarity, and nucleate microtubules in dendrites in response to axon injury. The required proteins include the seven transmembrane domain Frizzled (Fz) proteins (Wnt receptors), Arrow (Arr, a Wnt coreceptor), heterotrimeric G proteins, Dishevelled (Dsh), Casein kinase I (CK1)γ, and Axin. Axin seems to be the key output protein of this pathway because it was sufficient to recruit γTub to ectopic sites in dendrites. Within branch points, Fz, Axin, and Dsh were found on puncta that colocalized with Rab5. In addition, new end-binding protein 1 (EB1) comets at polymerizing microtubule plus ends initiated from puncta marked with Fz, Arr, Dsh, Axin, and Rab5. It is proposed that Wnt signaling proteins localize to early endosomes at dendrite branch points and function there to control local microtubule nucleation. Although it has previously been shown that Wnt signaling proteins can function from endosomes, identification of microtubule nucleation as an output of endosomal Wnt proteins is quite unexpected (Weiner, 2020).

It was particularly intriguing to find integral membrane signaling proteins required for noncentrosomal microtubule nucleation. Although Wnt signaling has been linked to microtubule plus-end regulation in axon growth cones and regulation of microtubule stability and spindle orientation, the only connection to the minus end is localization of some cytoplasmic Wnt signaling proteins like Axin to the centrosome in dividing cells. This study demonstrated that a Wnt signaling pathway acts upstream of microtubule nucleation in a postmitotic cell. Not only were many canonical Wnt signaling proteins required for γTub-GFP to accumulate at branch points, but Axin and Dsh themselves concentrated at branch points. In addition, the scaffolding protein Axin was able to recruit γTub-GFP and the nucleation activator Cnn to mitochondria when tethered to them. Moreover, reduction of Wnt signaling proteins phenocopied loss of γTub in two functional nucleation assays, indicating that most or all dendritic nucleation occurs downstream of this pathway. Although this pathway seems to be the major regulator of dendritic nucleation, neurons are quite resilient to its loss under baseline conditions, and the simple ddaE neurons have normal arbor shape. This is likely because parallel pathways can be used to generate new minus ends. For example, microtubule severing can be used to generate new plus and minus ends and amplify microtubule number. In many cell types, minus ends generated when a microtubule is severed are recognized by minus-end binding proteins in the calmodulin-regulated spectrin-associated protein (CAMSAP)/Patronin family. In C. elegans, γTub-mediated microtubule nucleation has been shown to act in parallel and quite redundantly with Patronin to regulate microtubule organization. Recent work has shown that Patronin-mediated minus-end growth is an important regulator of dendritic microtubules in Drosophila, so it is possible that microtubule severing in conjunction with Patronin recruitment to minus ends can compensate for nucleation under most normal circumstances. Consistent with this hypothesis, phenotypes from reduction of nucleation or Patronin become more evident after severe stress, including axon [29] or dendrite [77, 98] injury (Weiner, 2020).

Although it was consistently found that partial loss of function (RNAi or heterozygous mutants) for fz, fz2, arr, dsh, Gao, Gas, and Axin reduced γTub localization and/or function, no evidence was found that β-catenin/Arm, the key transcription factor that is the output of canonical Wnt signaling, was involved. In addition, an Arm protein trap showed clear expression in epidermal cells but was not seen in dendritic arborization neurons (da neurons). Because Axin itself was sufficient to recruit γTub, there was no strong rationale for a transcriptional regulator to mediate signaling between fz/arr and microtubule nucleation. It is proposed that canonical Wnt signaling proteins are co-opted in dendrites to directly recruit nucleation complexes to endosomes. Because this is a variant of canonical Wnt signaling that unexpectedly seems not to involve β-catenin, this pathway is termed apocryphal Wnt signaling in reference to the Apocrypha, ancient writings found in only some versions of the Bible (Weiner, 2020).

The involvement of arr as well as dsh and Axin suggests that a signalosome might be involved in dendritic Wnt signaling. Signalosomes form when wnt ligands bind to Fz and LRP5/6 at the plasma membrane, triggering recruitment and multimerization of Dsh and Axin. The normal output of signalosome formation is release of β-catenin from the destruction complex and its subsequent stabilization and transit to the nucleus to activate transcription. Signalosomes assemble at the plasma membrane. Endocytosis generally seems to promote Wnt signaling, although in many contexts the signalosome itself is disassembled upon endocytosis. It is not clear whether signalosomes persist after endocytosis, though in some Drosophila cells, Dsh and Arr are localized to endosomes. In dendrites, puncta of Fz, Dsh, and Axin colocalized with Rab5, suggesting that a stable signaling complex is present on endosomes in mature neurons. The initiation of comets from these puncta indicates that endosomes are likely the key site where Wnt signaling proteins promote nucleation. Colocalization of tagged Golgi proteins with Rab5 suggests that the previous association between Golgi markers and nucleation could have been due to leakage into endosomes. In addition, the identification of plasma membrane proteins acting upstream of γTub in dendrites suggests a more general role for the Golgi in the cell body by controlling secretion of Arr and Fz (Weiner, 2020).

Wnt signaling receptors have been classically studied at the plasma membrane, where they bind extracellular ligands that can be autocrine or paracrine in nature. A requirement for arr and fz upstream of γTub in dendrites suggests that a Wnt ligand is likely involved. Failure of neuronal wntless knockdown to reduce γTub-GFP at branch points favors the hypothesis that the ligand may be secreted from a neighboring cell. In the embryo, wingless (wg)/Wnt-1 is made in a patch of epithelial cells adjacent to developing dendritic arborization neurons and helps pattern dendrite orientation in ddaE . It would be very interesting if surrounding cells influenced the microtubule cytoskeleton in mature neurons through Fz and Arr at the plasma membrane. This signaling pathway is particularly intriguing in the context of regeneration or during neurodegenerative disease. During axon regeneration, the initial injury response involves a nucleation-dependent increase in microtubule dynamics, which serves a neuroprotective role. Modulating Wnt signaling could therefore influence neuroprotection in dendrites. In addition, this study found that this pathway is required during dendrite regeneration to position nucleation sites in regrowing dendrites. Interestingly, G protein coupled receptors (GPCRs) represent 33% of all Food and Drug Administration-approved drug targets, and as part of this family, Fz presents a possible target (Weiner, 2020).

Local microtubule nucleation also occurs in axons. As Rab5 endosomes are present throughout axons, it will be interesting to determine whether Wnt signaling proteins can be recruited to axonal early endosomes and whether they recruit nucleation proteins in this part of the cell. It is also possible that a link between Wnt signaling, endosomes, and nucleation could exist more broadly in other cell types. Indeed, the localization of Axin to centrosomes suggests that even in mitotic cells, parts of this relationship are conserved. Intriguingly, endosomal membranes are concentrated around the centrosome, and Rab5 reduction disrupts mitosis, so it is possible that Wnt signaling proteins, endosomes, and nucleation function together at centrosomes (Weiner, 2020).

GENE STRUCTURE

cDNA clone length - 1553

Bases in 5' UTR - 90

Exons - 4

Bases in 3' UTR - 89

PROTEIN STRUCTURE

Amino Acids - 457

Structural Domains and Evolutionary Homologs

Information about gammaTubulin structural domains and evolutionary homologs can be found at the gammaTubulin at 23C site.

date revised: 20 December 2002

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