betaTubulin56D (ß1 tubulin)

DEVELOPMENTAL BIOLOGY

Embryonic

Maternally expressed beta1 tubulin is incorporated into mitotic spindles. Later in development a strong expression in the CNS is observed. Furthermore, all chordotonal organs and the apodemes are marked by beta1 tubulin. Nuclear run-on assays and stage specific in vitro transcription show a zygotic expression of the beta1 tubulin gene from the extended germ-band stage onwards. beta1 tubulin gene is a very early effector gene, starting its expression shortly after the commitment of neuroblast cell fate. This gene offers an excellent model system for the identification of neural and apodeme specific transcription factors (Buttgereit, 1991).

Maternally encoded ß1 tubulin is incorporated into zygotic mitotic spindles. Thus ß1 tubulin is the primary ß tubulin component of the mitotic apparatus. However, gamma tubulin has an important role as a nucleating substrate for ß1 tubulin polymerization in the mitotic apparatus. Later in development a strong expression of ß1 is observed in the central nervous system and in the epidermis. All chordotonal organs and apodemes (muscle attachment sites within the epidermis) are marked by ß1 tubulin. In Drosophila testis stem cells ß1 tubulin is found in mitotically active germ cells and all somatic parts of the testis, but starting with early spermatocytes, the ß1 isotype is switched off and all microtubular arrays contain ß2 tubulin (Raff, 1982, Bialojan, 1984, Gasch, 1988, Buttgereit, 1991 and Buttgereit, 1993).

Cytoskeletal changes occur during the delamination of precursors of the peripheral nervous system (microchaete precursors in the pupal notum) and central nervous system (embryonic SI neuroblasts). The patterns of delamination and mitosis are closely correlated: delamination occurs either immediately after a cell has divided (in the case of microchaete precursors) or shortly before the division (in the case of the neuroblasts). In addition, cytoskeletal changes similar to those occurring during mitosis can be seen in delaminating neuronal precursors. Thus, during both mitosis and delamination, the discrete apicobasally oriented microfilament-tubulin bundles break down. Microfilaments form a dense, diffuse cortical layer surrounding the entire cell body. Microtubules are concentrated at the apically located centrosome. The relationship between mitosis and delamination is supported by the finding that the neurogenic gene Notch and segment polarity gene wingless affect both proliferation and delamination in the ventral neurectoderm (Hartenstein, 1994).

The stripe gene is both necessary and sufficient to initiate the developmental program of epidermal muscle attachment (EMA or segmental border) cells. In stripe mutant embryos, these cells do not differentiate correctly. Ectopic expression of Stripe in various epidermal cells transforms these cells into muscle-attachment cells expressing an array of epidermal muscle attachment cell-specific markers. These markers include goovin, delilah, and ß1 tubulin. The EMA-specific genes induced by Stripe can be divided into two groups: genes that follow Stripe ectopic expression in all embryonic stages or genes that can not be detected in early (stage 10-11) or late (older than stage 14) developmental stages, but only in intervening stages. groovin and alien represent the first group (all stages) and delilah and ß1 tubulin represent the second group (intervening stages) and are expressed only in stages 12-14 (Becker, 1997).

The ectopic epidermal muscle attachment cells are capable of attracting somatic myotubes from a limited distance, providing that the myotube has not yet been attached to or been influenced by a closer wild-type attachment cell. Analysis of the relationships between muscle binding and differentiation of the epidermal muscle attachment cell has been performed in mutant embryos in which either loss-of-muscles or ectopic muscles were induced. This analysis indicates that although the initial expression of epidermal muscle-attachment cell-specific genes including stripe and groovin is muscle independent, continuous gene expression is maintained only in epidermal muscle attachment cells that are connected to muscles. Normally, the expression of ß1 tubulin is restricted to the final stage of gene expression in tendon-like cells, supporting the idea of a distinct mechanism regulating gene expression within the tendon cells as a result of muscle interactions. These results suggest that the binding of a somatic muscle to an epidermal muscle attachment cell triggers a signal affecting gene expression in the attachment cell. Thus there exists a reciprocal signaling mechanism between the approaching muscles and the epidermal muscle attachment cells. First the epidermal muscle attachment cells signal the myotubes and induce myotube attraction and adhesion to their target cells. Following this binding, the muscle cells send a reciprocal signal to the epidermal muscle attachment cells inducing their terminal differentiation into tendon-like cells (Becker, 1997).

In the Drosophila embryo, the correct association of muscle cells with their specific ectodermally derived tendon cells, also known as epidermal muscle attachment or EMA cells, is achieved through reciprocal interactions between these two distinct cell types. Vein, a neuregulin-like factor secreted by the approaching myotube, activates the EGF-receptor signaling pathway within the tendon cells to initiate tendon cell differentiation. kakapo is expressed in the tendons and is essential for muscle-dependent tendon cell differentiation. Kakapo is a large intracellular protein and contains structural domains also found in cytoskeletal-related vertebrate proteins (including plakin, dystrophin, and Gas2 family members). kakapo mutant embryos exhibit abnormal muscle-dependent tendon cell differentiation. The expression of delilah, stripe, and beta1 tubulin is induced in the epidermal attachment cells as a result of the EGF-receptor pathway activation by the neuregulin-like growth factor, Vein. Vein is secreted by mesodermal cells underlying the EMA cells. Vein protein localization is restricted to the muscle-tendon junctional site in wild-type embryos. However, in kak mutant embryos, Vein protein is not localized and appears rather diffuse. This altered pattern of Vein may explain the multiple number of cells expressing delilah and stripe: since Vein is not strictly localized at a given muscle-tendon junction site, it apparently weakly activates the EGF-receptor pathway in neighboring cells as well. It is presumed that the only cells that can respond to the ectopic Vein protein are the competent population of EMA cells, defined by the early expression of stripe. These cells express stripe during early developmental stages in a muscle-independent manner and normally lose their stripe expression by stage 16 of embryonic development. When these competent EMA cells receive the muscle-derived Vein signal, the expression of stripe and delilah is reactivated. It appears that only this population of cells is capable of responding to Vein, since the pattern of the ectopic Stripe- or Delilah-expressing cells in the kak mutant embryos resembles that of the early population of Stripe-expressing cells. The reduced levels of beta1 tubulin mRNA in the mutant tendon cells may also result from the abnormal pattern of Vein localization, since lower levels of Vein may not be sufficient to induce maximal beta1 tubulin expression. It therefore appears that the primary defect in kak mutant embryos stems from the lack of Vein accumulation at the muscle-tendon junctional site (Strumpf, 1998).

Microtubules in the syncytial blastoderm embryo

Cyclic reorganizations of filamentous actin, myosin II and microtubules in syncytial Drosophila blastoderms have been studied using drug treatments, time-lapse movies and laser scanning confocal microscopy of fixed stained embryos (including multiprobe three-dimensional reconstructions). These observations imply interactions between microtubules and the actomyosin cytoskeleton. They provide evidence that filamentous actin and cytoplasmic myosin II are transported along microtubules towards microtubule plus ends, with actin and myosin exhibiting different affinities for the cell's cortex. These studies further reveal that cell cycle phase modulates the amounts of both polymerized actin and myosin II associated with the cortex. Pseudocleavage furrow formation in the Drosophila blastoderm is analogous to how the mitotic apparatus positions the cleavage furrow for standard cytokinesis, and these findings are related to polar relaxation/global contraction mechanisms for furrow formation (Foe, 2000).

Laser scanning confocal microscope (hereafter LSCM) sectional views are provided of anterior ends of Drosophila embryos showing anaphase and interphase of cycle 9 (before centrosomes, microtubules and nuclei reach the cortex) and cycle 10 (the first round of bud formation and breakdown). In cycle 9, myosin II staining concentrates in a cortical rim during interphase but leaves the cortex during anaphase. Likewise, F-actin concentrates during interphase 9 in a cortical rim; the concentration attenuates greatly during anaphase 9. Throughout interphase 9, with no nuclei/asters near the cortex, cortical myosin II and F-actin co-localize. Similar waxing and waning of cortical F-actin and myosin II, co-localized and synchronized with globally synchronous mitotic cycles, is observed in cycle 8. Migrating nuclei with microtubule arrays reach the cortex 1 minute after interphase 10 begins. As telophase 9 ends and interphase 10 begins, F-actin and myosin II re-accumulate co-localized to high levels in a spatially uniform cortical rim. Two minutes after nuclei reach the cortex, cortical F-actin and myosin II are no longer co-localized but occur in the complementary patterns. Myosin II occurs at high levels between buds, but vacates the cortex where buds now protrude, while F-actin attains high levels precisely on the domes of the buds that myosin II has vacated. During anaphase 10, cortical levels of F-actin and myosin II are globally low. Cortical F-actin re-accumulation begins first near centrosomes at anaphase/telophase. Regardless of cortical fluctuations, high levels of myosin II staining occur throughout the embryo interior. When myosin dissociates from the cortex, it transiently boosts the concentration of myosin immediately beneath the cortex, but does not significantly boost the concentration of internal myosin globally, presumably because it is dispersing into an ocean of cytoplasmic myosin filling these large cells. Throughout the interior cytoplasm, F-actin occurs diffusely, and additionally in particles, but at lower levels than cortically (Foe, 2000).

When migrating nuclei with associated microtubule arrays first reach the periphery early in interphase 10, myosin II staining disappears from a small region immediately above the nuclei and, during the next 3 minutes of interphase and prophase, the holes vacated by myosin II staining enlarge into oblong holes. During metaphase and anaphase, the cortical myosin staining dims, then, during telophase, myosin staining returns brightly to the cortex except near centrosomes where it remains dim. Holes in the cortical myosin pattern, appearing in the cortex precisely when/where microtubule arrays first contact the cortex, are interpreted as implying an interaction between myosin II and microtubules (Foe, 2000).

Four hypotheses have been proposed about consecutive and simultaneous mitotic-cycle-modulated interactions between the cell cortex (the approximately 3 mm deep zone immediately underlying the plasma membrane), F-actin, myosin II, centrosomes and microtubules. When working together, they can explain these experimental results (Foe, 2000).

• Hypothesis H1: mitotic phase modulates the amount of myosin II associated with the cortex and the amount of actin polymerized into filaments at the cortex. Independent of the proximity of centrosomes and/or microtubules, both cortical myosin II and cortical F-actin increase from telophase through interphase then fall through metaphase and anaphase.
• Hypothesis H2: centrosomes, via the polymerization of the microtubule asters that they nucleate, boost the rate of actin polymerization nearby.
• Hypothesis H3: a kinesin-like activity attaches actin filaments to adjacent microtubules and transports actin filaments toward microtubule plus ends; this activity is weaker than the attachment of F-actin to the cortex.
• Hypothesis H4: a kinesin-like activity moves myosin II filaments towards microtubule plus ends; this is stronger than the attachment of myosin to the cell's cortex so that appropriately oriented microtubule arrays contacting the cortex strip myosin II off the plasma membrane.

The same four mechanical hypotheses that are proposed to explain pseudocleavage furrow and bud formation in the fly syncytial blastoderm, if operative in dividing mononucleate cells, could time and initiate localization of the actomyosin components of the contractile ring for cytokinesis. H1, by melting down prior interphase F-actin structures during metaphase, and H2 by causing F-actin to re-polymerize near centrosomes beginning in anaphase, in effect force a redeployment of the cell's actin just prior to beginning the specialized task of cytokinesis. By H1, the cortical concentrations of F-actin and myosin II, having fallen to low concentrations in metaphase-anaphase, rebuild in telophase. But the expanding telophase microtubule asters approaching the cortex at opposite poles of the cell would trigger, via H4, depletion of cortical myosin II filaments near the spindle poles where microtubules impinge, while simultaneously concentrating myosin II filaments by moving them through the cytoplasm towards the cell mid-zone. H4 will thus eventually concentrate myosin II in a three-dimensional disk whose perimeter will become the contractile ring. By H2, actin polymer will form coincidentally with microtubule outgrowth initially most concentrated near centrosomes, followed later in telophase by a migration of F-actin along astral microtubules away from centrosomes (by H3) and toward concentration in a three-dimensional disc-shaped volume centered at the equator where it will co-localize with myosin II. The 'rings' of cortical F-actin seen in late interphase, correspond to where these equatorial disc volumes intersect the cortex in the dividing mononucleate cell. The regions between buds where both F-actin and myosin II are present together at the cortex during interphase (though concentrations of F-actin may be higher elsewhere) constitute the so-called pseudocleavage furrows in the fly syncytial blastoderm. This region would be homologous to the cortex of the cleavage furrow, which constricts during cytokinesis (Foe, 2000 and references therein).

H1-H4 imply a bipolar 'global contraction-polar relaxation' mechanism for positioning the contractile apparatus for cytokinesis. Transient 'polar relaxation' would convert a global cortical contraction into a self-amplifying equatorial contraction. Computer simulations have shown how contracting an initially isotropic actomyosin meshwork into an equatorial belt aligns the filaments parallel to the equator, as in a contractile ring, positioning them to cleave a cell in two by a purse-string contraction. Plus-end transport along astral microtubules of cortical F-actin (H3) and of myosin II filaments (H4) could provide a mechanistic explanation for this oft-hypothesized polar relaxation, while simultaneously causing an increase in equatorial tension (assuming that cortical contractile strength is proportional to co-localized F-actin and myosin II concentrations). Either polar relaxation, or equatorial strengthening, or both together, can set the stage for the kind of actomyosin contraction-based cytokinesis that has been proposed (Foe, 2000 and references therein).

The establishment of the contractile furrow is a mechanism requiring two independent steps: the release in tension at the poles, preceded by (or coincident with) a global increase in cortical tensioning. The global loss of actin and myosin from the cortex during metaphase/anaphase and their return beginning in telophase, which is observed in Drosophila embryos (H1), if phylogenetically general, could underlie the cyclic changes in cortical contractility. Operating together, H1-H4 are potentially capable of implementing a 'global contraction-polar relaxation' mechanism of furrow initiation for cytokinesis. If cortical contractile strength is proportional to co-localized F-actin and myosin II concentrations, then H3 and H4 would bring about equatorial strengthening of the cortical actomyosin meshwork, in principal also implementing 'equatorial stimulation'. Note that, in flattened cells with small asters, the microtubules in the mid-zone between spindle poles are positioned to execute the same actomyosin rearrangements as astral microtubules in spherical cells. Note also that H3 plus H4 can cause equatorial concentration of whatever cytoplasmic actomyosin network a cell contains, focusing internal forces on the furrow cortex with the potential to aid furrow invagination in non-spherical cells. Homologies between cleavage and pseudocleavage, while attractive, come with the caveat that cortical tensioning and microtubule outgrowth both occur earlier in the mitotic cycle during cytokinesis in echinoderms than during pseudocleavage furrow formation in Drosophila syncytia (Foe, 2000 and references therein).

In summary, this study has aimed to deduce, from descriptions of wild-type and drug-perturbed cytoskeletal kinematics of microtubules, F-actin and myosin II in syncytial Drosophila embryos, the specific ways that these filament systems must be interacting. The speculative mechanistic hypotheses that were deduced, H1-H4, are consistent with a large body of circumstantial and partial evidence reviewed above. This machinery, if phylogenetically general, could unify old ideas about cytokinesis with new molecular findings, reconcile polar relaxation with equatorial stimulation models of furrow formation, and homologize cytoskeletal pseudocleavage furrow formation in syncytia with cleavage furrow formation in mononucleate cells. Future revelations can be expected of the molecular details by which the products of an ensemble of key genes (e.g. anillin, centrosomin, Diaphanous, KLP-3A, pavarotti, polo kinase, Rac 1, septins, etc.) collaborate to bring about and regulate the interactions between F-actin, myosin II, centrosomes and microtubules (Foe, 2000).

Transiently reorganized microtubules are essential for zippering during dorsal closure

There is emerging evidence that microtubules in nondividing cells can be employed to remodel the intracellular space. This study demonstrates an essential role for microtubules in dorsal closure, which occurs toward the end of Drosophila melanogaster embryogenesis. Dorsal closure is a morphogenetic process similar to wound healing, whereby a gap in the epithelium is closed through the coordinated action of different cell types. Surprisingly, this complex process requires microtubule function exclusively in epithelial cells and only for the last step, the zippering, which seals the gap. Preceding zippering, the epithelial microtubules reorganize to attain an unusual spatial distribution, which is described with subcellular resolution in the intact, living organism. This study provides a clearly defined example where cells of a developing organism transiently reorganize their microtubules to fulfill a specialized morphogenetic task (Jankovics, 2006).

To date, the functional analysis of spatial MT organization in nondividing cells of multicellular organisms has been carried out using isolated, cultured cells. This study performed such analysis in vivo, by using intact, living dorsal closure (DC) stage embryos of Drosophila. DC is a morphogenetic process where an eye-shaped gap in the dorsal epithelium that is occupied by amnioserosa cells is closed. DC is orchestrated by the Jun kinase, the Dpp, and the Wg signaling pathways. These control two major events that bring about DC. First, the two lateral epithelial cell layers approach each other by moving dorsally, which is termed convergence. Secondly, the epithelial cells of each layer sequentially meet and connect to each other at the anterior- and posterior-most ends of the opening, a process called zippering. Convergence requires the apical constriction of the amnioserosa cells, which generates a force that pulls the lateral epithelial cell sheets toward the midline. At the same time, the epithelial cells change shape and elongate along their dorsal/ventral (D/V) axis. In addition, the dorsal-most row of epithelial cells (DME cells) polarize parallel to the D/V axis, excluding proteins of the cell-cell adhesion complexes and accumulating actin and myosin II at their dorsal surface. This allows contraction of the dorsal side of the DME cells, which further narrows the epithelial gap. DME cells also execute zippering by forming cellular protrusions, lamellipodia and filopodia, at their dorsal membrane. The protrusions provide the initial contacts for connecting cells, and by interdigitating, they promote their association (Jankovics, 2006 and references therein).

DC is interesting to study due to its similarities to wound-healing processes and because it shows several important cellular behaviors such as cooperative cell movement, tissue force generation, and cell shape changes. While much is known about the requirements of the actin cytoskeleton during DC, the role of the MTs has not been addressed. Previous observations in fixed DC-stage embryos showed that MTs are aligned parallel to the D/V cell axis in epithelial cells. This study used real-time fluorescence imaging to describe the spatial distribution and the dynamic behavior of these MTs and to study their role during DC. A novel spatial MT distribution is described that is established in the cells of the converging epithelium. Thereby, multiple, antiparallel MTs form bundles at the apical cell cortex that align with the D/V cell axis. Although bundles are stable, individual MTs remain highly dynamic. MTs are not anchored at centrosomes but are associated individually with the apical cell cortex. Surprisingly, solely these MTs are essential for DC whereas MTs in all other relevant cells are dispensable. Epithelial MTs control exclusively a single essential step, the final zippering, and evidence is shown that this may be linked to a role in promoting cell-protrusion formation. The results provide the first detailed in vivo analysis of spatial MT organization and function in nondividing cells of an intact multicellular organism and reveal a surprisingly specific role for the MTs during a complex morphogenetic process (Jankovics, 2006).

MT reorganization in nondividing cells occurs during developmental differentiation processes or if cells react to environmental changes for example after wounding of a tissue. The reason for such MT reorganization is often not known. In vivo studies of MT reorganization events in cells of multicellular organisms were mostly limited to large, autonomously functioning cells such as oocytes or to cultured cells or cell layers. This study showa that Drosophila epithelial cells during DC represent an excellent experimental system, which provides sufficient subcellular resolution for the in vivo study of the nature and function of a transient MT reorganization event. Using time-lapse imaging of DC-stage embryos expressing GFP-tagged forms of tubulin or the plus end tracking protein EB1, it is shown that during DC, antiparallel MTs form stable bundles that align parallel to the D/V cell axis at the apical cell cortex. Within bundles, the MTs remain highly dynamic, which allows them to grow into the cellular protrusions that form at the dorsal surface of the DME cells. Surprisingly, elimination of these MTs by injection of MT depolymerizing drugs into the embryo or by expressing an MT-severing protein in a subset of epithelial cells inhibited exclusively the zippering process that completes DC. All other essential processes, the convergence of the two lateral epithelial cell layers, D/V cell polarization of the DMEs, or their actin-based dorsal constriction were not altered (Jankovics, 2006).

How could MT function be linked to zippering at the molecular level? One obvious scenario is that MTs are required for the local delivery of adhesion proteins. MT-based, localized delivery of factors was already shown to be crucial for proper morphogenesis in fission yeast cells and for a number of processes during early Drosophila development. However, zippering is not a simple one-step process. It is proposed to start with the interdigitation of the cell protrusions that form at the dorsal side of the DME cells and that establish the first contacts between equivalent cells of the two opposing epithelial cell layers. Consistent with this, the protrusions are essential for zippering. The initial cell-cell contacts subsequently develop into the known cell-adhesion structures. The possibility for initial interdigitation occurs at the anterior and posterior ends of the dorsal opening where the two cell layers meet. As this possibility also exists in embryos that cannot zipper due to a lack of MTs, it is conceivable that MTs are required for the interdigitation of protrusions. Intriguingly, the absence of MTs considerably affects the number and appearance of cellular protrusions known to be essential for zippering, which provides another possible explanation for the inability of these cells to interact with each other. An MT-mediated increase in protrusion formation could provide DME cells with sufficient interactive surface or interaction time to enable interdigitation. Consistent with this, the stripes of cells lacking MTs in Spastin-overexpression experiments were unable to zipper on their own but eventually managed to establish cell adhesion when forced into close proximity by zippering of the neighboring wild-type cells. To understand MT function during zippering, one may therefore need to ask how MTs modify cell protrusions. Since protrusions can still form in the absence of MTs, these do not seem to control the on/off activity of the protrusion-forming machinery but may rather modulate its activity. MTs have been shown to affect protrusion formation in several cultured cell types but the molecular mechanisms are not clear. It has been speculated that MTs may modulate the actin machinery by delivering regulatory factors such as guanine nucleotide exchange factors or GTPase-activating proteins that modulate the actin organizing activities of the Rac1, Cdc42, and Rho GTPases. It is also possible that growing MTs produce pushing forces that support protrusion growth (Jankovics, 2006).

Why do MTs reorganize in such a specific way? It was not possible to answer this question. If delivery of adhesion factors or promoting protrusion formation were indeed the critical functions of MTs, then their orientation parallel to the D/V cell axis would certainly improve delivery to the relevant site. However, because of the antiparallel arrangement, transport of factors is bidirectional, and therefore the factors would also be delivered to the wrong cell ends. In addition, MTs reorganize in all epithelial cells, although most of them are not involved in zippering and therefore do not need delivery of the proposed factors. It is possible that MTs reorganize to fulfill additional, nonessential functions that optimize the DC process. For example, the MTs are required for proper epithelial cell morphology. During convergence, these cells gradually elongate along the D/V axis while thinning out in the apical-basal direction. Elongation coincides with the D/V alignment of the MTs, implying that MT reorganization may be the consequence of cell shape changes. However, cells lacking MTs cannot maintain their shape after an initial elongation phase, suggesting that proper cell morphology is dependent on MT function. Notably, this is not the consequence of defects in polarization, since cell polarity is not affected in the absence of MTs. The role of epithelial cell shape changes in DC is not clear. Their dorsalward stretching may contribute to gap closure, but recent data indicated that it is not essential. The finding that the shape abnormalities resulting from MT depletion did not affect convergence of epithelial cell layers is consistent with this view (Jankovics, 2006).

These results provide further evidence that MT reorganization is not only crucial when cells change from an interphase to a mitotic state but also when they change behavior during development. It is important to understand how such rearrangements are controlled at the molecular level and also how cell-type-specific differences are achieved. DC in Drosophila provides an excellent experimental system in which the molecular mechanisms controlling MT organization can be studied in vivo by live imaging with appropriate subcellular resolution in combination with classical genetics (Jankovics, 2006).

Oogenesis

During oogenesis in Drosophila, determinants that will dictate abdomen and germline formation are localized to the 'polar plasm' in the posterior of the oocyte. Assembly of the polar plasm involves the sequential localization of several messenger RNAs and proteins to the posterior of the oocyte, beginning with the localization of Oskar mRNA and Staufen protein during stages 8 and 9 of oogenesis. The mechanism by which these two early components accumulate at the posterior is not known. Directed transport along microtubules could be used to accomplish this localization. A fusion protein composed of the bacterial beta-galactosidase enzyme as a reporter was used, joined to kinesin, part of the plus-end-directed microtubule motor. The fusion protein transiently localizes to the posterior of the oocyte during stages 8 and 9 of oogenesis. Treatment with the microtubule-depolymerizing agent colchicine prevents both the localization of the fusion protein and the posterior transport of Oskar mRNA and Staufen protein. Furthermore, the fusion protein localizes normally in oocytes mutant for either oskar and staufen, but not in other mutants in which Oskar mRNA and Staufen protein are mislocalized. Thus, association with a plus-end-directed microtubule motor can promote posterior localization of a reporter protein during oogenesis. The genetic requirements for this localization and its sensitivity to colchicine, both of which are shared with the posterior transport of Oskar mRNA and Staufen protein, suggest that a similar mechanism may function in both processes (Clark, 1994).

Marker studies show that regardless of the temperature at which mago nashi females are reared, posterior follicle cells are specified properly. This suggests that the earlier gurken signaling from the posterior of the oocyte occurs normally and indicates that mago functions within the oocyte to mediate the return signal(s) sent from the posterior follicle cells to the oocyte. In the absence of wild-type mago function, the reorganization of the microtubule network, essential for relocation of the nucleus from the posterior to the anterior/dorsal part of the oocyte, fails to occur, and axis formation and subsequent germ-plasm assembly is defective (Newmark, 1997).

A mutant, maelstrom (mael), is described that disrupts a previously unobserved step in mRNA localization within the early oocyte, distinct from nurse-cell-to-oocyte RNA transport. Mutations in maelstrom disturb the localization of mRNAs for Gurken (a ligand for the Drosophila Egf receptor), Oskar and Bicoid at the posterior of the developing (stage 3-6) oocyte. maelstrom mutants display phenotypes detected in gurken loss-of-function mutants: posterior follicle cells with anterior cell fates, Bicoid mRNA localization at both poles of the stage 8 oocyte and ventralization of the eggshell. These data are consistent with the suggestion that early posterior localization of Gurken mRNA is essential for activation of the Egf receptor pathway in posterior follicle cells. mael mutation affects the distribution and dynamics of oocyte microtubules. grk and mael mutants have a defective microtubule cytoskeleton similar to that previously described for the oocyte polarity mutants PKA and mago nashi; however, the grk and mael cytoskeletons are not identical. Both mutants have a high concentration of microtubules at the posterior of the oocyte in stages 8 and 9 when microtubules are normally concentrated at the oocyte anterior. In stage 7 however, mael microtubules are tightly bundled around the cortex, while grk mutants have a more diffuse network. This bundling is similar to the continous subcortical array of microtubules in wild-type stage 10b oocytes. Time-lapse videomicroscopy indicates that the cytoplasm undergoes premature streaming. Posterior localization of mRNA in stage 3-6 oocytes could be one of the earliest known steps in the establishment of oocyte polarity. The maelstrom gene encodes a novel protein with a punctate distribution in the cytoplasm of the nurse cells and the oocyte until the protein disappears in stage 7 of oogenesis (Clegg, 1997).

In the Drosophila ovary, membrane skeletal proteins such as the adducin-like Hts protein(s), Spectrin, and Ankyrin are found in the spectrosome, an organelle in germline stem cells (GSC) and their differentiated daughter cells (cystoblasts). These proteins are also components of the fusome, a cytoplasmic structure that spans the cystoblast's progeny that develop to form a germline cyst consisting of 15 nurse cells and an oocyte. Spectrosomes and fusomes are associated with one pole of spindles during mitosis and are implicated in cyst formation and oocyte differentiation. The asymmetric behavior of the spectrosome persists throughout the cell cycle of GSC. Eliminating the spectrosome by the htsl mutation leads to randomized spindle orientation, suggesting that the spectrosome anchors the spindle to ensure the asymmetry of GSC division; eliminating the fusome in developing cysts results in defective spindles and randomized spindle orientation as well as asynchronous and reduced cystocyte divisions. These observations suggest that fusomes are required for the proper formation and asymmetric orientation of mitotic spindles. Moreover, they reinforce the notion that fusomes are required for the four synchronous divisions of the cystoblast leading to cyst formation. In htsl cysts that lack fusomes and fail to incorporate an hts gene product(s) into ring canals following cyst formation, polarized microtubule networks do not form, the dynamics of cytoplasmic dynein are disrupted, and Oskar and Orb RNAs fail to be transported to the future oocyte. These observations support the proposed role of fusomes and ring canals in organizing a polarized microtubule-based transport system for RNA localization that leads to oocyte differentiation (Deng, 1997).

Anterior patterning of the Drosophila embryo depends on localization of Bicoid mRNA to the anterior pole of the developing oocyte: BCD mRNA localization requires both the exuperantia (exu) gene and an intact microtubule cytoskeleton. A GFP-Exu fusion protein supports BCD mRNA localization and complements the embryonic anterior axis defects produced by exu mutations. During mid-oogenesis, the GFP-Exu fusion protein assembles into particles that are concentrated around the nurse cell nuclei; these particles cluster at the ring canals that link the germline cells of the egg chamber, and accumulate at the anterior pole of the oocyte. At these stages, BCD mRNA also shows a perinuclear or apical distribution in nurse cells, and accumulates at the anterior cortex of the oocyte. These observations suggest that the GFP-Exu particles are transport riboneclear proteins containing BCD mRNA, and that these particles are targeted to the anterior cortex of the developing oocyte. Although direct proof for an association of BCD mRNA with these particles has not yet been obtained, microtubule depolymerization disrupts both BCD mRNA and Exu protein localization, suggesting that these two components utilize a similar, if not identical, anterior localization pathway (Theurkauf, 1998 and references).

To gain insight into the mechanism of anterior patterning, time lapse laser scanning confocal microscopy was used to analyze transport of particles containing a Green Fluorescent Protein-Exu fusion (GFP-Exu), and to directly image microtubule organization in vivo. These observations indicate that microtubules are required for three forms of particle movement within the nurse cells, while transport through the ring canals linking the nurse cells and oocyte appears to be independent of both microtubules and actin filaments. As particles enter the oocyte, a final microtubule-dependent step directs movement to the oocyte cortex. Exu protein and BCD mRNA are synthesized in a cluster of 15 nurse cells that are linked to the oocyte by ring canal bridges. The first cytoplasmic transport steps in anterior patterning therefore take place within the nurse cells. The analysis indicates that transport within the nurse cell cytoplasm is composed of at least three microtubule-dependent steps that produce a net movement of GFP-Exu particles toward the oocyte. The majority of the individual GFP-Exu particles within the nurse cell cytoplasm move rapidly and with no apparent net directionality with respect to the egg chamber axis. These movements are reversibly inhibited by the microtubule-disrupting drug colcemid. Microtubules throughout the nurse cell cytoplasm that lack clear orientation with respect to the egg chamber axis have been directly observed. Based on these observations, it is concluded that microtubules mediate random particle movements within the nurse cells. In the absence of microtubules, no movement or redistribution of GFP-Exu particles was observed. Simple diffusion thus appears to be insufficient to efficiently disperse these large particles. It is therefore speculated that the random microtubule-dependent particle movements are essential to dispersing GFP-Exu particles. It is proposed that this particle dispersal is required for efficient net particle transport through the nurse cells (Theurkauf, 1998).

Vectorial particle transport is observed in the region near the ring canals linking the nurse cells with the oocyte. In this region, particles tend to move directly to the cell-cell junctions. These movements, like the random movements observed in bulk nurse cell cytoplasm, are reversibly inhibited by colcemid. In addition, a dynamic population of microtubules associated with the nurse cell-oocyte ring canals is directly observed. It is therefore proposed that GFP-Exu particles approach the ring canal junctions by a microtubule-dependent random walk. The particles then associate with microtubules that are organized around the ring canals, and are transported to the nurse cell-oocyte junctions. It is this second step that imparts net directionality on particle transport through the nurse cell cytoplasm (Theurkauf, 1998).

Previous ultrastructural analysis of Exu distribution failed to identify microtubules in direct association with Exu-containing structures, termed sponge bodies. However, the current in vivo analysis indicates that at least some of the microtubules in the nurse cells turn over within 10 to 20 seconds. These microtubules are therefore likely to be difficult to preserve by standard fixation procedures. The failure to identify microtubules directly associated with sponge bodies may reflect the dynamic nature of these filaments. Perinuclear particle clustering in the nurse cells also appears to be microtubule-dependent. This process is reversibly disrupted by colcemid, and microtubules are associated with the surface of the nurse cell nuclei. The function of microtubule-dependent perinuclear clustering is not yet clear, although it seems unlikely that this process contributes directly to movement through the nurse cells. It has been proposed that Exu particles are RNPs that contain BCD mRNA, as well as other proteins. If so, these particles could form in the perinuclear region as BCD mRNA exits the nurse cell nuclei, and microtubule-dependent transport could facilitate complex formation by concentrating cytoplasmic components of the particles in this region. Consistent with this suggestion, GFP-Exu particle size is decreased by microtubule depolymerization, and particles appear to increase in size and fluorescence intensity on microtubule repolymerization (Theurkauf, 1998).

Essentially all of the GFP-Exu particle movements in the nurse cell cytoplasm are microtubule-dependent: these movements are presumably mediated by microtubule motor proteins. It is speculated that several different microtubule motors function in Exu particle motility. Alternatively, the variability in the rates and directionality of particle movements in the nurse cells could reflect complexities in the underlying microtubule cytoskeleton or particle-specific differences in the regulation of a single motor. However, the use of multiple motors for this transport process would serve to isolate axis specification from complete disruption by mutations in single motor protein genes. Mutations in known motor proteins have not yet been identified that disrupt these transport steps. Once GFP-Exu particles are localized to the nurse cell-oocyte ring canals, a distinct transport process appears to drive movement through the cell-cell junctions. These movements are uniform in direction and velocity, raising the possibility that they reflect a very local flow of cytoplasm through the ring canal junctions. The apparent absence of bulk movement through the ring canal suggests that this step in the transport pathway is not due to cytoplasmic flow, but reflects the action of a more selective mechanism. The best characterized specific transport processes require microtubules or actin filaments, yet movement through the ring canals is relatively insensitive to microtubule and actin assembly inhibitors. These observations raise the possibility that this transport step is independent of both actin filaments and microtubules. However, cytochalasin D and colcemid only affect dynamic filaments that are in equilibrium with subunits in the cytoplasm. It is therefore possible that stable actin filaments or microtubules mediate transport through the ring canals. The inhibitor data reported here, combined with previously published data, suggest that GFP-Exu transport through the nurse cell-oocyte ring canals is independent of both microtubules and actin filaments (Theurkauf, 1998).

This suggests a multi-step model for transport and anterior localization of Exu during stages 9 and 10 of oogenesis. It is speculated that Exu protein assembles into particles within the nurse cell cytoplasm, perhaps at the nuclear periphery, where these particles are localized by a microtubule-dependent process. Particles dissociate from the perinuclear regions, and random microtubule-dependent movements then distribute these large particles throughout the nurse cell cytoplasm. As particles approach the posterior of the nurse cell, they interact with microtubules originating near the nurse cell-oocyte ring canals, and are transported to the cell-cell junctions along these microtubules. Particles are then transported through the ring canals in a second vectorial process that appears to be independent of both actin filaments and microtubules. In the final transport step, particles entering the oocyte interact with microtubules originating at the anterior cortex of the oocyte, and are localized to the anterior in a microtubule-dependent step. At the cortex, particle may associate with asymmetrically localized binding sites that stabilize the asymmetric distribution. These observations and previous studies suggest that the polarity of the oocyte microtubule network is not in itself sufficient to generate anterior asymmetry, and that additional factors are required to restrict morphogens to the anterior pole. Based on these observations, a multi-step anterior localization pathway is proposed (Theurkauf, 1998).

Several observations indicate that factors in addition to egg chamber geometry and microtubule-dependent transport play a role in anterior axis specification. For example, BCD mRNA that is ectopically localized to the posterior of mago nashi and PKA mutant oocytes is dispersed as ooplasmic streaming begins at stage 10b, while the anteriorly localized transcripts in these oocytes are stable in spite of cytoplasmic streaming. Stable association of BCD mRNA with the cortex thus appears to be restricted to the anterior pole. In addition, several mRNAs are localized with BCD to the oocyte anterior during stages 9 and 10 of wild-type oogenesis. However, unlike BCD mRNA, these transcripts are dispersed upon ooplasmic streaming at stage 10b. These observations indicate that anterior transcript binding sites are specific for BCD mRNA, or a complex containing this mRNA. It has been suggested that localization of BCD mRNA depends on both microtubule-dependent movement to the cortex and transcript stabilization by a microtubule-independent mechanism. In a modified model for BCD mRNA transport, BCD mRNA binding activity is restricted to the anterior cortex, where it mediates pole-specific stabilization of transcript accumulation (Theurkauf, 1998 and references).

The centrosome-nucleus complex and microtubule organization in the Drosophila oocyte

Molecular motors transport the axis-determining mRNAs oskar, bicoid and gurken along microtubules (MTs) in the Drosophila oocyte. However, it remains unclear how the underlying MT network is organized and how this transport takes place. A centriole-containing centrosome has been detected close to the oocyte nucleus. Remarkably, the centrosomal components, gamma-tubulin and Drosophila Pericentrin-like protein also strongly accumulate at the periphery of this nucleus. MT polymerization after cold-induced disassembly in wild type and in gurken mutants suggests that in the oocyte the centrosome-nucleus complex is an active center of MT polymerization. The MT network comprises two perpendicular MT subsets that undergo dynamic rearrangements during oogenesis. This MT reorganization parallels the successive steps in localization of gurken and oskar mRNAs. It is proposed that in addition to a highly polarized microtubule scaffold specified by the cortex oocyte, the repositioning of the nucleus and its tightly associated centrosome could control MT reorganization and, hence, oocyte polarization (Januschke, 2006).

Both the nature and the localization of the MTOC beyond stage 6 of Drosophila oogenesis have not yet been clarified. Up to stage 6, gamma-tubulin has been shown to closely associate with the nucleus at the posterior of the oocyte. In addition, electron microscopy studies have demonstrated the presence of centrioles close to the oocyte nucleus up to stage 4. Thus, until stage 6, the centrosome associates with the nucleus at the posterior of the oocyte. In Drosophila females, meiosis takes place in the absence of centrosomes. It has therefore been speculated that, at stage 6, centrosome organization changes, involving the disappearance of centrioles and the generation of MTs from a diffuse organizing center. To better understand this process, the distribution of gamma-tubulin in the oocyte was re-investigated. Before repolarization of the MT cytoskeleton, it was found that gammaTub23C and gammaTub37C localize in a layer around the nucleus, with an enrichment at the posterior pole of the oocyte. This is in agreement with the location of the MTOC at this stage. After repolarization of the MT cytoskeleton, both gamma-tubulin isoforms remain located in a perinuclear manner. Interestingly, gammaTub37C, but not gammaTub23C, labels a small body close to the oocyte nucleus. In addition, gammaTub37C and gammaTub23C also exhibit differential expression patterns in embryos: gammaTub37C is located with the centrosomes of mitotic cells, whereas gammaTub23C is not. Thus, gamma-tubulin is distributed in close association with the nucleus periphery and possibly on a centrosome-like structure. Pericentrin/AKAP450 is another major component of the centrosome. Green fluorescence protein (GFP) fusion of the C-terminal part of Pericentrin/AKAP450 and its Drosophila homolog pericentrin-like protein (D-PLP) have been shown to localize to the centrosomes respectively in cultured human cells, Drosophila embryos and spermatocytes. Using the UAS/Gal4 system, GFP-cter-D-PLP was specifically expressed in the germline and a bright dot was detected in the vicinity of the nucleus before and after nuclear migration. GFP-cter-D-PLP was also detected in all germline nuclei, as has been observed previously. From stage 7 onward, the bright dot remained in the immediate vicinity of the oocyte nucleus (<1 µm distance). Furthermore, both GFP-cter-D-PLP and gammaTub37C co-localize to this discrete body, indicating that this structure could correspond to a centrosome. In G2 centriole, tubulin is highly polyglutamylated. The ID5 antibody labels basal bodies and centrioles in several species. Using this antibody, a dot was detected close to the nucleus throughout oogenesis that remained detectable up to stage 10A. This suggests that the dot represents a centriole-containing centrosome. Indeed, using electron microscopy, two to possibly four centrioles were clearly detected closely associated with the nucleus in stage 9 oocytes. This demonstrates the existence of centrioles associated with the nucleus at least up to stage 9. MT fibers emanating from those centrioles could not be unambiguously detected. Then the link between centrosome and nucleus was examined using colchicine. In flies fed with colchicine, MTs in the germline were completely depolymerized, and the oocyte nucleus was mispositioned. In the oocyte, it was observed that the nucleus and the centrosome were significantly separated, the distance between them increasing during oocyte growth. In a few cases, it was noticed that the nucleus could reach the anterior cortex without the centrosome; however, a centrosome was never observed at the anterior without the nucleus. It is concluded that the close localization of the centriole-containing centrosome to the nucleus depends on MTs (Januschke, 2006).

The structure of the MT network during mid-oogenesis is dynamic. At stage 7, MTs are visible as a mesh at the anterior cortex. Later, at stage 10, MT bundles are observed that promote cytoplasmic streaming. In-between MT distribution has been described as an AP gradient. However, high-resolution images of oocyte MTs are lacking. Therefore, a protocol frequently used to increase the detection of the MT cytoskeleton in cell culture was modified for the Drosophila egg chamber to characterize MT organization in the oocyte during the crucial period in which bcd, grk and osk mRNAs are localized. MTs were detected throughout oogenesis using alpha-Tubulin but also with a Kinesin heavy chain antibody (alpha-Khc), which revealed the MT array and its complexity in unprecedented definition. It was noticed that the range of detected details was increased and more reproducible with alpha-Khc antibody. To control the specificity of Khc detection, germline and follicle cell mutant clones were generated homozygous for khc^7.288. In such mutant cells, no Khc was detected, indicating that the detection is specific. Labeling with antibodies directed against aromatic C-terminal amino acid residues (Tyr or Phe) of alpha-tubulin and against Khc largely overlapped. This confirmed that the structures revealed by Khc were MTs. A Khc fraction was also detected at the posterior of the oocyte. That Khc accumulates along MTs may be due to permeabilization before fixation, which could cause rigor binding of Khc to MTs. This detection procedure may also permit the extraction of a soluble pool of Khc and reveal the remaining fraction distributed along the MTs. With this detection procedure, Khc revealed by Kinesin-ßgal exhibited a more restricted distribution compared with alpha-Khc antibody. This is probably due to the substitution of the C-terminal part of Khc by the ß-galactosidase in the reporter construct, impairing the recycling of the chimeric Kinesin motor leading to its accumulation exclusively at the posterior. With this detection method, the MT minus-end marker, Nod-ßgal, was detected in the antero-dorsal corner above the oocyte nucleus as well as in the opposite antero-ventral corner. Moreover, localized determinants such as Osk and Grk were correctly positioned in the oocyte (Januschke, 2006).

To confirm that the detection method does not alter MT organization, MT distribution was analyzed in follicle cells, which should be sensitive to the extraction procedure, since they are more directly exposed than the oocyte. MT distribution in different follicle cell types was unchanged, when comparing living and fixed egg chambers. The main body follicle cell MTs seemed unchanged. Main body follicle cell MTs have been shown to be highly stable, and might therefore reflect the sensitivity of the protocol with limitations. Nevertheless, stretched follicle cells showed strikingly similar MT patterns in living and fixed conditions as well. Apicalbasal polarity was not affected in follicle cells, as demonstrated by the correct apical localization of atypical protein kinase C. Importantly, the MT distribution of living egg chambers expressing GFP-alpha-Tubulin at stage 7 and stage 9 was similar to the one observed using anti-alpha-Tubulin and Khc antibodies. Therefore it seems that the fixation conditions preserve the wild-type MT organization and that Khc can be suitable to label bulk MTs (Januschke, 2006).

When fixed wild-type oocytes were analyzed by confocal microscopy, MT organization in the oocyte appeared unchanged from stage 2 to stage 6. With stage 7, MT organization was modified and two MT subsets became apparent. This organization was more evident at stage 8. A first subset consisted of cortical MTs oriented along the dorso-ventral (DV) axis parallel to the oocyte nurse cell border, and juxtaposed to the lateral cortices, wrapping the oocyte from stage 7 to 9. At least some MT bundles of this subset could be traced back to the oocyte nucleus. The DV orientation of MT bundles, depicted as black fibers in the schematic representations, was highly reproducible for all stages and persisted throughout mid-oogenesis (Januschke, 2006).

A second MT subset was present in the center of the oocyte. Although there was some variability in the patterns observed, it was found that each developmental stage showed a characteristic MT distribution. During stage 6, MTs from this subset were cortical and extended from the nucleus at the posterior to the anterior cortex, compact bundles of MTs formed a circle-like structure resembling a diaphragm. This subset was formed by long MT bundles that extended (once or more) along the entire cortex. By stage 8, the oocyte had considerably grown and individual MT bundles were therefore easier to track. MT bundles emanated from the anterior and the nucleus to point toward the posterior. MTs extended again along the entire cortex, after which they turned to the central cytoplasm. This, in turn, generated free MT (plus) ends in the center of the oocyte. By stage 9, the central MT network was clearly oriented along the oocyte AP axis. One or two thick MT bundles extended from the anterior, pointing toward the posterior pole. These bundles formed a structure resembling a horseshoe, with its open side facing the posterior. Importantly, both subsets could also be detected in living egg chambers, as shown for the DV subset and the AP subset. Thus, MTs show strong rearrangements throughout mid-oogenesis, which results in two perpendicular MT arrays reflecting the two axes of the oocyte (Januschke, 2006). An ex-vivo assay was developed to localize MT nucleation sites by dissecting ovaries and placing them on ice for 30 minutes. This treatment resulted in complete depolymerization of MTs. When allowed to recover at 25°C for 30 minutes, MT distribution could be re-established to the wild-type situation, in which both the cortical and the central subsets of MTs were detectable. gamma-Tubulin distribution was not affected by cold-induced MT depolymerization. When short periods of regrowth were analyzed, MT nucleation appeared limited to the close vicinity of the nucleus and was often asymmetric, suggesting a centrosome-associated nucleation activity. MT regrowth appeared to be stepwise, since after 15 minutes only the DV cortical subset was established. MTs clustered around the oocyte nucleus and aligned along the cortex in the DV direction. The cortical location of these fibers was clearly revealed by the presence of Khc-positive dots at either the dorsal or the ventral side. This indicates that the DV MT subset is the first to regrow. The regrowth experiment was repeated using colchicine. After the drug was washed out, MT repolymerization was observed at the oocyte nucleus. Taken together, these results indicate that, at least with the detection method that was used, the oocyte nucleus and its immediate surroundings have the capacity to nucleate MTs (Januschke, 2006).

To test whether the centrosome-nucleus complex could direct the repolarization of the MT network, how MTs distribute in grk mutant oocytes was examined. In this mutant, the nucleus frequently remains at the posterior of the oocyte due to a failure in the signaling cascade that induces the repolarization of the cytoskeleton. In grk mutant oocytes similar in size to wild-type stage 8, the MT distribution was dramatically affected. Specifically, MT organization appeared completely reversed compared with wild type, in which the nucleus is at the anterior and MT plus-ends are located toward the posterior at stage 8. In slightly older oocytes, MTs remain stretched out along the cortex from the posterior toward the anterior, where they fold back to the center of the oocyte. MT ends in the center are most probably plus-ends, since the pool of Khc (localized at the posterior of wild-type oocytes, co-localizes with Kinesin-ßGal to the center of the oocyte, between the flanking MT ends. Interestingly, MT distribution in grk oocytes was strikingly similar to MTs of wild-type egg chambers before the migration of the oocyte nucleus. Likewise, the centrosome, as revealed by gamma-tubulin, which is found at the posterior of stage 6 wild-type oocytes, stays at the posterior in grk mutants. Thus, in grk mutants, distribution of MT and MTOC seemed similar to their distribution in wild-type stage 6 (Januschke, 2006).

grk mutant oocytes, having mispositioned nuclei, provide an ideal basis to test the MT nucleating capacity of the centrosome-nucleus complex using the cold-shock assay. After cold-shock treatment of grk oocytes, complete MT depolymerization was checked for. As in the wild type, during the initial period of recovery at 25°C, MT polymerization took place only in the immediate vicinity of the mispositioned oocyte nucleus. Therefore, as in wild-type oocytes, MT nucleation is often asymmetric and restricted to the area surrounding the nucleus. This result strengthens the possibility that the centrosome-nucleus complex is an active MTOC (Januschke, 2006).

Thus, in the Drosophila oocyte a centriole-containing centrosome is present in close association with the nucleus, which itself is covered by PCM components until late in oogenesis. In addition, MTs can nucleate from this centrosome-nucleus complex. The MTs appear to form two orthogonal MT populations that develop through several steps during mid-oogenesis. It is proposed that the migration of the nucleus in the oocyte could control the reorganization of the MT network (Januschke, 2006).

In region 2 of the germarium, nurse cell centrosomes migrate toward the oocyte. Later, in region 3, these centriole-containing centrosomes become located as an aggregate between the oocyte nucleus and the follicle cell border. Pericentriolar material closely associated with the oocyte nucleus can be clearly detected until stage 6 with several centrosomal markers, such as gamma-tubulin, Centrosomin and D-Tacc. From stage 4 onward, the fate of the centriole cluster has been unknown. This study shows that both gammaTub37C and gammaTub23C are localized in a perinuclear manner throughout oogenesis. gammaTub37C highlights a discrete body close to the nucleus. This body is similarly detected by the centrosomal marker D-PLP and by a specific antibody for polyglutamylated Tubulin, which detects centrioles. Consistent with this, two to possibly four centrioles were detected in the immediate vicinity of the nucleus in stage 9 oocytes. This result demonstrates that at least until stage 9, a centriole-containing centrosome is present in the oocyte. Currently, it is not known whether they are still present at the onset of meiosis I during stage 13, since it has previously been proposed that the meiotic spindle is achieved without centrosomes. During skeletal muscle morphogenesis, myotube centrosomes dissociate from their nuclei, centrioles disappear and the centrosomal matrix is redistributed to the nucleus periphery. Similarly, during oogenesis, centrioles from nurse cell centrosomes may disappear. However, their pericentriolar material may relocate to the oocyte nucleus periphery. This would explain the specific enrichment of the oocyte nucleus with perinuclear MTOC material. The only centrosome remaining associated with a nucleus is that of the oocyte. Furthermore, the structure of this centrosome remains intact. It is concluded that the four centrioles found close to the nucleus in stage 9 may correspond to the initial oocyte centrosome in the duplication phase observed in G2 (Januschke, 2006).

Effects of Mutation or Deletion

Axonemes are ancient organelles that mediate motility of cilia and flagella in animals, plants, and protists. The long evolutionary conservation of axoneme architecture, a cylinder of nine doublet microtubules surrounding a central pair of singlet microtubules, suggests all motile axonemes may share common assembly mechanisms. Consistent with this, alpha- and ß-tubulins utilized in motile axonemes fall among the most conserved tubulin sequences, and the ß-tubulins contain a sequence motif at the same position in the carboxyl terminus. Axoneme doublet microtubules are initiated from the corresponding triplet microtubules of the basal body, but the large macromolecular 'central apparatus' that includes the central pair microtubules and associated structures is a specialization unique to motile axonemes. In Drosophila spermatogenesis, basal bodies and axonemes utilize the same alpha-tubulin but different ß-tubulins. ß1 is utilized for the centriole/basal body, and ß2 is utilized for the motile sperm tail axoneme. ß2 contains the motile axoneme-specific sequence motif, but ß1 does not. The 'axoneme motif' specifies the central pair. ß1 can provide partial function for axoneme assembly but cannot make the central microtubules. Introducing the axoneme motif into the ß1 carboxyl terminus, a two amino acid change, confers upon ß1 the ability to assemble 9 + 2 axonemes. This finding explains the conservation of the axoneme-specific sequence motif through 1.5 billion years of evolution (Nielsen, 2001).

Attempts were made to identify motile axoneme-specific sequences in Drosophila ß2 tubulin by constructing chimeric ß-tubulins in which selected residues in ß1 were changed to ß2 identity. Chimeras were expressed in ß2's normal domain in the postmitotic male germ cells and tested for their ability to support axoneme assembly. Previous experiments had showen that, when tested in such conditions, ß1 can not make a motile axoneme with central pair microtubules. Instead of the canonical wild-type 9 + 2 axoneme architecture, ß1-mediated axonemes have 9 + 0 architecture. Only 25 of 446 amino acids differ between ß1 and ß2; thus, small changes in primary structure must mediate ß2's ability to make motile 9 + 2 axonemes. The last 15 amino acids in the ß-tubulin protein comprise the highly variable carboxyl terminus, which has been identified as an isotype-defining region of the molecule and is important for axoneme morphogenesis. One third of the amino acid differences between ß1 and ß2 lie within the carboxyl terminus. Moreover, in the absence of the ß-tubulin carboxyl terminus, coherent axonemes can not be initiated at the basal body at all. Therefore, in the first chimera tested (ß1-ß2i), the ß1 carboxyl terminus was replaced in its entirety with that of ß2. ß1-ß2i supports assembly of 9 + 2 axonemes, demonstrating that the ß2 carboxyl terminus carries information sufficient for assembly of the central pair microtubules. This observation led the authors to test the specific role of the axoneme motif. Remarkably, introducing the axoneme motif into the ß1 carboxyl terminus -- a two amino acid change -- allowed ß1 to make 9 + 2 axonemes (ß1-ß2ii) (Nielsen, 2001).

Although ß1-ß2i and ß1-ß2ii support assembly of 9 + 2 axonemes, neither generate functional sperm. Axonemes assembled from these chimeras fail to maintain structural integrity for the full length of the sperm tail. There are thus two separable aspects of axoneme-specific function intrinsic to the Drosophila ß2 isoform. The axoneme motif specifies the central pair, but other features of the ß2 molecule are required for distal axoneme integrity. Comparison of the overall phenotypes of ß1-ß2i and ß1-ß2ii shows that distal axoneme structure is better in ß1-ß2i males, including retention of central pairs. In middle and distal cross sections, 16 of 34 intact axonemes in males with ß1-ß2i (as the sole source of ß-tubulin) had central pairs, but, in ß1-ß2ii males, only 10 of 71 intact axonemes retained central pairs. Thus, features in the ß2 carboxyl terminus other than the axoneme motif, as well as features of the ß2 molecule other than the carboxyl terminus, are required for axoneme structural integrity (Nielsen, 2001).

Additional changes were made in ß1-ß2i to test the function of internal ß2 residues in axoneme morphogenesis. The internal variable region (amino acids 55-57) was tested because previous work showed it to be important for morphology of axoneme doublet microtubules. Amino acid 349 was tested because the tubulin crystallographic structure revealed that this residue contacts the alpha subunit and cysteine occurs at this position only in Drosophila ß2, its identical D. hydeii homolog, and in the moth Heliothis virescens testis-specific ß-tubulin. Like ß1-ß2i, chimeras carrying internal ß2-specific residues are able to make each component of the normal 9 + 2 axoneme architecture but cannot maintain a complete sperm tail (Nielsen, 2001).

All of the ß1-ß2 chimeras are compatible with axoneme motility and full male fertility when they are coexpressed with ß2 and comprise 50% or less of the postmitotic ß-tubulin pool. However, coexpression of any of the chimeric ß-tubulins at a ratio of 2:1 with endogenous ß2, causes defects in axoneme morphogenesis and male sterility. Quantitation of axoneme phenotypes in 2:1 genotypes allowed subtle differences in the functional properties of the different chimeras to be distinguised. ß1-ß2i is most effective at maintaining axoneme structure, but the other chimeras exhibit differential capacity relative to ß1 for retention of the central pair microtubules (ß1-ß2i > ß1-ß2iv > ß1-ß2iii > ß1) and maintenance of the organization of the outer nine doublet microtubules (ß1-ß2i > ß1 > ß1-ß2iv > ß1-ß2iii). Thus, despite increasing identity to ß2, the internal changes introduced into ß1-ß2i decrease functionality in axoneme assembly. In addition, like ß1, the chimeric ß-tubulins cause insertion of additional doublets into the axoneme to generate 10-doublet axonemes, as well as assembly of ectopic cytoplasmic doublet microtubules. The ß1-ß2 proteins are thus truly chimeric -- they possess ß2-specific features required for correct initiation of 9 + 2 axonemes but, nonetheless, retain essential ß1-like features incompatible with ß2 function, reflected in the capacity for de novo generation of doublet microtubules (Nielsen, 2001).

The proportion of axoneme defects increases with distance from the basal body, indicating either that axonemes become progressively defective as they grow or that distal structure fails to be maintained after assembly. The possibility that ß2-containing dimers might be preferentially incorporated early in axoneme morphogenesis was considered. In this model, an increasing gradient of chimeric ß1-ß2 dimers would be generated along the length of the axoneme, corresponding to the progressive loss of distal structure observed. As the most stringent test of this hypothesis, immunolocalization of ß1 along the length of axonemes was examined in males with two copies of ß1 and one of ß2. ß1 is uniformly distributed. Thus, loss of distal axoneme integrity does not result from differential usage of tubulin heterodimers during assembly but more likely reflects accumulation of slight differences in axoneme geometry over the long length of the Drosophila sperm tail (Nielsen, 2001).

The axoneme motif most likely mediates central pair assembly through isotype-specific interactions with other proteins. The carboxyl terminus is a surface feature both in the dimer and in microtubules; thus, sequence changes are unlikely to influence tubulin function by changes in the 3D structure. The carboxyl terminus is a site both for MAP binding and for posttranslational modifications. For example, ß-tubulin carboxyl terminus polyglycylation is necessary for motility in Tetrahymena. The observation that altering internal ß1 residues to ß2 identity can decrease functionality argues that their normal function requires amino acid interactions that obtain only in the ß2 protein. Amino acid residues 55-57 as well as 349 are involved in interprotofilament contacts; an alteration in these contacts could potentially affect the geometry of the entire axoneme (Nielsen, 2001).

The data presented here show that specific changes in ß-tubulin residues produce discrete effects on axoneme morphogenesis. A deeply conserved feature of ß-tubulins used in motile axonemes, the axoneme motif, specifies an equally ancient structural feature, the central pair microtubules. In contrast, sequences in variable regions and other internal residues affect an evolutionarily labile feature, the length of the axoneme, and may have coevolved in ß2 to support the exceptionally long sperm tails within the genus Drosophila: from 2 mm in D. melanogaster up to the giant 5.8 cm in D. bifurca (Nielsen, 2001).

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