Polyclonal antibodies were generated against a EB1-GST fusion protein. The affinity-purified antibodies recognized a protein with a molecular weight of 31 kD on immunoblots of extracts from Drosophila embryos and Schneider (S2) tissue culture cells. The intracellular localization of Dm EB1 was examined in S2 cells. However, under routine growth conditions, these cells adopt a spherical morphology and possess a thin rim of cytoplasm that encircles the nucleus. As a result, S2 cells have been considered relatively poor for cytological examination. When these cells were examined by immunofluorescence, the microtubule cytoskeleton appeared as a dense basket-like network without visible organization; Dm EB1 also was difficult to visualize, but it clearly colocalized along short (1-2 µm) stretches of microtubules. The crowded packing of microtubules, however, made it difficult to discern unambiguously whether the colocalization corresponded to the microtubule plus ends (Rogers, 2002).

To improve the cytology of the S2 cells, various substrates were tested for their ability to promote cell adhesion and spreading. One of the substrates tested, concanavalin A, promoted S2 cell attachment to coverslips and caused them to adopt a flattened, discoid morphology (~20 µm in diameter) within 1-2 h. In these preparations, S2 cells elaborated a well-developed, radial interphase microtubule network with readily discernible tips extending toward the cell periphery. Because of the considerable improvement in cytology, this cell preparation was employed for subsequent examination of EB1 and microtubules (Rogers, 2002).

In concanavalin A-treated cells, EB1 staining clearly coincided with individual microtubules and exhibited a comet-like gradient of staining, with the greatest intensity at the most distal tip of the microtubule. During all stages of mitosis, EB1 also was localized at microtubule plus ends. Additionally, puncta of Dm EB1 staining were found at the duplicated centrosomes of prophase cells as they began to separate from one another. During metaphase, EB1 localization to the tips of astral microtubules was particularly prominent. In addition, as cells progressed to telophase, EB1 staining was enriched on the interpolar microtubule bundles that separated each chromosomal mass. The distribution of EB1 in S2 cells is, therefore, very similar to the localization that has been described in vertebrate cell lines (Morrison, 1998; Tirnauer, 1999; Mimori-Kiyosue, 2000b; Rogers, 2002 and references therein).

The distribution of EB1 was examined in synctitial blastoderm embryos. Consistent with observations in S2 cells, antibodies against Dm EB1 decorated the mitotic spindle and showed prominent staining of the spindle poles and astral microtubules. Embryos in late anaphase and telophase also showed a dramatic accumulation of EB1 staining on interpolar microtubule bundles and midbodies (Rogers, 2002).

EB1 is ubiquitiously expressed during Drosophila development. Total protein extracts were prepared from various developmental stages. To see the protein expression profile in different tissue types in adult flies, total protein samples were prepared from head, thorax, and abdomen of adult Drosophila. These body parts are enriched in neural cells, muscle cells, and visceral/reproductive cells, respectively, and therefore serve as populations of contrasting cell types. The immunoblots showed that EB1 protein is equally well expressed in all of the body parts. These results indicated that DmEB1 is expressed ubiquitously during development (Elliott, 2005).

The localization of EB1 was examined in the chordotonal sensory organs in wild-type flies. A pair of chordotonal organs was chosen located at the anterior ventral part of the abdomen because of its suitability for immunostaining. Fixed samples were immunostained with antibodies against DmEB1 andα-tubulin as well as a monoclonal antibody (mAb) 22C10, which recognizes a neuron-specific marker, the MAP1 homologue Futsch (Elliott, 2005).

Each sensory unit in the chordotonal organs is comprised of a monociliated neuron and support cells and aligned in parallel in a highly organized manner. Cap cell and ligament structures were strongly stained with the anti-α-tubulin antibody. EB1 is concentrated in the distal ends of cap cells, which are connected to body walls, and subregions of scolopale cells and ligament cells. Magnified images surrounding neuron dendrites revealed that EB1 was localized to the scolopale, spindle-shaped structure enclosing the cilia, and regions of cells surrounding the inner dendritic segment of the neurons. No significant signals were seen in cap structures or ciliary endings (Elliott, 2005).

Mechanisms for focusing mitotic spindle poles by minus end-directed motor proteins

During the formation of the metaphase spindle in animal somatic cells, kinetochore microtubule bundles (K fibers) are often disconnected from centrosomes, because they are released from centrosomes or directly generated from chromosomes. To create the tightly focused, diamond-shaped appearance of the bipolar spindle, K fibers need to be interconnected with centrosomal microtubules (C-MTs) by minus end-directed motor proteins. This study characterized the roles of two minus end-directed motors, dynein and Ncd, in such processes in Drosophila S2 cells using RNA interference and high resolution microscopy. Even though these two motors have overlapping functions, Ncd is primarily responsible for focusing K fibers, whereas dynein has a dominant function in transporting K fibers to the centrosomes. A novel localization of Ncd to the growing tips of C-MTs is reported, that is shown is mediated by the plus end-tracking protein, EB1. Computer modeling of the K fiber focusing process suggests that the plus end localization of Ncd could facilitate the capture and transport of K fibers along C-MTs. From these results and simulations, a model is proposed on how two minus end-directed motors cooperate to ensure spindle pole coalescence during mitosis (Goshima, 2005; full text of article).

EB1 is a highly conserved microtubule plus end-tracking protein that binds various cargo proteins (e.g., APC [adenomatous polyposis coli protein]). Ncd also was recently observed to bind to an EB1 affinity column (Rogers, 2004). It was therefore of interest investigate whether the plus end accumulation of Ncd-GFP is mediated by EB1. After EB1 depletion by RNAi in Ncd-GFP-NES cell line, no plus end accumulation of Ncd-GFP-NES was seen; instead the microtubules were evenly labeled with this protein. After EB1 RNAi, microtubules become less dynamic and frequently enter a pause state where they exhibit minimal growth or shrinkage. However, even the subset of growing microtubule never accumulated Ncd-GFP-NES at their tips (Goshima, 2005).

Next, an in vitro interaction between purified Ncd and EB1 was tested using a GST pull-down assay. It was found that the nonmotor 'tail' domain (aa 1-290) of Ncd can bind directly to the COOH terminus domain of EB1 (EB1-C; aa 208-278), albeit weakly. This binding was competed by addition of a fragment of human APC protein (2744-2843 aa) that binds to EB1's COOH-terminal domain, suggesting that the Ncd tail and APC bind to the same site on EB1. However, expression of the Ncd tail domain (1-290 aa) fused to GFP did not track along microtubule plus ends in vivo, suggesting that the motor domain may augment affinity for the microtubules and thereby aid plus end localization (Goshima, 2005).

No specific mutagenesis strategy was availabe for eliminating plus end tracking of Ncd while retaining its other critical mitotic activities such as microtubule cross-bridging. However, the consequences were tested of pole focusing and Ncd-GFP localization in mitosis after RNAi of EB1. Time-lapse imaging of mitotic EB1 RNAi cells showed no plus end microtubule enrichment, as expected from the observed interphase results. However, Ncd-GFP still strongly and dynamically (revealed by FRAP) localized to spindle MTs, indicating that K fiber binding does not require EB1. In this setting of EB1 RNAi where Ncd was mislocalized from microtubule plus ends but not the spindle, centrosome detachment and K fiber focusing was examined. Qualitative study of EB1 RNAi found both centrosome detachment and pole defocusing. When these phenotypes were examined quantitatively, it was found that the EB1 phenotype consists of pronounced K fiber defocusing and has less pronounced centrosome detachment, which is more similar to the Ncd RNAi than the dynein RNAi phenotype. Although not definitive proof because EB1 RNAi causes microtubule dynamics defects in addition to displacing Ncd from the microtubule plus end, this result is consistent with a functional link between EB1-dependent localization of Ncd to microtubule tips and K fiber coalescence (Goshima, 2005).

Based on live cell imaging and computer simulation analyses, it is proposed that the coalescence of spindle poles involves the following steps: (1) inter-K fiber cross-linking, (2) 'search and capture' of K fibers by the tip of growing C-MTs, and (3) K fiber transport on C-MTs. RNAi analysis of dynein and Ncd as well as live cell imaging of Ncd-GFP has provided insight into the roles of these minus end-directed motor proteins in these processes. By quantitatively comparing Ncd and dynein knockdown phenotypes in the same cell type, it was found that these Ncd and dynein have distinct but overlapping functions in the three steps of pole focusing (Goshima, 2005).

RNAi results suggest that Ncd plays a role in K fiber focusing through three mechanisms described above. Ncd's major role is likely to be in inter-K fiber cross-linking, as evidenced by the splaying of K fibers after Ncd RNAi and the prominent localization of Ncd-GFP to K fibers. This process most likely involves cross-linking of microtubules by Ncd's force generating motor domain and its positively charged 'tail' domain that also binds to microtubules independently of the motor. This process occurs in the absence of centrosomes and C-MTs, because acentrosomal spindles created by centrosomin RNAi also show severe K fiber unfocusing when Ncd is also knocked down by RNAi. It is also showm that the process of lateral K fiber interactions is highly dynamic, because K fibers are continually splaying and coalescing. Such observations are also consistent with FRAP measurements of Ncd-GFP, which show that these motors are associating and dissociating with K fibers on a rapid time scale and thus are not behaving as static cross-linkers. Albeit less efficient than dynein, Ncd also likely contributes to minus end-directed transport of K fibers along C-MTs, because the centrosome to K fiber distance is somewhat greater in the Ncd/Dhc64C double RNAi compared with Dhc64C alone. Finally, it is believed that Ncd at the tips of C-MTs may act to capture K fibers and facilitate subsequent minus end transport of the K fiber (Goshima, 2005).

Cytoplasmic dynein in S2 cells plays a dominant role in transporting K fibers along microtubules, as evidenced by finding that Dhc64C RNAi causes detachment of centrosomes from the minus ends of K fibers. Although secondary to Ncd, dynein also contributes to the focusing of the minus ends of K fibers. This role of dynein becomes particularly clear after Ncd depletion, since Ncd/Dhc64c double RNAi causes very severe splaying of K fibers. It is believed that this K fiber focusing effect also primarily involves dynein's role as a transporter of K fiber bundles along C-MTs, which causes the coalescence of most peripheral K fibers toward the centrosome as shown in computer simulations. However, dynein may have other roles in K fiber coalescence, such as potentially transporting and concentrating cross-linking proteins at minus ends of K fibers. Indeed, synthetic effects of kinesin-14 and dynein motors on pole focusing have been reported in centrosome-free spindles reconstituted in Xenopus egg extract (Goshima, 2005).

The molecular properties of kinesin-14/Ncd and cytoplasmic dynein are well designed to support the above proposed functions of these two motors in pole focusing. Cytoplasmic dynein is a fast, processive motor. Thus, small numbers of dyneins could rapidly transport K fibers along C-MTs. In contrast, Ncd is nonprocessive, slow motor that is not designed for cargo transport. Instead, its ability to bind two microtubules and its slow motor activity makes it an effective cross-bridger between microtubules in the spindle. These properties are likely to be advantageous for inter-K fiber cross-linking, as well as for crossbridging of C-MTs plus ends to K fiber. However, the rapid on-off rates of these cross-bridges, as shown by FRAP data, would still enable dynein to effectively transport the K fibers along the C-MTs (Goshima, 2005).

Phenotypic RNAi analyses may account for differences in the pole unfocusing phenotypes of Ncd or dynein depletions that have been described in the literature. Specifically, spindle architecture in the given system (e.g., the presence or absence of centrosome and differences in microtubule dynamics) may determine whether Ncd or dynein acts as the essential contributor to pole coalescence. For example, Ncd/kinesin-14 function, is particularly important for K fiber focusing, may become more crucial when centrosomes are detached or absent from the spindle. Consistent with this idea, the most dramatic pole unfocusing phenotypes for kinesin-14 mutations/depletions have been described in plant mitosis and animal meiosis, systems in which spindle assembly occurs through a centrosome-independent mechanism and in which interactions between C-MTs and K fibers are simply absent. However, dynein also is likely to play crucial roles in pole coalescence in some acentrosomal spindles, as shown convincingly in Xenopus extract system. In contrast, somatic animal cell mitosis utilizes centrosomes, and kinesin-14 is less important for pole focusing in such cells (e.g., Ncd is nonessential in fly development). Microtubule dynamics, specifically the relative number of K- to C-MTs in the bipolar spindle, also may alter the relative contribution of the two motors. For example, if C-MTs are very abundant, the high probability of close approximation of K- and C-MTs may enable dynein to easily link these two networks without any assistance from kinesin-14 motors at microtubule plus ends (Goshima, 2005).

An unexpected finding of this study is the microtubule plus end tracking of Ncd. Localization of a kinesin-14 motor protein to the plus end of interphase microtubules has been recently reported in plants. The accumulation of kinesin-14 at the plus end overlap zone in mitotic spindle has been shown but whether this localization reflects localization of plus ends of individual microtubules is not known. Nevertheless, this work does suggest that the plus end tracking in mitotic microtubules might be a broadly conserved feature of kinesin-14 motors. Yeast Kar3p also was shown to accumulate at the plus ends of microtubules at the shmoo, but it is more enriched on the depolymerizing microtubules, which is not observed for Ncd. Even though it was found that C-MTs are still dynamic after Ncd RNAi, it is possible that Ncd at the plus end also modulates microtubule dynamics, as does EB1 or other tip-localized proteins (Goshima, 2005).

The enhanced K fiber unfocusing in EB1 RNAi-treated cells, which displaces Ncd from plus ends but not K fibers, suggests that plus end tracking of Ncd may serve a function in pole focusing. It is proposed that plus end tracking of Ncd on newly nucleated C-MTs, as a 'capture factor,' facilitates their connection to K fibers, possibly using its second microtubule binding site located in its NH2-terminal tail domain. This idea is analogous to a 'search and capture' model for how C-MTs find chromosomes. In this case, the tip-localized motor Ncd enables C-MTs to 'search' for and then 'capture' a second major microtubule network in the spindle, the K fibers. Ncd may generate a connection between K fiber and C-MTs temporary, and thereby facilitate the recruitment of minus end-directed transporter (primarily dynein but Ncd contributing as well) for the transport of K fibers. Additionally, Ncd at the plus end may act as a K fiber transporter once it binds, although this transport would be less efficient than that produced by fast and processive dynein motors. It is also noted that the simulations are two-dimensional and encounters between C-MTs and K fibers would become less likely in three dimensions, and one might expect the effect of a C-MT-mediated capture/transport mechanism to become more important under such circumstances (Goshima, 2005).

The microtubule plus end search-and-capture mechanism might apply to other aspects of metazoan cell division. For example, cross-linking interactions between antiparallel microtubules occurs at overlap zone of microtubules, and genetic study demonstrates that Ncd produces an inward force on antiparallel microtubules during early mitosis. Ncd at the tips of growing microtubules may act to capture microtubules that arise from the opposite pole. Another possible target of tip-localized Ncd may be free microtubules, which are either released from centrosomes or generated de novo in cytoplasm and are eventually incorporated into the spindle by a dynein-dependent transport process (Goshima, 2005).

Adherens junctions inhibit asymmetric division in the Drosophila epithelium: EB1 homologs are required for the symmetric epithelial division along the planar axis

Asymmetric division is a fundamental mechanism for generating cellular diversity. In the central nervous system of Drosophila, neural progenitor cells called neuroblasts undergo asymmetric division along the apical-basal cellular axis. Neuroblasts originate from neuroepithelial cells, which are polarized along the apical-basal axis and divide symmetrically along the planar axis. The asymmetry of neuroblasts might arise from neuroblast-specific expression of the proteins required for asymmetric division. Alternatively, both neuroblasts and neuroepithelial cells could be capable of dividing asymmetrically, but in neuroepithelial cells other polarity cues might prevent asymmetric division. This study shows that by disrupting adherens junctions the symmetric epithelial division can be converted into asymmetric division. It was further confirmed that the adenomatous polyposis coli (APC) tumour suppressor protein is recruited to adherens junctions, and demonstrated that both APC and microtubule-associated EB1 homologs are required for the symmetric epithelial division along the planar axis. These results indicate that neuroepithelial cells have all the necessary components to execute asymmetric division, but that this pathway is normally overridden by the planar polarity cue provided by adherens junctions (Lu, 2001).

Drosophila neuroblasts delaminate from a polarized epithelial layer in the ventral neuroectoderm and divide asymmetrically along the apical-basal axis to produce larger apical neuroblasts and smaller basal ganglion mother cells. Previous studies identified Inscuteable (Insc) as a central protein in organizing neuroblast division. Insc provides positional information that couples mitotic spindle orientation with the basal localization of cell-fate determinants such as Numb and Prospero together with their respective adaptor proteins Partner of Numb (Pon) and Miranda (Lu, 2001).

The apical localization of Insc involves both a Baz-dependent initiation step and a maintenance step that requires Baz and Partner of Inscuteable (Pins). The expression of Baz and Pins in both neuroblasts and neuroepithelial cells suggests that these cells share certain apical-basal polarity information. Consistent with this notion is the observation that, when Pon is expressed ectopically in epithelial cells it is localized to the basal cortex, as in neuroblasts. Unlike neuroblasts, however, epithelial cells divide symmetrically along the planar axis and segregate ectopic Pon equally between the two daughter cells. These observations raise further questions: do epithelial cells have the ability to couple spindle orientation with protein localization, and segregate proteins asymmetrically between two unequally sized daughter cells? If so, what prevents them from executing this asymmetric division (Lu, 2001)?

To characterize epithelial division by monitoring it in live embryos, transgenic embryos were used expressing Pon and tau proteins fused with green fluorescent protein (GFP). During epithelial cell cycle, tau-GFP-labelled mitotic spindle is formed along the planar axis of the embryo, and Pon-GFP is initially uniformly associated with the cortex and then localized to a basal crescent. The mitotic spindle remains orientated along the planar axis throughout mitosis. After cytokinesis, the Pon-GFP crescent is bisected by the cleavage furrow and is equally distributed between two equally sized daughter cells. This in vivo analysis shows that the machinery for basal protein localization is intact in epithelial cells, but it is uncoupled from spindle orientation (Lu, 2001).

The uncoupling of spindle orientation with asymmetric protein localization in epithelial cells might be due to either a lack of such a coupling mechanism or the dominance of the coupling mechanism by yet another spindle-positioning mechanism. One of the hallmarks of epithelial cells is the adherens junction, which is composed of the cadherin-catenin complex and other associated proteins, is connected to the cytoskeleton, and is thought to be important in maintaining the planar organization of the epithelial monolayer. Therefore the possible role of adherens junction in orientating epithelial division was tested. The formation of adherens junction requires genes such as shotgun, crumbs (crb) and stardust. RNA interference (RNAi) was used to disrupt Crb function and analysed the effect on epithelial division (Lu, 2001).

Double-stranded (ds) crb RNA was injected into transgenic embryos expressing Pon-GFP and tau-GFP. In about 70% of crb(RNAi) embryos, the organization of the ectodermal epithelium was disrupted, with epithelial cells losing their columnar shape, adopting rounded morphology, and becoming separated from each other. Live imaging of epithelial divisions in these embryos revealed that nearly all the epithelial cells show a tight coupling between the positioning of Pon-GFP crescents and the orientation of the mitotic spindle. Pon-GFP crescents were found at basal and lateral positions and less frequently at apical positions on the cell cortex, and one of the spindle poles was positioned underneath the Pon-GFP crescent (Lu, 2001).

After cytokinesis, Pon-GFP was segregated to one of the two similarly sized daughter cells. Asymmetric segregation of Pon-GFP to one of two similarly sized daughter cells was also observed in crb zygotic mutant embryos. Immunostaining of crb(RNAi) embryos with antibodies against Asense, Prospero and Insc indicated that epithelial cells do not express these neuronal markers, suggesting that the ability of these cells to undergo asymmetric division is not a result of cell-fate change (Lu, 2001).

Overexpression of the membrane-bound cytoplasmic tail of Crb (Crb-intra) causes similar disorganization of the epithelium as seen in crb mutants. Therefore the effect of overexpressing Crb-intra on epithelial division was examined. As observed in crb(RNAi) embryos, epithelial cells overexpressing Crb-intra showed coupling of the mitotic spindle with the Pon-GFP crescent and asymmetric segregation of Pon-GFP to one of the daughter cells. Thus, when the formation of the adherens junction is disrupted, epithelial cells switch from a symmetric to an asymmetric division pattern (Lu, 2001).

In addition to its function in localizing Insc and regulating division axis in the neuroblasts, Baz is also required for the formation of adherens junction and the maintenance of epithelial polarity. Nextthe function of Baz in epithelial division was investigated. The baz(RNAi) embryos showed overall disruption of epithelium organization similar to that observed in crb(RNAi) embryos. Unlike in crb(RNAi) embryos, however, epithelial cells in baz(RNAi) embryos divide in a symmetric fashion, with Pon-GFP distributed uniformly around the cell cortex throughout mitosis and the mitotic spindle orientated in random directions. After cytokinesis, two equally sized daughter cells were produced and Pon-GFP was equally distributed between them (Lu, 2001).

Daughter cell size asymmetry in neuroblast division is largely unaffected in baz(RNAi) embryos. It was also observed that in crb(RNAi) epithelial cells Baz can still be localized into a crescent but the crescent is mispositioned and that Pon-GFP is always localized to the opposite side of the Baz crescent. This suggests that, although mispositioned, Baz is still functional in directing Pon-GFP localization in crb(RNAi) embryos. To test whether the coupling of Pon-GFP localization with spindle orientation observed in crb(RNAi) embryos is Baz dependent, double RNAi was performed by co-injecting a mixture of baz and crb dsRNAs. Epithelial divisions in the co-injected embryos looked similar to baz single-injected embryos, with Pon-GFP segregated equally between two equally sized daughter cells. It is therefore concluded that epithelial cells depend on Baz to couple spindle orientation with protein localization when the adherens junction is disrupted (Lu, 2001).

To investigate the molecular mechanism underlying the planar positioning of spindles by the adherens junction, the function of proteins associated with the adherens junction was examined. A ubiquitously expressed, epithelial-cell-enriched APC (E-APC) is localized to the adherens junction, and, in shotgun and crb mutants, this adherens junction localization of E-APC is disrupted. The human APC protein interacts with a microtubule-associated EB1 protein, and the yeast homologue of EB1 (Bim1), together with the cortical marker Kar9, has been implicated in a search-and-capture mechanism of spindle positioning. Therefore the function of E-APC in epithelial cell division was tested (Lu, 2001).

In about 60 of the E-APC(RNAi) embryos, the positioning of Pon-GFP crescent and orientation of mitotic spindle became tightly coupled during epithelial division. At cytokinesis, epithelial cells divided asymmetrically to produce two unequally sized daughter cells, and Pon-GFP was always segregated to the smaller daughter cell. The asymmetric segregation of Pon-GFP and the ability to undergo unequal cytokinesis all depend on Baz, because in baz and E-APC double RNAi embryos, Pon-GFP is equally segregated to two similarly sized daughter cells . Therefore, in the absence of E-APC, epithelial cells divide asymmetrically in a Baz-dependent fashion. This suggests that adherens-junction-associated E-APC promotes spindle positioning along the planar axis and prevents the coupling of spindle positioning with asymmetric basal protein localization (Lu, 2001).

To test whether E-APC functions with EB1 to orientate the mitotic spindle, RNAi was performed on a closely related fly homologue of EB1 (dEB1 ). In dEB1(RNAi) embryos, the epithelial divisions were also asymmetric, producing two unequally sized daughter cells, with Pon-GFP segregated to the smaller cell. It was observed that the penetrance of dEB1(RNAi) phenotype (~20%) is lower than that of E-APC(RNAi). Since there is strong maternal contribution of dEB1, the low penetrance might be due to a perdurance of maternal dEB1 protein. Alternatively, it might be due to functional compensation by two other distantly related EB1 homologues in the fly genome. It has been noted that E-APC lacks the carboxy-terminal domain that is required for interaction with EB1, and no direct interaction was observed between E-APC and EB1 in in vitro binding assays. It therefore remains to be determined whether the two are functionally linked together in vivo through some cofactor(s), or whether E-APC functions mainly to maintain adherens junction integrity and EB1 interacts with other unidentified molecules to orientate spindles (Lu, 2001).

These results indicate that two sets of polarity cues exist for spindle positioning in epithelial cells: a planar polarity cue mediated by the adherens junction and an apical-basal polarity cue regulated by Baz. The division pattern of wild-type epithelial cells suggests that the planar polarity cue is normally dominant over the apical-basal polarity cue. Epithelial cells within the procephalic neurogenic region (PNR) that express endogenous Insc or epithelial cells outside of the PNR that express ectopic Insc are known to orientate their mitotic spindle along the apical-basal axis during division. This suggests that the dominance of planar polarity over apical-basal polarity can be overcome by the expression of Insc. The normal appearance of the adherens junction in epithelial cells in the PNR, together with the observation that these cells divide along the planar axis and maintain their normal monolayer organization in insc mutant, suggests that Insc functions by strengthening the apical-basal polarity instead of weakening the planar polarity through changing the behaviour of the adherens junction (Lu, 2001).

When neuroblasts delaminate from the epithelium layer, they undergo morphological changes from columnar to round shape, lose their contacts with the surrounding cells and thus the adherens junction structures. This situation may be reminiscent of epithelial cells in adherens-junction mutants in which the planar polarity cue is lost. In both cases, the Baz-mediated polarity pathway takes over. That one polarity cue can dominate over another cue in orientating division axis may have its precedents in other organisms. Budding yeast can divide in either an axial or a bipolar pattern. Mutations in genes such as AXL1, BUD3, BUD4 and BUD10/AXL2 result in loss of polarity cue for axial bud formation and the cells divide in a bipolar fashion. This suggests that axial and bipolar cues coexist and that the axial cue is normally dominant over the bipolar cue. During mammalian cortical neurogenesis, neural progenitors switch from early symmetric divisions to later asymmetric divisions. It will be interesting to determine whether similar mechanisms and molecules are used to control this division symmetry switch in mammals. These results on E-APC highlights the importance of tumour suppressors in regulating not only cell growth but also polarity and asymmetric division (Lu, 2001).

A functional link between localized Oskar, dynamic microtubules, and endocytosis

Many cell types including developing oocytes, fibroblasts, epithelia and neurons use mRNA localization as a means to establish polarity. The Drosophila oocyte has served as a useful model in dissecting the mechanism of mRNA localization. The polarity of the oocyte is established by the specific localization of three critical mRNAs - oskar, bicoid and gurken. The localization of these mRNAs requires microtubule integrity, and the activity of microtubule motors. However, the precise organization of the oocyte microtubule cytoskeleton remains an open question. In order to examine the polarity of oocyte microtubules, the localization of canonical microtubule plus end binding proteins, EB1 and CLIP-190, was visualized. Both proteins were enriched at the posterior of the oocyte, with additional foci detected within the oocyte cytoplasm and along the cortex. Surprisingly, however, it was found that this asymmetric distribution of EB1 and CLIP-190 was not essential for oskar mRNA localization. However, Oskar protein was required for recruiting the plus end binding proteins to the oocyte posterior. Lastly, these results suggest that the enrichment of growing microtubules at the posterior pole functions to promote high levels of endocytosis in this region of the cell. Thus, multiple polarity-determining pathways are functionally linked in the Drosophila oocytes (Sanghavi, 2012).

Although posterior EB1 and CLIP-190 are not essential for oskar mRNA localization, Oskar protein is required for recruiting EB1 and CLIP-190 to the posterior pole. The mechanism by which Oskar performs this function is less clear. Oskar protein has been shown to induce the formation of long F-actin fibers at the posterior of the oocyte. It was therefore hypothesized that these actin fibers might recruit growing microtubule plus ends. Consistent with this hypothesis, treatment of ovaries with Lat-A resulted in loss of EB1 and CLIP-190 foci from the posterior pole. Under these treatment conditions however, Oskar protein was still detected at the posterior. This suggests that EB1 and CLIP-190 recruitment to the posterior pole occurs downstream of Oskar protein localization and requires integrity of the actin cytoskeleton. How might actin function in recruiting EB1 and CLIP-190 to the posterior pole? One possibility is via the activity of cross-linking proteins that have been shown to bind microtubule plus ends as well actin. Another possibility is that the actin network might be required for polarizing oocyte microtubules. Consistent with this notion, mutations in the actin nucleators, capu and spire, result in oocytes that lack microtubule polarity (Sanghavi, 2012).

The results also suggest that Oskar is not sufficient for recruiting EB1 and CLIP-190. Ectopic expression of Oskar protein along the entire oocyte cortex or within a central focus failed to efficiently recruit EB1 and CLIP-190. It is therefore likely that multiple factors function to recruit these plus end binding proteins to the posterior pole. Curiously, however, mis-expression of Oskar reduced the posterior accumulation of EB1. Posterior localization of CLIP-190 was also affected under these conditions, but to a lesser degree. The reason for this phenotype is unclear, but it nonetheless suggests that mis-expression of Oskar affects the dynamics of microtubules within the oocyte (Sanghavi, 2012).

In contrast to EB1 and CLIP-190, the ectopic Oskar focus contained high levels of Khc. A similar result was also observed by Z using Kin:βgal as a reporter. At present it is unclear whether Khc and Kin:βgal localization are truly indicative of microtubule plus ends. In addition to the ectopic Oskar focus, Khc was found in the center of par-1 mutant oocytes, but EB1 and CLIP-190 were not. Similarly, Khc, but not EB1 and CLIP-190, was localized to the posterior in oskar protein-null mutants. As mentioned previously, Khc and Kin:βgal might label a population of microtubules that are polarized, yet relatively static in vivo. Further work is needed to explore this possibility (Sanghavi, 2012).

It has been demonstrated that endocytosis within the oocyte is asymmetric, occurring at a higher level at the posterior). Oskar protein is required for maintaining this polarized distribution of endocytosis. The mechanism by which Oskar performs this function, however, is largely unknown (Sanghavi, 2012).

The current findings indicate that growing microtubule plus ends are recruited to the oocyte posterior by Oskar. In this regard, it is interesting to note that CLIP-170, the vertebrate homolog of CLIP-190, was initially identified as a protein that directly bound to endocytic vesicles as well as microtubule plus ends. More recently, CLIP-170 was also shown to function at microtubule plus ends to stimulate phagocytosis in macrophage cells and to promote the capture and minus end transport of melanophores in Xenopus). These findings highlight the importance of microtubule plus ends and CLIP-170 in vesicle transport (Sanghavi, 2012).

Based on these observations, it is hypothesized that Oskar promotes endocytosis by recruiting microtubule plus end binding proteins to the posterior pole. Consistent with this hypothesis, EB1 and CLIP-190 localization, as well as endocytosis was drastically reduced at the oocyte posterior in oskar protein-null and staufen mutant. Treatment of egg chambers with a microtubule-stabilizing drug also had a similar effect on microtubule plus end localization and endocytosis. These results suggest an intimate relationship between dynamic microtubules and efficient endocytosis at the posterior pole. One possibility is that the dynamic microtubules themselves are important for posterior endocytosis. Alternatively, EB1 and CLIP-190 might deliver factors to the posterior that facilitate high endocytic activity (Sanghavi, 2012).

These results indicate that oocyte microtubules are polarized, with an enrichment of plus ends at the posterior pole. The results also suggest that the primarily function of the polarized microtubule network is to promote high endocytic activity at the oocyte posterior (Sanghavi, 2012).

Effects of Mutation or Depletion

RNAi and antibody depletion of EB1

To gain insight into the cellular functions of EB1, whether RNAi depletion of EB1 affected microtubule organization was examined by fluorescence microscopy. Six days of dsRNA treatment was sufficient to reduce EB1 protein to very low levels. When plated on concanavalin A-coated coverslips, EB1 dsRNA-treated cells attached and spread as well as control cells and displayed no obvious morphological abnormalities. Tubulin staining revealed that the interphase microtubule organization in these cells was indistinguishable from controls (Rogers, 2002).

To probe the effects of EB1 depletion more carefully, live cell fluorescence microscopy was used to observe microtubule behavior in control and EB1 dsRNA-treated S2 cells transfected with GFP-tubulin. Microtubules in untreated control cells exhibited dynamic instability, asynchronously transiting between phases of elongation and shrinkage. The rates of microtubule growth and shrinkage were 3.8 ± 0.9 µm/min and 8.7 ± 2.8 µm/min, respectively. The rate of catastrophe (the switch from growth, or pause to shrinkage) was 0.021 transitions per second, whereas the rate of rescue was 0.029 transitions from shrinkage (or pause) to growth per second. The populations of microtubules in control cells spent, on average, 55.2% of the time in growth, 27.5% of the time in shrinkage, and 17.3% of the time in a paused state (neither growing nor shrinking). These parameters of dynamic instability measured for S2 cell microtubules are similar to those measured in other cell types using GFP-tagged tubulin, with velocities of growth and shrinkage intermediate to those measured in mammalian cells and yeast (Tirnauer, 1999; Yvon, 1999; Rusan, 2001). These results represent the first direct measurements of microtubule dynamic instability in Drosophila cells (Rogers, 2002).

Microtubule behavior was very different in cells depleted of EB1 by dsRNA. Rates of microtubule growth (3.7 ± 1.0 µm/min) and shrinkage (8.6 ± 3.1 µm/min) were similar compared with untreated control cells. However, the frequencies of catastrophe in EB1-depleted cells were approximately threefold lower compared with control cells. The most notable effect of EB1 depletion was that microtubules spent the majority (55.2%) of their lifetimes in a paused state relative to growth (30%) or shrinkage (9.3%). These results indicate that EB1 promotes microtubule dynamics in Drosophila cells. The effects of EB1 RNAi on microtubule dynamics in S2 cells are qualitatively similar to interphase microtubule behavior observed in bim1Delta S. cerevisiae as reported by Tirnauer (1999). In both cases, microtubule catastrophe and rescue frequencies were decreased in the absence of EB1/Bim1p and microtubules spent the majority of their lifetimes in a state of pause (Rogers, 2002).

Given the role of EB1 family members in mitosis in yeast, how RNAi inhibition of EB1 expression affected mitosis was examined in Drosophila cells. Mitosis in untreated or GFP dsRNA-treated cells progressed in a very reproducible manner. At prophase, the two spindle poles were in close proximity to condensing chromosomes and always nucleated asters of long, radial microtubules. As the cells proceed to prometaphase, the spindles assumed a typical bipolar organization and chromosomes are positioned between each pole. At this stage, and for all successive stages, bipolar spindles nucleate highly developed radial arrays of astral microtubules, many of which extended to the cell cortex. The chromosomes congressed to the metaphase plate, and subsequently migrated to the spindle poles during anaphase and telophase, the two incipient cells assumed a more rounded shape (Rogers, 2002).

In cells lacking EB1, defects in microtubule organization are readily apparent. During preprophase, EB1-deficient cells duplicate centrosomes normally and the two centrosomes migrated to opposite sides of the nucleus as in control cells. At this stage, each centrosome nucleated a normal radial array of long microtubules that extended toward the cell periphery. However, when dsRNA-treated cells progress to prophase, the long cytoplasmic microtubules disappear, and instead, only very short (<1 µm) astral microtubules are observed clustered around the two poles. Short microtubule fragments unattached to the poles are often present in the cytoplasm of EB1-deficient cells. These phenotypes are observed in 74% of the EB1-deficient prophase cells examined, but were never observed in untreated or GFP RNAi control cells. From these observations, it is concluded that EB1 is required for stabilizing microtubules and creating astral arrays in mitosis (Rogers, 2002).

The loss of EB1 also produced aberrant spindle phenotypes in metaphase cells that could be classified into four general categories. The most common defect was a complete loss of astral microtubules. These spindles maintain their bipolar symmetry, but commonly exhibit detachment of centrosomes from the spindle. The second class of defects (observed in 33% of the cells) lacked astral microtubules and exhibited an overall compaction of the spindle into a basket-like meshwork of microtubules surrounding the chromosomes. In these structures, the poles could not be clearly distinguished, but mitotic chromosomes maintained their position at the center of the spindle. The third type of defect was a detachment of a spindle pole from the bundles of microtubules that were connected to the kinetochores. These spindles exhibited a 'splayed' morphology. The fourth category of defect was 'barrel-shaped' spindles that maintained their symmetry, but failed to focus the microtubules at the poles and also lacked astral microtubules. These phenotypes did not appear to be due to gross centrosome defects, as immunofluorescent staining with antibodies against centrosomin protein revealed spindle poles to be present and intact. In all four classes of defective spindles, the distance from pole to pole was significantly smaller (5.4 ± 1.1 µm) than in GFP dsRNA-treated cells (7.7 ± 0.9 µm). These results indicate that EB1 plays a critical role during spindle assembly (Rogers, 2002).

The mitotic defects observed in the EB1 RNAi-treated cells were severe enough that it was thought that they might affect cell cycle progression by activating the spindle checkpoint. To test this possibility, fixed cells were stained for DNA and the number of cells with mitotic figures was scored as a percentage of the entire cell population. In EB1 dsRNA-treated cultures, the mitotic index was 5.9%, approximately double that of control cultures at 2.7%. Although significant, this difference was not as dramatic as might be expected if mitotic progression were completely blocked. If the mitotic checkpoint were activated for prolonged periods of time, an increase in apoptotic cells might be expected. However, S2 cells exhibit macrophage-like properties, and it was observed that they consume their apoptotic neighbors, as judged by nuclear morphology. This property of S2 cells could give rise to artificially low mitotic index measurements. To determine at which stage of the cell cycle mitotic progression is interrupted, all of the mitotic cells in these samples were categorized according to their stage of mitosis. In control-treated cultures, cells appeared to spend approximately the same amount of time in each stage of mitosis. In EB1-depleted cells, however, there is an accumulation at metaphase (~40% compared with 22% in controls) and in telophase (~43% compared with 38% in controls). These data suggest that inhibition of EB1 activates the spindle checkpoint. Further work will be required to understand potential checkpoint activation in response to loss of EB1 function, perhaps by live cell imaging (Rogers, 2002).

During normal mitosis, the mitotic spindle positions itself at the geometric center of the cell. In S2 cells lacking EB1, however, the spindle is frequently mispositioned. To quantitate this effect, the spindle center was determined by measuring the distance between the two poles in cells that had their mitotic spindle aligned parallel to the coverslip. Then, the centroid of the cell ws calculated and the offset distance between the cell centroid and the spindle center was determined for untreated cells and for EB1- and GFP RNAi-treated cells. In untreated and GFP dsRNA-treated cells, the average offset distances between the centroid and the spindle center were 0.42 µm ± 0.17 and 0.35 µm ± 0.15, respectively. In contrast, the average offset distance in EB1-deficient cells was significantly greater, 1.93 µm ± 0.57. From these data, it is concluded that loss of EB1 function causes mispositioning of the mitotic spindle (Rogers, 2002).

Although the Schneider cell system provided a convenient method to generate loss-of-function phenotypes for EB1, extended live cell imaging of the spindle proved technically difficult. To study the role of EB1 in spindle dynamics, the Drosophila synctitial blastoderm was used as a model, because thousands of synchronous spindles that divide within a two-dimensional plane can be readily observed by confocal microscopy. Furthermore, because preblastoderm embryos are not governed by the same mitotic checkpoint mechanisms as differentiated cells, the later stages of mitosis can be observed after treatments that might otherwise induce mitotic arrest by activation of the spindle checkpoint. To study the role of EB1 in spindle dynamics in vivo, anti-EB1 antibodies were microinjected into transgenic preblastoderm embryos expressing GFP-tubulin. Using time-lapse spinning disk confocal microscopy, the effects were observed of EB1 inhibition on mitotic spindle formation, spindle elongation, and chromosomal segregation (Rogers, 2002).

In control embryos, dynamics of the mitotic spindles followed a well-characterized, documented progression. During interphase of cycle 12, duplicated centrosomes move to opposite sides of the nucleus to positions separated by ~120°. Upon entry into prometaphase, the nuclear envelope break down and the nuclear space is invaded by microtubules emanating from opposite poles. These microtubules form attachments either with chromosomes to form kinetochore fibers or intercalate with microtubules of opposite polarity to form interpolar bundles. A few minutes after chromosomes congress to the metaphase plate, the spindles transits to anaphase and sister chromosomes segregate to opposite poles to complete mitosis. The pole-to-pole distances are highly reproducible in spindles throughout cycle 12. After nuclear envelope breakdown of cycle 12, the length of the spindle is ~8 µm. As the cells progress to metaphase, spindles elongate at a rate of ~0.03 µm/s until reaching a separation of ~12 µm. Upon anaphase onset, spindles further elongate at a rate ~0.07 µm/s until reaching a maximal length of ~16.5 µm. These measurements are in close agreement with a previous description of Drosophila embryo spindle dynamics (Rogers, 2002).

To investigate whether EB1 plays a role in mitosis in this system, EB1 antibodies were microinjected into Drosophila embryos expressing GFP-tubulin. The injected antibodies produced a readily apparent gradient of effects on spindle structure and behavior, with the most severe defects centered around the injection site. As these embryos approach cycle 12 mitosis, it becames readily apparent that the spindles closest to the injection site have fewer microtubules and have a shorter pole-to-pole distance than controls. This phenotype is similar to that produced by RNAi of EB1 in S2 cells. Given the general similarity in phenotypes observed with antibody microinjection and RNAi, it is believed that the antibodies are manifesting their effects through EB1. However, the possibility cannot be excluded of effects manifest through other polypeptides (e.g., the weakly reactive 75-kD polypeptide observed after long exposure of immunoblots). The exact mechanism of the antibody-induced defect is not known, although the similarity to the RNAi phenotype makes gives rise to the suspicition that the antibody injection leads to a loss of EB1 function (Rogers, 2002).

Spindles further from the injection site exhibited a pole-to-pole distance that more closely resembled controls, but also frequently displayed structural defects such as frayed and monopolar half spindles that had both centrosomes present at a single pole. Observation of these defects over time revealed that spindle structure was dynamic and these frayed and monopolar spindles could sometimes correct themselves and complete mitosis. Regions of these embryos distal to the injection site supported formation of morphologically normal spindles that progress through mitosis similar to controls (Rogers, 2002).

The effects of EB1 inhibition on spindle elongation were quantitated by measuring the pole-to-pole distances of spindles proximal to the injection site over time. During the prophase-to-metaphase transition, spindles elongate twofold slower (~0.015 µm/s) and achieve a shorter length (8.1 ± 0.5 µm) at metaphase. At anaphase, spindles elongate threefold slower (~0.01 µm/s) and elongate to a maximal length 40% less than controls (9.2 ± 0.6 µm). In addition to reduced rates of elongation, spindles proximal to the injection site exhibit a striking overall reduction in associated microtubules and fail to form normal interpolar microtubule bundles or a midbody at the end of anaphase (Rogers, 2002).

If interference with EB1 activity disrupts normal spindle elongation at anaphase, it was speculated that proper chromosome segregation could be affected as well. To test this hypothesis, EB1 antibodies and rhodamine-labeled histones were coinjected into embryos expressing GFP-tubulin to simultaneously observe the behaviors of chromosomes and microtubules. In control embryos, fluorescent histones incorporated into chromatin and allowed observation of chromosome condensation at prometaphase, chromosome congression to the metaphase plate, and sister chromatid separation and segregation to each pole during anaphase. Injection of EB1 antibodies, however, disrupts chromosome segregation and produces a range of phenotypes. The mildest defect caused by EB1 antibody injection was the generation of lagging chromosomes during anaphase (30%). More deleterious effects are produced when the chromosomes begin to segregate but fail during anaphase, producing bilobed (8.6%) or stretched (31%) chromosomal masses that fail to segregate and decondense midway between the poles at the end of mitosis. The most extreme defect observed is complete inhibition of chromosomal segregation, leading to the formation of a tetraploid nucleus in between two spindle poles. Taken together, these results from microinjecting anti-EB1 antibodies into preblastoderm embryos indicate that EB1 plays a crucial role in mitotic spindle formation and elongation and is needed for the proper segregation of mitotic chromosomes during anaphase (Rogers, 2002).

EB1 is essential during Drosophila development and plays a crucial role in the integrity of chordotonal mechanosensory organs

EB1 is a conserved microtubule plus end tracking protein considered to play crucial roles in microtubule organization and the interaction of microtubules with the cell cortex. Despite intense studies carried out in yeast and cultured cells, the role of EB1 in multicellular systems remains to be elucidated. This study describes the first genetic study of EB1 in developing animals. One of the multiple Drosophila EB1 homologues, DmEB1, is ubiquitously expressed and has essential functions during development. Hypomorphic DmEB1 mutants show neuromuscular defects, including flightlessness and uncoordinated movement, without any general cell division defects. These defects can be partly explained by the malfunction of the chordotonal mechanosensory organs. In fact, electrophysiological measurements indicated that the auditory chordotonal organs show a reduced response to sound stimuli. The internal organization of the chordotonal organs also is affected in the mutant. Consistently, DmEB1 is enriched in those regions important for the structure and function of the organs. Therefore, DmEB1 plays a crucial role in the functional and structural integrity of the chordotonal mechanosensory organs in Drosophila (Elliott, 2005; Full text of article).

In this study, Drosophila is used as a model system to examine in vivo roles of one of the EB1 homologues in a developmental context. In Drosophila, proteins encoded by up to six genes share some homology to EB1 in the N-terminal region (microtubule binding domain. The similarity of three of them to EB1 also is extended to the C-terminal region. Among them, Drosophila EB1 is the most similar to human EB1. This study shows that the Drosophila EB1 protein is ubiquitously expressed during Drosophila development. Other EB1 homologues also are expressed during development, although their expression is rather limited. Three mutant alleles of Drosophila EB1 were identified, one of which is lethal and the other two are semilethal. Transgenic wild-type EB1 genes expressed from the ubiquitin promotor fully rescues these DmEB1 mutants. Therefore, the DmEB1 gene has a unique and essential function that is not complemented by the other homologues during Drosophila development. Inability to complement the DmEB1 defects could be due to differences in expression or protein activity of the other EB1 homologues (Elliott, 2005).

Previous studies using RNA interference (RNAi) or antibody injection indicated that DmEB1 is required for the regulation of microtubule dynamics and spindle organization/positioning. Therefore, it is rather puzzling at first sight that the EB1 mutants showed limited defects without significant abnormalities in universal cell functions. However, it should be emphasized that the EB1 mutations examined in this study are hypomorphic and a low level of active protein produced in the mutants may be sufficient to support spindle formation. Moreover, EB1 protein has a large maternal contribution that may mask the requirement in early development. It is also possible that other EB1 homologues may have redundant functions that compensate for loss of EB1 in Drosophila. Therefore, it is likely that the phenotype observed in EB1 mutants represents a subset of EB1 functions in vivo. Nevertheless, the hypomorphic mutations uncovered the neuromuscular involvement of EB1 without interference by more universal cellular roles (Elliott, 2005).

Although the EB1 alleles do not show significant cell division or morphological defects, studies on semilethal alleles of EB1 revealed defective neuromuscular functions. These adult flies are flightless and hold their wings in the wrong position when resting. Moreover, they show uncoordinated body movements when correcting the body position after being overturned. Consistently, rare escapers of a stronger allele show much more severe body coordination defects (Elliott, 2005).

These behavioral phenotypes of DmEB1 mutants share some characteristics with the phenotypes of mutants defective in chordotonal sensory organ function. Chordotonal organs are mechanosensory organs that act as stretch receptors and also form the fly's auditory systems. DmEB1P mutant is indeed defective in the function of auditory chordotonal organs. Therefore, malfunction of chordotonal organs can, in part, explain the EB1 mutant defects. However, mutants completely lacking chordotonal organs show better viability than the DmEB1P homozygotes, indicating that the EB1 mutants have additional, unidentified defects (Elliott, 2005).

Mechanotransduction typically requires intact mechanical linkages between stimulus-delivering structures and neuronal transducing elements. Each sensory unit of a chordotonal organ is comprised of one or a few ciliated neurons and several supporting cells, including cap cells, ligament cells, and scolopale cells. The central structure is the scolopale, a spindle-shaped structure encasing a sensory cilium. Sensory cilia are connected at their tips to the extracellular dendritic cap, at their base to a ciliary rootlet, and via transcellular attachments to the actin-based rods of the scolopale. These structures are connected to other body structures through cap cells and neurons/ligament cells. Disruption of any of these linkages could result in a reduced response (Elliott, 2005).

The immunolocalization study of EB1 indicated that, rather than EB1 distributing uniformly to all cells in the chordotonal organs, it is concentrated at high levels in subregions of supporting cells in the chordotonal organs. These coincide with structurally important regions for chordotonal functions. They include (1) anterior ends of cap cells that are in contact with the body walls, (2) scolopale region, (3) regions of cells surrounding the inner segment of neuron dendrites, and (4) part of the ligament cells. These cell structures probably require a high level of EB1 and are sensitive to a reduction of the protein level in the mutant, although all cells are likely to express and require it at some level. It also should be noted that EB1 does not have significant concentration at ciliary endings, although in Chlamydomonas (Pedersen, 2003) the EB1 homologue is localized to the flagellar tip and implicated in intraflagellar transport in (Elliott, 2005).

Immunostaining of mutant chordotonal organs in the abdomen revealed that, although the overall arrangement of the chordotonal organs and the number of cells are not altered, the alignment of the dendrites is disrupted. This misalignment is often associated with unusually stretched dendrite morphology. Electron microscopy of mutant chordotonal organs revealed ultrastructures with normal morphology, such as cilia, dendritic cap, scolopale structures, basal bodies, and ciliary rootlets. Comparing the mutant phenotype with the immunolocalization results, one possibility is that this misalignment may represent an underlying weakness of cell structures such as the area supporting inner segment of neuron dendrites. In addition, the defects of other EB1-rich regions may well contribute functional and structural defects of chordotonal organs in the mutant (Elliott, 2005).

How then does the loss of EB1 cause the defects in function and organization of chordotonal organs at the molecular level? RNAi in Drosophila revealed that EB1 plays roles in regulating the mitotic spindle organization and spindle positioning/orientation. Any defects in chromosome segregation may lead to a failure to produce the sufficient number of cells comprising the chordotonal organs. Any defects in spindle positioning or orientation disrupt asymmetric divisions and result in a failure of correct fate determination among cells comprising chordotonal organs. However, no general cell division defects or significant alteration of the cell numbers were observed in chordotonal organs of the mutant (Elliott, 2005).

Instead, observations on mutant chordotonal organs are consistent with roles of EB1 on microtubule cytoskeleton. DmEB1 and homologues in other organisms are shown to regulate interphase microtubule dynamics and thought to be involved in interactions between microtubule plus ends and the cell cortex. Cells comprising chordotonal organs are highly polarized and have specialized cytoskeletons both molecularly and morphologically. Furthermore, chordotonal organs require robust mechanical linkages between stimulus-delivering structures and neuronal transducing elements to sense tensions effectively (Elliott, 2005).

The actin-microtubule linker Short stop, which localizes to the cell cortex, interacts with EB1 and is shown to be required for chordotonal organ integrity. The chordotonal phenotype of the short stop mutant is similar to defects seen in the DmEB1 mutant. Therefore, it is possible that DmEB1 may link microtubules to the cell cortex via interactions with Short stop. Although the linkage between microtubules and the cell cortex is important for most cell types, chordotonal organs are highly polarized and are required to withstand strong mechanical forces; therefore, they may be particularly sensitive to the reduction in EB1 activity. Immunofluorescence microscopy using antibodies against α-tubulin and Futsch (the MAP1 homologue) or electron microscopy could not resolve the precise defect of the cytoskeleton. Further studies are required to understand exact roles of DmEB1 in chordotonal organ function (Elliott, 2005).

Directed microtubule growth, +TIPs, and kinesin-2 are required for uniform microtubule polarity in dendrites

In many differentiated cells, microtubules are organized into polarized noncentrosomal arrays, yet few mechanisms that control these arrays have been identified. For example, mechanisms that maintain microtubule polarity in the face of constant remodeling by dynamic instability are not known. Drosophila neurons contain uniform-polarity minus-end-out microtubules in dendrites, which are often highly branched. Because undirected microtubule growth through dendrite branch points jeopardizes uniform microtubule polarity, this system was used to understand how cells can maintain dynamic arrays of polarized microtubules. It was found that growing microtubules navigate dendrite branch points by turning the same way, toward the cell body, 98% of the time and that growing microtubules track along stable microtubules toward their plus ends. Using RNAi and genetic approaches, it was shown that kinesin-2, and the +TIPS EB1 and APC, are required for uniform dendrite microtubule polarity. Moreover, the protein-protein interactions and localization of Apc2-GFP and Apc-RFP to branch points suggests that these proteins work together at dendrite branches. The functional importance of this polarity mechanism is demonstrated by the failure of neurons with reduced kinesin-2 to regenerate an axon from a dendrite. It is concluded that microtubule growth is directed at dendrite branch points and that kinesin-2, APC, and EB1 are likely to play a role in this process. It is proposed that kinesin-2 is recruited to growing microtubules by +TIPS and that the motor protein steers growing microtubules at branch points. This represents a newly discovered mechanism for maintaining polarized arrays of microtubules (Mattie, 2010).

Using Drosophila dendrites as a model system, this study demonstrates that growing microtubule plus ends almost always turn toward the cell body at branch points and that they track stable microtubules through branches. Kinesin-2, EB1, and APC are all required for maintaining microtubule polarity and are linked in an interaction network (Mattie, 2010).

On the basis of these results, a model for directed growth of microtubules in dendrites is proposed. Apc2 most likely contains a branch localization signal because Apc2-GFP localizes well to dendrite branches even when overexpressed. Localization of Apc2 to dendrite branch points can recruit Apc to the branch. Apc can interact with the Kap3 subunit of kinesin-2 and so could increase concentration of the motor near the branch. EB1-GFP does not concentrate at branches, therefore it is proposed that a growing microtubule plus end coated with EB1 is transiently linked, through the interaction between Apc and the EB1 tail, to kinesin-2 as it passes through the branch. SxIP motifs in Apc and Klp68D could also contribute to this interaction. Because both Kap3 and the SxIP motif in Klp68D are in the kinesin-2 tail, the motor domain would be free to walk along a nearby stable microtubule toward the plus end and cell body (Mattie, 2010).

Even a very brief application of force pulling the growing microtubule toward the cell body should be sufficient to steer growth toward the cell body. Once the tip of the microtubule turns, growth should be constrained by the dendrite walls. The association of the growing plus end with a stable microtubule would probably only need to be maintained over a distance of a micron. This model is consistent with the observations that kinesin-2 has shorter run lengths than kinesin-1 and that individual EB1 interactions with the microtubule plus end persist for less than a second (Mattie, 2010).

Observations of plus-end behavior in vivo also favor a model in which only transient interactions of the motor and microtubule plus end are involved; frequently plus ends are seen turning sharply. Stable microtubules do not accommodate such sharp turns, so the sharp turns of plus ends are not likely to occur while the plus end is tracking along a stable microtubule. Instead, they probably represent a switch from a freely growing plus end to one that is following a track (Mattie, 2010).

Directed growth of microtubules at dendrite branch points allows dendrites to maintain uniform minus-end-out polarity despite continued microtubule remodeling. This mechanism is also probably necessary to establish uniform microtubule polarity in branched dendrites, but it probably cannot account for minus-end-out polarity on its own. It is hypothesized that directed microtubule growth is used in concert with an unidentified mechanism to control microtubule polarity in dendrites. Transport of oriented microtubule pieces has been proposed to contribute to axon microtubule polarity, and a similar mechanism could play a role in dendrites. For example, a kinesin anchored to the cell cortex by its C terminus could shuttle minus-end-out microtubule seeds into dendrites. Alternatively, polarized microtubule nucleation sites could be localized within dendrites. Identifying directed growth as one mechanism that contributes to dendrite microtubule polarity should facilitate identification of the missing pieces of this puzzle (Mattie, 2010).

Because kinesin-2, APC, EB1, and polarized microtubules are found in many cell types, directed growth of microtubules along stable microtubule tracks could be very broadly used for maintaining microtubule polarity. This is a newly identified function for both the +TIP proteins and kinesin-2. APC has previously been localized to junctions between a microtubule tip and the side of another microtubule at the cortex of epithelial cells, and sliding of microtubule tips along the side of microtubules was also seen in this system, but there was no association with a particular direction of movement or overall microtubule polarity (Mattie, 2010).

Kinesin-2 has previously been shown to be enriched in the tips of growing axons in cultured mammalian neurons, and it is possible that they could mediate tracking of growing microtubules along existing microtubule tracks in the growth cone. In fact, this type of microtubule tracking behavior has been observed in axonal growth cones under low-actin conditions. Thus, directed growth of microtubules could also be important during axon outgrowth. Indeed, the same principle of directed growth by a motor protein connected to a growing microtubule plus end could be involved in aligning microtubules in many circumstances (Mattie, 2010).


Search PubMed for articles about Drosophila Eb1

Alves-Silva, J., et al. (2012). Spectraplakins promote microtubule-mediated axonal growth by functioning as structural microtubule-associated proteins and EB1-dependent +TIPs (tip interacting proteins). J. Neurosci. 32(27): 9143-58. PubMed Citation: 22764224

Applewhite, D. A., et al. (2010). The spectraplakin Short stop is an actin-microtubule cross-linker that contributes to organization of the microtubule network. Mol. Biol. Cell 21(10): 1714-24. PubMed Citation: 20335501

Askham, J. M., Vaughan, K. T., Goodson, H. V. and Morrison, E. E. (2002). Evidence that an interaction between EB1 and p150(Glued) is required for the formation and maintenance of a radial microtubule array anchored at the centrosome. Mol. Biol. Cell 13: 3627-3645. Medline abstract: 12388762

Banerjee, B., Kestner, C. A. and Stukenberg, P. T. (2014). EB1 enables spindle microtubules to regulate centromeric recruitment of Aurora B. J Cell Biol 204: 947-963. PubMed ID: 24616220

Beinhauer, J. D., et al. (1997). Mal3, the fission yeast homologue of the human APC-interacting protein EB-1 is required for microtubule integrity and the maintenance of cell form. J. Cell Biol. 139: 717-728. Medline abstract: 9348288

Berrueta, L., et al. (1999). The APC-associated protein EB1 associates with components of the dynactin complex and cytoplasmic dynein intermediate chain. Curr. Biol. 9: 425-428. Medline abstract: 10226031

Bloom, K. 2000. It's a kar9ochore to capture microtubules. Nat. Cell Biol. 2: E96-E98. Medline abstract: 10854334

Bouissou, A., Verollet, C., de Forges, H., Haren, L., Bellafche, Y., Perez, F., Merdes, A. and Raynaud-Messina, B. (2014). γ-Tubulin ring complexes and EB1 play antagonistic roles in microtubule dynamics and spindle positioning. EMBO J 33: 114-128. PubMed ID: 24421324

Browning, H. and Hackney, D. D. (2005). The EB1 homolog Mal3 stimulates the ATPase of the kinesin Tea2 by recruiting it to the microtubule. J. Biol. Chem. 280: 12299-12304. Medline abstract: 15665379

Busch, K. E., Hayles, J., Nurse, P. and Brunner, D. (2004a). Tea2p kinesin is involved in spatial microtubule organization by transporting tip1p on microtubules. Dev. Cell 6(6): 831-43. Medline abstract: 15177031

Busch, K. E. and Brunner, D. (2004b). The microtubule plus-end-tracking proteins mal3p and tip1p cooperate for cell-end targeting of interphase microtubules. Curr. Biol. 14: 548-559. Medline abstract: 15062095

Currie, J. D., et al. (2011). The microtubule lattice and plus-end association of Drosophila mini spindles is spatially regulated to fine-tune microtubule dynamics. Mol. Biol. Cell 22(22): 4343-61. PubMed Citation: 21965297

Cuschieri, L., Miller, R. and Vogel, J. (2006). Gamma-tubulin is required for proper recruitment and assembly of Kar9-Bim1 complexes in budding yeast. Mol. Biol. Cell 17(10): 4420-34. Medline abstract: 16899509

Dzhindzhev, N. S., Rogers, S. L., Vale, R. D. and Ohkura, H. (2005). Distinct mechanisms govern the localisation of Drosophila CLIP-190 to unattached kinetochores and microtubule plus-ends. J. Cell Sci. 118(Pt 16): 3781-90. Medline abstract: 16105886

Elliott, S. L., Cullen, C. F., Wrobel, N., Kernan, M. J. and Ohkura, H. (2005). EB1 is essential during Drosophila development and plays a crucial role in the integrity of chordotonal mechanosensory organs. Mol. Biol. Cell 16(2): 891-901. Medline abstract: 15591130

Goshima, G., Nedelec, F. and Vale, R. D. (2005). Mechanisms for focusing mitotic spindle poles by minus end-directed motor proteins. J. Cell Biol. 171(2): 229-40. Medline abstract: 16247025

Green, R. A., Wollman, R., Kaplan, K. B. (2005). APC and EB1 function together in mitosis to regulate spindle dynamics and chromosome alignment. Mol. Biol. Cell 16: 4609-4622. Medline abstract: 16030254

Gu, C., Zhou, W., Puthenveedu, M. A., Xu, M., Jan, Y. N. and Jan, L. Y. (2006). The microtubule plus-end tracking protein EB1 is required for Kv1 voltage-gated K+ channel axonal targeting. Neuron 52(5): 803-16. Medline abstract: 17145502

Hayashi, I. and Ikura, M. (2003). Crystal structure of the amino-terminal microtubule-binding domain of end-binding protein 1 (EB1). J. Biol. Chem. 278: 36430-36434. Medline abstract: 12857735

Hayashi, I., Wilde, A., Mal, T. K. and Ikura, M. (2005). Structural basis for the activation of microtubule assembly by the EB1 and p150Glued complex. Mol. Cell 19: 449-460. Medline abstract: 16109370

Heinemann, S. H. and Hoshi, T. (2006). Multifunctional potassium channels: electrical switches and redox enzymes, all in one. Sci. STKE pe33. Medline abstract: 16940439

Honnappa, W., et al. (2005). Structural insights into the EB1-APC interaction. EMBO J. 24: 261-269. Medline abstract: 15616574

Hwang, E., Kusch, J., Barral, Y., and Huffaker, T. C. (2003). Spindle orientation in Saccharomyces cerevisiae depends on the transport of microtubule ends along polarized actin cables. J. Cell Biol. 161: 483-488. Medline abstract: 12743102

Li, W., Moriwaki, T., Tani, T., Watanabe, T., Kaibuchi, K. and Goshima, G. (2012). Reconstitution of dynamic microtubules with Drosophila XMAP215, EB1, and Sentin. J Cell Biol 199: 849-862. PubMed ID: 23185033

Juwana, J. P., (1999). EB/RP gene family encodes tubulin binding proteins. Int. J. Cancer. 81: 275-284. Medline abstract: 10188731

Kerres, A., Vietmeier-Decker, C., Ortiz, J., Karig, I., Beuter, C., Hegemann, J., Lechner, J. and Fleig, U. (2004). The fission yeast kinetochore component Spc7 associates with the EB1 family member Mal3 and is required for kinetochore-spindle association. Mol. Biol. Cell 15: 5255-5267. Medline abstract: 15371542

Kita, K., Wittmann, T., Nathke, I. S., Waterman-Storer, C. M. (2006). Adenomatous polyposis coli on microtubule plus ends in cell extensions can promote microtubule net growth with or without EB1. Mol. Biol. Cell 17: 2331-2345. Medline abstract: 16525027

Komarova, Y., Lansbergen, G., Galjart, N., Grosveld, F., Borisy, G. G. and Akhmanova, A. (2005). EB1 and EB3 control CLIP dissociation from the ends of growing microtubules. Mol. Biol. Cell 16: 5334-5345. Medline abstract: 16148041

Korinek, W. S., et al. (2000). Molecular linkage underlying microtubule orientation toward cortical sites in yeast. Science 287: 2257-2259. Medline abstract: 10731146

Kronja, I., et al. (2009). XMAP215-EB1 interaction is required for proper spindle assembly and chromosome segregation in Xenopus egg extract. Mol. Biol. Cell. 20: 2684-2696. PubMed Citation: 19369422

Lee, L., et al. (2000). Positioning of the mitotic spindle by a cortical-microtubule capture mechanism. Science 287: 2260-2262. Medline abstract: 10731147

Li, W., et al. (2011). EB1 promotes microtubule dynamics by recruiting Sentin in Drosophila cells. J. Cell Biol. 193(6): 973-83. PubMed Citation: 21646401

Ligon, L. A., Shelly, S. S., Tokito, M., Holzbaur, E. L. (2003). The microtubule plus-end proteins EB1 and Dynactin have differential effects on microtubule polymerization. Mol. Biol. Cell 14: 1405-1417. Medline abstract: 12686597

Louie, R. K., Bahmanyar, S., Siemers, K. A., Votin, V., Chang, P., Stearns, T., Nelson, W. J., Barth, A. I. M. (2004). Adenomatous polyposis coli and EB1 localize in close proximity of the mother centriole and EB1 is a functional component of centrosomes. J. Cell Sci. 117: 1117-1128. Medline abstract: 14970257

Lu, B., Roegiers, F., Jan, L. and Jan, Y. (2001). Adherens junctions inhibit asymmetric division in the Drosophila epithelium. Nature 409: 522-525. Medline abstract: 11206549

Mattie, F. J., et al. (2010). Directed microtubule growth, +TIPs, and kinesin-2 are required for uniform microtubule polarity in dendrites. Curr. Biol. 20(24): 2169-77. PubMed Citation: 21145742

Meireles, A. M., Dzhindzhev, N. S. and Ohkura, H. (2011). Kebab: kinetochore and EB1 associated basic protein that dynamically changes its localisation during Drosophila mitosis. PLoS One 6(9): e24174. PubMed Citation: 21912673

Mennella, V., et al. (2005). Functionally distinct kinesin-13 family members cooperate to regulate microtubule dynamics during interphase. Nat. Cell Biol. 7(3): 235-45. Medline abstract: 15723056

Miller, R.K., Cheng, S. C. and Rose, M. D. (2000). Bim1p/Yeb1p mediates the Kar9p-dependent cortical attachment of cytoplasmic microtubules. Mol. Biol. Cell. 11: 2949-2959. Medline abstract: 10982392

Mimori-Kiyosue, Y., Shiina, N. and Tsukita, S. (2000a). Adenomatous polyposis coli (APC) protein moves along microtubules and concentrates at their growing ends in epithelial cells. J. Cell Biol. 148: 505-518. Medline abstract: 10662776

Mimori-Kiyosue, Y., Shiina, N. and Tsukita, S. (2000b). The dynamic behavior of the APC-binding protein EB1 on the distal ends of microtubules. Curr. Biol. 10: 865-868. Medline abstract: 10899006

Morrison, E. E., et al. (1998). EB1, a protein which interacts with the APC tumor suppressor, is associated with the microtubule cytoskeleton throughout the cell cycle. Oncogene 17: 3471-3477. Medline abstract: 10030671

Moseley, J. B., et al. (2007). Regulated binding of adenomatous polyposis coli protein to actin. J. Biol. Chem. 282(17): 12661-8. Medline abstract: 17293347

Muhua, L., et al. (1998). A cytokinesis checkpoint requiring the yeast homologue of an APC- binding protein. Nature 393: 487-491. Medline abstract: 9624007

Nakata, T. and Hirokawa, N. (2003). Microtubules provide directional cues for polarized axonal transport through interaction with kinesin motor head. J. Cell Biol. 162: 1045-1055. Medline abstract: 12975348

Pedersen, L. B., Geimer, S., Sloboda, R. D., and Rosenbaum, J. L. (2003). The Microtubule plus end-tracking protein EB1 is localized to the flagellar tip and basal bodies in Chlamydomonas reinhardtii. Curr. Biol. 13: 1969-1974. Medline abstract: 14614822

Peth, A., Boettcher, J. P. and Dubiel, W. (2007). Ubiquitin-dependent proteolysis of the microtubule end-binding protein 1, EB1, is controlled by the COP9 signalosome: possible consequences for microtubule filament stability. J. Mol. Biol. 368(2): 550-63. Medline abstract: 17350042

Rehberg, M. and Graf, R. (2002). Dictyostelium EB1 is a genuine centrosomal component required for proper spindle formation. Mol. Biol. Cell 13(7): 2301-10. Medline abstract: 12134070

Rogers, S. L., Rogers, G. C., Sharp, D. J. and Vale, R. D. (2002). Drosophila EB1 is important for proper assembly, dynamics, and positioning of the mitotic spindle. J. Cell Biol. 158(5): 873-84. Medline abstract: 12213835

Rogers, S. L., Wiedemann, U., Hacker, U., Turck, C. and Vale, R. D. (2004). Drosophila RhoGEF2 associates with microtubule plus ends in an EB1-dependent manner. Curr. Biol. 14(20): 1827-33. Medline abstract: 15498490

Sandblad, L., et al. (2006). The Schizosaccharomyces pombe EB1 homolog Mal3p binds and stabilizes the microtubule lattice seam. Cell 127(7): 1415-24. Medline abstract: 17190604

Sanghavi, P., Lu, S. and Gonsalvez, G. B. (2012). A functional link between localized Oskar, dynamic microtubules, and endocytosis. Dev Biol 367: 66-77. PubMed ID: 22561189

Schroder, J. M., Schneider, L., Christensen, S. T. and Pedersen, L. B. (2007). EB1 is required for primary cilia assembly in fibroblasts. Curr. Biol. 17(13): 1134-9. Medline abstract: 17600711

Schuyler, S. C. and Pellman, D. (2001). Microtubule 'plus-end-tracking proteins': the end is just the beginning. Cell 105: 421-424. Medline abstract: 11371339

Shaw, R. M., et al. (2007). Microtubule plus-end-tracking proteins target gap junctions directly from the cell interior to adherens junctions. Curr. Biol. 16: 564-569. Medline abstract: 17289573

Slep, K. C., Rogers, S. L., Elliott, S. L., Ohkura, H., Kolodziej, P. A. and Vale, R. D. (2005). Structural determinants for EB1-mediated recruitment of APC and spectraplakins to the microtubule plus-end. J. Cell Biol. 168: 587-598. Medline abstract: 15699215

Slep, K. C. and Vale, R. D. (2007). Structural basis of microtubule plus end tracking by XMAP215, CLIP-170, and EB1. Mol. Cell 27(6): 976-91. Medline abstract: 17889670

Su, L. K., et al. (1995). APC binds to the novel protein EB1. Cancer Res. 55: 2972-2977. Medline abstract: 7606712

Su, L. K., and Qi, Y. (2001). Characterization of human MAPRE genes and their proteins. Genomics 71: 142-149. Medline abstract: 11161807

Subramanian, A., et al. (2003). Shortstop recruits EB1/APC1 and promotes microtubule assembly at the muscle-tendon junction. Curr. Biol. 13: 1086-1095. Medline abstract: 12842007

Tirnauer, J. S., et al. (1999). Yeast Bim1p promotes the G1-specific dynamics of microtubules. J. Cell Biol. 145: 993-1007. Medline abstract: 10352017

Tirnauer, J. S. and Bierer, B. E. (2000). EB1 proteins regulate microtubule dynamics, cell polarity, and chromosome stability. J. Cell Biol. 149: 761-766. Medline abstract: 10811817

Tirnauer, J. S., Grego, S., Salmon, E. D., and Mitchison, T. J. (2002a). EB1-microtubule interactions in Xenopus egg extracts: role of EB1 in microtubule stabilization and mechanisms of targeting to microtubules. Mol. Biol. Cell 13: 3614-3626. Medline abstract: 12388761

Tirnauer, J. S., Canman, J. C., Salmon, E. D., and Mitchison, T. J. (2002b). EB1 targets to kinetochores with attached, polymerizing microtubules. Mol. Biol. Cell 13; 4308-4316. Medline abstract: 12475954

Toyoshima, F. and Nishida. E. (2007). Integrin-mediated adhesion orients the spindle parallel to the substratum in an EB1- and myosin X-dependent manner. EMBO J. 26(6): 1487-98. Medline abstract: 17318179

van der Vaart, B., et al. (2011). SLAIN2 links microtubule plus end-tracking proteins and controls microtubule growth in interphase J. Cell Biol. 193: 1083-1099. PubMed Citation: 21646404

Vaughan, K. T. (2005). TIP maker and TIP marker; EB1 as a master controller of microtubule plus ends. J. Cell Biol. 171: 197-200. Medline abstract: 16247021

Wang, S., Reuveny, A. and Volk, T. (2015). Nesprin provides elastic properties to muscle nuclei by cooperating with spectraplakin and EB1. J Cell Biol 209: 529-538. PubMed ID: 26008743

Watson, P., Stephens, D. J. (2006). Microtubule plus-end loading of p150Glued is mediated by EB1 and CLIP-170 but is not required for intracellular membrane traffic in mammalian cells. J. Cell Sci. 119: 2758-2767. Medline abstract: 16772339

Wen, Y., et al. (2004). EB1 and APC bind to mDia to stabilize microtubules downstream of Rho and promote cell migration. Nat. Cell Biol. 6: 820-830. Medline abstract: 15311282

Widlund, P. O., et al. (2011). XMAP215 polymerase activity is built by combining multiple tubulin-binding TOG domains and a basic lattice-binding region. Proc. Natl. Acad. Sci. 108(7): 2741-6. PubMed Citation: 21282620

Wu, X. S., Tsan, G. L., Hammer, J. A. (2005). Melanophilin and myosin Va track the microtubule plus end on EB1. J. Cell Biol. 171: 201-207. Medline abstract: 16247022

Wu, X., Kodama, A. and Fuchs, E. (2008). ACF7 regulates cytoskeletal-focal adhesion dynamics and migration and has ATPase activity. Cell 135: 137-148. PubMed Citation: 18854161

Yin, H., Pruyne, D., Huffaker, T. C. and Bretscher, A. (2000). Myosin V orientates the mitotic spindle in yeast. Nature 406: 1013-1015. Medline abstract: 10984058

Eb1: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation

date revised: 15 June 2015

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