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Vol. 16, Issue 2, 891-901, February 2005
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* Wellcome Trust Centre for Cell Biology, Institute of Cell Biology, School of Biological Sciences, The University of Edinburgh, Edinburgh EH9 3JR, United Kingdom;
Department of Neurobiology and Behavior, The State University of New York at Stony Brook, Stony Brook, NY 11794
Submitted July 27, 2004;
Revised November 22, 2004;
Accepted November 28, 2004
Monitoring Editor: Lawrence Goldstein
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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A number of microtubule-associated proteins (MAPs) and motors have been shown to modify the behavior of microtubules (Cassimeris and Spittle, 2001
). An interesting class of MAPs are the plus end tracking proteins that preferentially bind to plus ends of growing microtubules (for review, see Schuyler and Pellman, 2001). Microtubule plus ends interact with kinetochores or particular regions of the cell cortex. A "search and capture" mechanism is proposed to achieve such interactions, which involves selective stabilization of the microtubules contacting kinetochores or specific molecules at the cell cortex (Kirschner and Mitchison, 1986). Motor-guided mechanisms also may be involved (Yin et al., 2000; Hwang et al., 2003). Molecular mechanisms of interactions between dynamic microtubule ends and kinetochores/the cell cortex are still mysterious, but plus end tracking proteins are considered to play a central role in this process.
One of the most studied family of plus end tracking proteins is the EB1 family. EB1 was first identified as an interacting protein of the adenomatous polyposis coli tumor suppressor protein (Su et al., 1995). EB1 homologues are present in all eukaryotes and preferentially localize to the plus ends of growing microtubules (Tirnauer et al., 1999; Mimori-Kiyosue et al., 2000; Rehberg and Gräf, 2002; Rogers et al., 2002). In mammalian cells, EB1 has been reported to interact with components of the dynein/dynactin complex (Berrueta et al., 1999). RNA interference of a Drosophila homologue, DmEB1, has shown that it affects the dynamics of interphase microtubules, as well as the organization and positioning/orientation of the mitotic spindle (Lu et al., 2001; Rogers et al., 2002). A study using Xenopus egg extracts has described EB1 as an antipause, anticatastrophe factor (Tirnauer et al. 2002a). In addition, a role for EB1 in microtubule–kinetochore attachment has been suggested (Rehberg and Gräf, 2002; Tirnauer et al., 2002b). Studies in budding yeast provide significant functional and mechanistic insights into EB1, which indicate critical roles in the spindle orientation by connecting an astral microtubule to the cell cortex. (Schwartz et al., 1997; Tirnauer et al., 1999; Korinek et al., 2000; Lee et al., 2000; Miller et al., 2000; Hwang et al., 2003; Liakopoulos et al., 2003).
Interactions between microtubules and the cell cortex are considered to be particularly important during development. However, the roles of EB1 in development have not been well investigated. In this study, we examine the role of EB1 in flies, particularly in the chordotonal sensory organs. A chordotonal sensory organ detects a relative position of body parts by acting as an internal stretch sensor as well as making up auditory receptors in flies (Eberl et al., 2000). Chordotonal organs belong to type I mechnoreceptors, which have monodendritic, ciliated neurons associated with supporting cells (Jarman, 2002). These neurons and supporting cells are highly polarized and contain specialized cytoskeletal systems (Dettman et al., 2001), but only limited information is available on the molecular basis of the cell architecture in this sensory organ.
Here, we report the first genetic study of EB1 in higher eukaryotes by using the fruit fly Drosophila melanogaster to elucidate its role in developing organisms. We have found that one of EB1 homologues, DmEB1, has an essential function during development. Loss of DmEB1 disrupts body coordination and compromises the function and structure of the chordotonal sensory organs.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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) were followed throughout. All stocks and crosses were grown at 25°C in cornmeal media. w1118 was used as wild type. Details of balancers and mutations are described in Lindsley and Zimm (1992) or FlyBase (The FlyBase Consortium, 2003). l(2)04524 (DmEB1P) was obtained from The Bloomington Stock Center (Indianapolis, IN). DmEB12 and DmEB15 were a generous gift from John Roote (Cambridge University, Cambridge, United Kingdom) and were studied over DmEB1P or each other because the chromosomes seem to have unrelated background lethal mutations. These mutations were kept over CyO or In(2LR)Gla, Bc Elp. Wild-type DmEB1 cDNA was cloned into a transformation vector (pUb) under the control of the ubiquitin promotor. The resulting plasmid was used for germline transformation. Transgenes on X and the third chromosome were used for rescue experiments. DmEB1P homozygote carrying one copy of the transgene produced completely viable, fertile, healthy adult flies without behavioral defects.
DNA Manipulation, Protein Preparation, and Immunoblots
Standard molecular techniques (Sambrook et al., 1989; Harlow and Lane, 1988) were followed throughout. Mutation sites in DmEB12 and DmEB15 were determined by direct sequencing of DmEB1 coding regions amplified from mutant genomic DNAs by polymerase chain reaction. Microtubule sedimentation experiments were carried out as described previously (Cullen et al., 1999). A total protein sample from each stage or adult body part of Drosophila was prepared as described previously (Cullen et al., 1999). The DmEB1 antibody used for these studies was generated at Diagnostic Scotland (Midlothian, United Kingdom) by immunization of a rabbit with full-length DmEB1 fused with glutathione S-transferase made in bacteria. The specificity of the antibody against Drosophila extract was confirmed by immunoblots.
Cytological Analysis
Immunostaining of fly tissues was carried out as described in Cullen et al. (1999) and Cullen and Ohkura (2001). Methanol fixation was used for embryos and nonactivated oocytes, and formaldehyde was used for other tissues. Samples were examined and images were collected using an Axioplan 2 microscope (Carl Zeiss, Thornwood, NY) attached with a charge-coupled device camera (Hamamatsu, Bridgewater, NJ) or confocal microscopes TCS (Leica Microsystems, Deerfield, IL) or LSM5.10 (Carl Zeiss). Figures were prepared using Photoshop (Adobe Systems, Mountain View, CA).
Preparation of abdominal chordotonal organs for immunostaining was carried out as follows. Pharate adults were used within 1 d after wings become darkened, to obtain wild-type and DmEB1P homozygotes of roughly the equivalent stage. These pharate adults were dissected from pupae cases and fixed with 3.4% of formaldehyde in phosphate-buffered saline immediately after the abdomen was cut open. Internal tissues were removed and subjected for immunostaining.
Various primary antibodies used in these studies were as follows: anti-Futsch (22C10; Fujita et al., 1982), anti-Msps (Cullen et al., 1999), anti-
-tubulin (TAT1, Woods et al., 1989; DM1A, Sigma-Aldrich, St. Louis, MO; and YOL1/34, SeraLab, Crawley, United Kingdom), and anti-DmEB1 (this study). Secondary antibodies were obtained from Jackson ImmunoResearch Laboratories (West Grove, PA) or Molecular Probes (Eugene, OR).
Orcein staining of central nervous systems and calculation of mitotic index and anaphase frequency were carried out as described previously (Cullen et al., 1999).
For electron microcopy of chordotonal organs, Johnston's organs from pharate adults were prepared according to Eberl et al. (2000). Sections of 90-nm thickness were made using a UCT Ultra microtome (Leica Microsystems) and examined using a transmission electron microscope CM120 Biotwin (Philips, The Netherlands).
Neurological Analysis
Electrophysiological measurements of chordotonal neurons in Johnston's organ in the antenna were recorded as described previously (Eberl et al., 2000). Recordings from DmEB1 homozygotes were usually preceded by and alternated with age-matched controls (w or heterozygous siblings). The response of each antenna was averaged to 10 trains of five pulses, and then the maximum peak-peak amplitude was taken from the averaged trace.
The behavior of adult flies was examined after sufficient time was given for recovery if they had been exposed to carbon dioxide. Flies were either directly observed or videorecorded by using a digital camera under a dissection microscope. For measuring the time taken to "get up," each adult fly was placed on a petri dish and turned over on their back by using a paint brush. Flight test was carried out according to Ashburner (1989) by using a vertical tube 8 cm in width and 40 cm in height.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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; Rogers et al., 2002), because it shows the highest similarity to human EB1 and seems to be most abundantly expressed. As a first step, we examined protein expression of DmEB1 during Drosophila development. Total protein extracts were prepared from various developmental stages. Immunoblots showed that DmEB1 protein is ubiquitously expressed throughout development (Figure 1A). To see the protein expression profile in different tissue types in adult flies, we prepared total protein samples 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 DmEB1 protein is equally well expressed in all of the body parts (Figure 1B). These results indicated that DmEB1 is expressed ubiquitously during development.
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DmEB1 Is Associated with Microtubule Structures
EB1 and its homologues have been shown to localize to various microtubule structures. However, localization studies have been limited mainly to cultured cells or unicellular systems. We examined subcellular localization of DmEB1 in various tissues during development.
In rapid syncytial embryonic cell cycles, DmEB1 colocalizes with spindle microtubules during mitosis (Rogers et al., 2002; Figure 1C). There is conflicting evidence in other organisms on whether EB1 also can localize to centrosomes without microtubules (Berrueta et al., 1998; Rehberg and Gräf, 2002; Louie et al., 2004). To test whether localization to spindle poles is dependent on microtubules, we depolymerized microtubules during syncytial division by incubation with colchicine. Tubulin staining indicates that microtubules were depolymerized. DmEB1 is diffused throughout the cytoplasm, and no accumulations were detected on centrosomes.
In human cultured cells, it is reported that EB1 fails to localize to taxol-stabilized microtubule structures (Morrison et al., 1998). We incubated Drosophila embryos with taxol and then subjected them to immunostaining. Taxol incubation promotes microtubule assembly mainly from centrosomes but also ectopic sites. DmEB1generally followed microtubule structures, indicating that DmEB1 is able to associate with taxol-stabilized microtubules in vivo (Figure 1E).
To examine the localization of DmEB1 in various cell cycles, we have examined DmEB1 distribution in later stages of development. In cellularized embryos, we found that DmEB1 localizes to spindle structures during mitosis (our unpublished data) and with cytoplasmic microtubules during interphase (Figure 1G). We also observed overlapped localization of DmEB1 with interphase microtubules in male premeiotic cells and meiotic spindles (Figure 1H). We cannot tell whether it is concentrated to plus ends of microtubules because we cannot resolve ends of single microtubules. We also examined female meiotic spindles, which are formed in a chromosome-driven manner without centrosomes or discrete microtubule organizing centers. DmEB1 localizes along the microtubules and no concentration was observed around the spindle poles (Figure 1F).
To assay the association of DmEB1 to microtubules in vitro, microtubule cosedimentation experiments were carried out. Soluble extracts from embryos were incubated with taxol to polymerize microtubules, and then microtubules were pelleted by centrifugation. Immunoblots indicated that a minor fraction of the protein coprecipitates with microtubules, whereas the majority of DmEB1 proteins stays in the supernatant (Figure 1I).
DmEB1 Is Essential during Drosophila Development
Although budding and fission yeast have only one EB1 homologue, it is not essential for viability. In higher eukaryotes, which have multiple genes, no genetic analysis has been reported. We asked questions as to whether DmEB1 is essential for viability during development and, if so, what would be the function.
As part of the genome project, one lethal mutant, l(2)04524, has been identified with a P-element insertion in proximity of the DmEB1 gene. Sequence analysis showed that a P-element of 
15 kb in length is inserted in the noncoding transcribed region (Figure 2A). Immunoblotting of total protein extracts from late third instars indicated that the amount of DmEB1 protein is greatly reduced in the l(2)04524 mutant (Figure 2C).
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The lethality of l(2)04524 is reverted at a high frequency by remobilization of the P-element, indicating that the P-element is responsible for the lethality (our unpublished data). To prove that the lethality is a direct consequence of disrupting DmEB1, we made a transgene of wild-type DmEB1 cDNA under the control of the ubiquitin promotor. This transgene completely rescued the lethality, fertility and any defects of the mutant (our unpublished data). Therefore, we concluded that DmEB1 is essential for viability in Drosophila and renamed l(2)04524 to DmEB1P.
Semilethal mutants that fail to complement l(2)04524 (DmEB1P) have been isolated (Roote, unpublished data). We studied two of the alleles, DmEB12 and DmEB15, at the molecular level. Sequencing of the DmEB1 coding region indicated that each allele has one point mutation resulting in a conversion of amino acid sequences (I93N for DmEB12 and C252Y for DmEB15; Figure 2B). From the primary sequence, DmEB1 can be divided into three domains: the N-terminal conserved region, the central nonconserved region, and the C-terminal conserved region. The mutated amino acids are not conserved in all EB1 homologues and mapped in different conserved regions of the proteins (Figure 2B). Therefore, the mutant proteins may still maintain some molecular functions that lead to the hypomorphic nature of the mutation. Immunoblotting indicated that the DmEB12 mutation does not affect the level of DmEB1 protein, whereas a mutation in DmEB15 reduced the amount of DmEB1 protein significantly (Figure 2C).
Mutations in DmEB1 Cause Neuromuscular Defects
DmEB1P homozygous larvae are fully viable and show normal motility and touch response. Dissection of third instars revealed fully formed imaginal discs and internal organs. To see possible defects at a cellular level, we examined chromosome and microtubule organization in dividing cells of the central nervous systems. The DmEB1P mutant had a similar mitotic index and frequency of anaphase to wild type (Figure 3, A and B) and did not show abnormal mitotic figures. Immunostaining indicated that the organization of mitotic spindles and interphase microtubules were not disrupted in the mutant (our unpublished data). Therefore, the residual amount of DmEB1 protein in the mutant seems to be sufficient for cell division.
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Homozygous larvae are able to pupate and develop into pharate adults but fail to eclose. Dissection of pupae indicated that a complete adult body was formed inside the pupal case. Examination of the external structure did not reveal morphological defects. Defects generally associated with a failure of cell division at a low frequency, such as roughened eyes or missing bristles, were not found. The planar polarity in wing cells or microchaetes was not affected. Visual inspection and immunostaining indicated that external sensory organs correctly formed in the DmEB1P mutant and neuron morphology was indistinguishable to that of wild type (our unpublished data). These results together with the observation from the larval central nervous systems indicated that there were no general cell division defects in this hypomorphic mutant. Cytological observation of testes did not reveal any defects (our unpublished data).
We found that very rare homozygotes of DmEB1P (i.e., escapers) were able to eclose. The escaper adults generally had severe problems in coordinating body movements. Legs shook and often legs of both sides crossed each other. They were barely able to walk and, once they fell upside down, could not coordinate body movements to correct the body position. These escapers usually died within a few days. Homozygotes dissected from the pupal cases showed a wide range of viability and body coordination defects.
Two other alleles, DmEB12 and DmEB15, showed reduced viability over DmEB1P. Heterozygotes between DmEB12 and DmEB15 showed reduced viability as well, whereas we did not examine the alleles as homozygotes due to unrelated background lethal mutations. Although viable escapers from these semilethal allelic combinations had no apparent morphological defects, their wings are held abnormally. All of DmEB15/DmEB1P adults held their wings downward (Figure 3D). In DmEB12/DmEB1P or DmEB12/DmEB15, this phenotype was seen at a lower frequency, and wings were often held up or parted. Such abnormal wing positioning also is seen in some mutants defective in neuronal activity (Kernan et al., 1994; Zhang et al., 2001). A flight test indicated that all of DmEB1 mutant flies were flightless. Even individuals that held their wings in the normal position were flightless, suggesting that the phenotype may be due to neuromuscular defects rather than morphological defects.
The most striking defect of these semilethal flies was a behavioral defect. Although they walked in a coordinated way, once the flies were turned upside down, they failed to recover their correct body position promptly (Figure 3E and Supplemental Movies 1 and 2). In wild type, recovery takes less than one second. In contrast, it took significantly longer and often >2 min in the DmEB12/DmEB1P mutant. This also was observed in individuals that held their wings in the correct position. This defect is not due to paralysis or a general failure of movements, because they could walk and frantically moved their legs and bodies when they were turned over.
Functional Defects in Chordotonal Sensory Organs in a DmEB1 Mutant
Some aspects of DmEB1 mutant behavior are similar to the behavior of mutants with defective chordotonal mechanosensory organs (Eberl et al., 2000), although a strong DmEB1 mutant is less viable than mutants completely lacking chordotonal function. Chordotonal organs are stretch receptors that transduce movement or vibration of limb and body segments. They comprise multiple sensory units or scolopidia, each of which includes one to three ciliated sensory neurons that are enclosed by a scolopale cell and attached to a cap cell (Jarman, 2002). Specialized actin- and microtubule-based cytoskeletal structures are prominent features of these support cells (Dettman et al., 2001). The largest chordotonal organ (Johnston's organ), located in the adult antenna, includes 100 scolopidia and responds to sound-induced vibration of distal antennal segments. (Eberl et al., 2000; Caldwell and Eberl, 2002).
To assay the function of chordotonal organs in the DmEB1P mutant, we recorded sound-evoked compound potentials from Johnston's organ. Individual wild-type and mutant flies were exposed to near-field sound pulse trains while extracellular potentials were recorded from their antennae (Figure 4A; Eberl et al., 2000). In wild-type controls, a standard pulse stimulus evoked compound potentials with an average peak-to-peak amplitude of 615 µV (Figure 4, B and C). In DmEB1P mutants, responses to stimuli of the same intensity were reduced to an average of 237 µV (Figure 4, B and C). Although many of the mutants showed reduced activity and severe uncoordination, the reduction in response amplitude is unlikely to be due to general "sickness," because four especially healthy and vigorous mutants gave similarly reduced responses (average amplitude 220 µV).
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Because antennal sound-evoked potentials represent an aggregate response from the many scolopidia in Johnston's organ, the response amplitude reduction may reflect either loss of function in a subset of scolopidia or an overall partial loss of function. Nevertheless, this result indicates that DmEB1 is required for normal electrophysiological function of auditory chordotonal organs.
DmEB1 Is Concentrated in a Subset of Cell Structures of Chordotonal Organs
To understand the role of chordotonal organs, we examined the localization of DmEB1 in the chordotonal sensory organs in wild-type flies. We chose a pair of chordotonal organs located at the anterior ventral part of the abdomen because of its suitability for immunostaining. Fixed samples were immunostained with antibodies against DmEB1 and -tubulin as well as a monoclonal antibody (mAb) 22C10, which recognizes a neuron-specific marker, the MAP1 homologue Futsch.
Each sensory unit in the chordotonal organs is comprised of a monociliated neuron and support cells (Figure 5C) and aligned in parallel in a highly organized manner (Figure 5B). Cap cell and ligament structures were strongly stained with the anti--tubulin antibody (Figure 5A). DmEB1 was 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 DmEB1 was localized to the scolopale, spindle-shaped structure enclosing the cilia (Figure 5D, left), and regions of cells surrounding the inner dendritic segment of the neurons (Figure 5D, right). We do not see significant signals in cap structures or ciliary endings.
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The Organization of Chordotonal Organs Is Disrupted in DmEB1 Mutant
To understand the basis of functional defects of chordotonal organs, we compared the morphology of chordotonal organs of the first abdominal segment from wild-type and mutant flies. Fixed samples were immunostained with an -tubulin antibody and the mAb 22C10.
The structures and overall organization of chordotonal organs were largely unaltered in the mutant (Figure 6, B and D). Quantitative comparison of the overall shape of chordotonal organs did not reveal differences between wild-type and DmEB1P mutant. Lengths and widths of chordotonal organs were not significantly different between wild-type and the mutant. In addition, the number of neurons (represented by the number of dendrites) are not significantly affected by the mutation (Figure 6D), and the number of other cell types seems unaltered. This is consistent with the previous observation that there are no general defects in cell division or fate determination in this allele.
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Next, we looked at the organization within the abdominal chordotonal organs. In wild-type flies, each chordotonal neuron, as revealed by the 22C10 antibody, has one extended dendrite with a bulged end (Figure 6A, arrow) to which the cilium is attached (the cilium itself does not stain with 22C10). In wild type, ends of the dendrites are generally well aligned within the organ (Figure 6A). In contrast, they are not well aligned in the chordotonal organs of DmEB1P mutant flies (Figure 6B). For consistent quantification, we looked at the alignment of the most anterior row of dendrites. In the mutant, most chordotonal organs show the ends of at least one dendrite ends is not aligned, whereas this kind of misalignment is much less often seen in wild type (Figure 6D; 77% in the mutant vs. 26% in wild type; p < 0.001). This misalignment is often associated with unusually stretched dendrite morphology (Figure 6A, arrowheads).
In conclusion, our immunostaining results suggest that the DmEB1 mutation does not affect cell division or fate determination to form chordotonal organs, but it disrupts the alignment and structural integrity of neuronal sensory processes within the organs.
Ultrastructures of Auditory Chordotonal Organs in DmEB1 Mutant
To complement the light microscopy analysis, we examined mutant chordotonal organs by electron microscopy. The Johnston's organ in the second antennal segment was used for this study. Each sensory unit in the Johnston's organ contains two monociliated neurons (Figure 7A).
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Longitudinal sections of the chordotonal organs from the DmEB1P mutant revealed prominent ultrastructures, including a dendritic cap, scolopale, cilia, and ciliary rootlets (Figure 7B). In each scolopidium, the dome-shaped scolopale surrounds cilia whose tips are connected to the dendritic cap. At the base, a pair of ciliary rootlets with regular repetitive structures is visible. In higher magnification, basal bodies are clearly identifiable at the base of cilia (Figure 7D). Comparison with wild-type organs indicates that these structures are morphologically normal. Cross-sections of a number of organs at different positions revealed regular structures with wild-type morphology (Figure 7C). Near the distal end, the extracellular dendritic cap is visible surrounding the cilia and interior to a circle of scolopale rods (Figure 7E). In a more proximal region, a pair of cilia are observed in the middle of a circle of scolopale rods without the cap (Figure 7F).
In summary, electron microscopy revealed that mutant chordotonal organs have all the basic inner structures with normal morphology.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Essential Roles of DmEB1 during Drosophila Development
EB1 is a fundamental protein in eukaryotes that is conserved from yeast to humans and plants (Tirnauer and Bierer, 2000). Yeasts have only one EB1 homologue, which has important roles in the organization of cytoplasmic microtubules, but it is not essential for viability (Beinhauer et al., 1997; Schwartz et al., 1997). In contrast, higher eukaryotes, such as humans and Drosophila, have multiple EB1 homologues (Juwana et al., 1999; Rogers et al., 2002). In these organisms, most studies have so far been concentrated on one of the homologues, and functional relationships among the other homologues have not been studied.
In this study, we use Drosophila 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; Juwana et al., 1999). The similarity of three of them to EB1 also is extended to the C-terminal region. Among them, DmEB1 is the most similar to human EB1. We showed that the DmEB1 protein is ubiquitously expressed during Drosophila development. Other EB1 homologues also are expressed during development, although their expression is rather limited (our unpublished data). In this study, we identified three mutant alleles of DmEB1, one of which is lethal and the other two are semilethal. Transgenic wild-type DmEB1 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.
Previous studies using RNA interference (RNAi) or antibody injection indicated that DmEB1 is required for the regulation of microtubule dynamics and spindle organization/positioning (Lu et al., 2001; Rogers et al., 2002). Therefore, it is rather puzzling at first sight that our DmEB1 mutants showed limited defects without significant abnormalities in universal cell functions. However, it should be emphasized that the DmEB1 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, DmEB1 protein has a large maternal contribution that may mask the requirement in early development. It is also possible that other EB1 homologues in Drosophila may have redundant functions that compensate for loss of DmEB1. Therefore, it is likely that the phenotype we observed in DmEB1 mutants represents a subset of DmEB1 functions in vivo. Nevertheless, our hypomorphic mutations uncovered the neuromuscular involvement of DmEB1 without interference by more universal cellular roles.
Roles of DmEB1 in Neural Function
Although our DmEB1 alleles do not show significant cell division or morphological defects, studies on semilethal alleles of DmEB1 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.
These behavioral phenotypes of DmEB1 mutants share some characteristics with the phenotypes of mutants defective in chordotonal sensory organ function (Eberl et al., 2000). Chordotonal organs are mechanosensory organs that act as stretch receptors and also form the fly's auditory systems. We showed that DmEB1P mutant is indeed defective in the function of auditory chordotonal organs. Therefore, malfunction of chordotonal organs can, in part, explain the DmEB1 mutant defects. However, mutants completely lacking chordotonal organs show better viability than the DmEB1P homozygotes, indicating that the DmEB1 mutants have additional, unidentified defects.
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 (Eberl et al., 2000; Jarman, 2002). 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.
Our immunolocalization study of DmEB1 indicated that, rather than DmEB1 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 DmEB1 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 DmEB1 does not have significant concentration at ciliary endings, although the EB1 homologue is localized to the flagellar tip and implicated in intraflagellar transport in Chlamydomonas (Pedersen et al., 2003).
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 DmEB1-rich regions may well contribute functional and structural defects of chordotonal organs in the mutant.
Molecular Function of DmEB1
How then does the loss of DmEB1 cause the defects in function and organization of chordotonal organs at the molecular level? RNAi in Drosophila revealed that DmEB1 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, we do not observe general cell division defects or significant alteration of the cell numbers in chordotonal organs of the mutant.
Instead, our observation on mutant chordotonal organs is consistent with roles of DmEB1 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 (Eberl et al., 2000; Dettman et al., 2001). Furthermore, chordotonal organs require robust mechanical linkages between stimulus-delivering structures and neuronal transducing elements to sense tensions effectively.
The actin-microtubule linker Short stop, which localizes to the cell cortex, interacts with DmEB1 (Subramanian et al., 2003) and is shown to be required for chordotonal organ integrity (Prokop et al., 1998). 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 DmEB1 activity. Immunofluorescence microscopy using antibodies against -tubulin and Futsch (the MAP1 homologue) or our 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.
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ACKNOWLEDGMENTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Footnotes |
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The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org). 
Present address: Department of Biological Sciences, Stanford University, Stanford, CA 94305
Present address: Medical School, The University of Edinburgh, Edinburgh EH8 9AG, United Kingdom
|| Corresponding author. E-mail address: h.ohkura{at}ed.ac.uk.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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