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

Abstract

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. Here, we describe the first genetic study of EB1 in developing animals. We show that 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.

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Molecular Biology of the Cell
Vol. 16, 891–901, February 2005
EB1 Is Essential during Drosophila Development and Plays
a Crucial Role in the Integrity of Chordotonal
Mechanosensory Organs□
V
Sarah L. Elliott,*†C. Fiona Cullen,* Nicola Wrobel,*‡Maurice J. Kernan,§and
Hiroyuki Ohkura*?
*Wellcome Trust Centre for Cell Biology, Institute of Cell Biology, School of Biological Sciences, The
University of Edinburgh, Edinburgh EH9 3JR, United Kingdom; and§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
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. Here, we describe the first genetic study of EB1 in developing
animals. We show that one of the multiple Drosophila EB1 homologues, DmEB1, is ubiquitously expressed and has
essential functions during development. Hypomorphic DmEB1 mutants show neuromuscular defects, including flight-
lessness 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.
INTRODUCTION
The microtubule network is one of the major cytoskeletal
systems in eukaryotes. Dynamic reorganization of the mi-
crotubule network is necessary for many diverse cellular
functions such as organelle and protein transport, cell archi-
tecture, cell polarity, and division. Microtubules alternate
between phases of rapid growth and disassembly. The mi-
crotubule dynamics are spatially and temporally regulated
within cells, both during the cell cycle and in development.
A central question is how microtubule dynamics and orga-
nization are controlled and linked to other cellular pro-
cesses.
A number of microtubule-associated proteins (MAPs) and
motors have been shown to modify the behavior of micro-
tubules (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 mole-
cules 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 interac-
tions 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 Gra ¨f, 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 ho-
mologue, 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 (Tir-
nauer et al. 2002a). In addition, a role for EB1 in microtubule–
kinetochore attachment has been suggested (Rehberg and
Gra ¨f, 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).
Article published online ahead of print in MBC in Press on Decem-
ber 9, 2004 (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.
E04-07-0633).
□
VThe online version of this article contains supplemental material
at MBC Online (http://www.molbiolcell.org).
Present addresses:
University, Stanford, CA 94305;‡Medical School, The University of
Edinburgh, Edinburgh EH8 9AG, United Kingdom.
†Department of Biological Sciences, Stanford
?Corresponding author. E-mail address: h.ohkura@ed.ac.uk.
© 2005 by The American Society for Cell Biology 891

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Interactions between microtubules and the cell cortex are
considered to be particularly important during develop-
ment. 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 support-
ing cells are highly polarized and contain specialized cy-
toskeletal 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 func-
tion during development. Loss of DmEB1 disrupts body
coordination and compromises the function and structure of
the chordotonal sensory organs.
MATERIALS AND METHODS
Drosophila Genetics
Standard techniques of fly manipulation (Ashburner, 1989) were followed
throughout. All stocks and crosses were grown at 25°C in cornmeal media.
w1118was 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 (Indi-
anapolis, IN). DmEB12and DmEB15were a generous gift from John Roote
(Cambridge University, Cambridge, United Kingdom) and were studied over
DmEB1Por 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. DmEB1Phomozygote carrying one copy of
the transgene produced completely viable, fertile, healthy adult flies without
behavioral defects.
DNA Manipulation, Protein Preparation, and Immuno-
blots
Standard molecular techniques (Sambrook et al., 1989; Harlow and Lane,
1988) were followed throughout. Mutation sites in DmEB12and DmEB15were
determined by direct sequencing of DmEB1 coding regions amplified from
mutant genomic DNAs by polymerase chain reaction. Microtubule sedimen-
tation 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 (Mid-
lothian, 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 em-
bryos and nonactivated oocytes, and formaldehyde was used for other tis-
sues. 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 DmEB1Phomozygotes of roughly
the equivalent stage. These pharate adults were dissected from pupae cases
and fixed with 3.4% of formaldehyde in phosphate-buffered saline immedi-
ately 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). Sec-
ondary antibodies were obtained from Jackson ImmunoResearch Laborato-
ries (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 Microsys-
tems) and examined using a transmission electron microscope CM120
Biotwin (Philips, The Netherlands).
Neurological Analysis
Electrophysiological measurements of chordotonal neurons in Johnston’s or-
gan in the antenna were recorded as described previously (Eberl et al., 2000).
Recordings from DmEB1 homozygotes were usually preceded by and alter-
nated 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.
RESULTS
A Drosophila EB1 Homologue, DmEB1, Is Ubiquitously
Expressed during Drosophila Development
To understand the role of EB1 in the context of developing
animals, we used D. melanogaster as a model organism.
Drosophila genome sequencing has identified three genes
(CG3265, CG18190, and CG32371) closely related to human
EB1 and a few others with limited similarity. We studied an
in vivo role of CG3265 (which is also called DmEB1 or dEB1;
Lu et al., 2001; 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 ab-
domen of adult Drosophila. These body parts are enriched in
neural cells, muscle cells, and visceral/reproductive cells,
respectively, and therefore serve as populations of contrast-
ing cell types. The immunoblots showed that DmEB1 pro-
tein is equally well expressed in all of the body parts (Figure
1B). These results indicated that DmEB1 is expressed ubiq-
uitously during development.
DmEB1 Is Associated with Microtubule Structures
EB1 and its homologues have been shown to localize to
various microtubule structures. However, localization stud-
ies 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 colocal-
izes with spindle microtubules during mitosis (Rogers et al.,
2002; Figure 1C). There is conflicting evidence in other or-
ganisms on whether EB1 also can localize to centrosomes
without microtubules (Berrueta et al., 1998; Rehberg and
Gra ¨f, 2002; Louie et al., 2004). To test whether localization to
spindle poles is dependent on microtubules, we depolymer-
ized microtubules during syncytial division by incubation
with colchicine. Tubulin staining indicates that microtubules
S. L. Elliott et al.
Molecular Biology of the Cell892

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were depolymerized. DmEB1 is diffused throughout the
cytoplasm, and no accumulations were detected on centro-
somes.
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 microtu-
bule 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 dur-
ing 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 dis-
crete 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 car-
ried out. Soluble extracts from embryos were incubated with
taxol to polymerize microtubules, and then microtubules
Figure 1.
during Drosophila development. Total protein samples were prepared from each stage of wild-type Drosophila. E1, 0- to 4-h embryos; E2, 4-
to 24-h embryos; L1, first instars; L2, second instars; L3, third instars; EP, early (white) pupae; LP, late (dark) pupae; M, adult male; and F,
adult female. From the top, an immunoblot probed with a DmEB1 antibody, an immunoblot using ?-tubulin antibody, and Coomassie Blue
staining. The dots on the left show the positions of molecular mass markers (48, 33, and 25 kDa [top]; 62, 48, and 33 kDa [middle]; and 175,
83, 62, 48, 33, 25, and 16 kDa [bottom]). (B) DmEB1 is ubiquitously expressed in different adult tissues. Protein samples were prepared from
head, thorax and abdomen of adult males and females. The dots on the left show the positions of molecular mass markers (33 and 25 kDa
[top]; 62 and 48 kDa [middle]; and 83, 62, 48, 33, and 25 kDa [bottom]). (C–G) DmEB1 associates with microtubule structures. Blue, DNA;
green, ?-tubulin; and red, DmEB1. Bar, 10 ?m for C–F and 100 ?m for G. (C) DmEB1 colocalizes with spindles during syncytial mitosis. (D)
Depolymerization of microtubules by colchicine delocalizes DmEB1. (E) DmEB1 is associated with taxol-stabilized spindle microtubules. (F)
DmEB1 colocalizes with spindle microtubules in female meiosis I, which lacks centrosomes. (G) DmEB1 colocalizes with interphase
microtubules in cellularized embryos. Lateral view of a stage 10 embryo. (H) DmEB1 colocalizes with a meiotic spindle and interphase
microtubules in spermatocytes. (I) A minor proportion of DmEB1 cosedimented with microtubules. A soluble Drosophila embryonic extract
(Ex) was incubated with paclitaxel and GTP to polymerize microtubules. Microtubules and associated proteins (Ppt) were separated from
soluble proteins (Sup). From the top, the immunoblot probed with DmEB1 antibody, the control immunoblot probed with antibody against
Msps (another microtubule-associated protein; Cullen et al., 1999), and immunoblot probed with ?-tubulin. The dots on the left show the
positions of molecular mass markers (33 and 25 kDa [from the top]; 175, 83, 62, and 48 kDa [from the bottom]).
DmEB1 is ubiquitously expressed and associates with microtubules during development. (A) DmEB1 is ubiquitously expressed
EB1 for Chordotonal Sensory Organs
Vol. 16, February 2005893

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were pelleted by centrifugation. Immunoblots indicated that
a minor fraction of the protein coprecipitates with microtu-
bules, 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 eu-
karyotes, 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).
The lethality of l(2)04524 is reverted at a high frequency by
remobilization of the P-element, indicating that the P-ele-
ment is responsible for the lethality (our unpublished data).
To prove that the lethality is a direct consequence of dis-
rupting 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, DmEB12and 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 DmEB12and
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 differ-
ent conserved regions of the proteins (Figure 2B). Therefore,
the mutant proteins may still maintain some molecular func-
tions that lead to the hypomorphic nature of the mutation.
Immunoblotting indicated that the DmEB12mutation does
not affect the level of DmEB1 protein, whereas a mutation in
DmEB15reduced the amount of DmEB1 protein significantly
(Figure 2C).
Mutations in DmEB1 Cause Neuromuscular Defects
DmEB1Phomozygous larvae are fully viable and show nor-
mal 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 chro-
mosome and microtubule organization in dividing cells of
the central nervous systems. The DmEB1Pmutant 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 dis-
rupted in the mutant (our unpublished data). Therefore, the
residual amount of DmEB1 protein in the mutant seems to
be sufficient for cell division.
Homozygous larvae are able to pupate and develop into
pharate adults but fail to eclose. Dissection of pupae indi-
cated 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 af-
fected. 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 to-
gether 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,
Figure 2.
of DmEB1 genomic region with mutations in DmEB1 alleles.
DmEB1P(P) has an insertion of 15-kb-long P-lacZ in the 5?-noncod-
ing transcribed region, whereas DmEB12(2) and DmEB15(5) have a
single point mutation (adenine to thymine and guanine to adenine,
respectively) in the coding region. (B) Amino acid conversions
caused by DmEB12and DmEB15. The EB1 family of proteins com-
monly has two conserved regions (gray shadows). DmEB12and
DmEB15mutations result in conversions I93N andC252Y, respec-
tively. Amino acid alignments of DmEB1, human (Hs) EB1, and
Saccharomyces cerevisiae Bim1p, Schizosaccharomyces pombe (Sp) Mal3
are shown below. Identical amino acids to DmEB1 were marked in
black. (C) DmEB1 proteins in DmEB1 alleles. Total protein samples
were prepared from third instars of wild-type (?), DmEB1P(P),
DmEB12(2), DmEB15(5), all over DmEB1P. The immunoblot was
probed with DmEB1 antibody. The dots on the left show the posi-
tions of molecular mass markers (48, 33, and 25 kDa from the top).
Molecular defects in DmEB1 alleles. (A) Molecular maps
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Molecular Biology of the Cell894

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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, DmEB12and DmEB15, showed reduced
viability over DmEB1P. Heterozygotes between DmEB12and
DmEB15showed 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/DmEB1Padults held their wings downward
(Figure 3D). In DmEB12/DmEB1Por 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 individ-
uals that held their wings in the normal position were flight-
less, suggesting that the phenotype may be due to neuro-
muscular 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/DmEB1Pmutant. 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 mech-
anosensory organs (Eberl et al., 2000), although a strong
DmEB1 mutant is less viable than mutants completely lack-
ing 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 sen-
sory neurons that are enclosed by a scolopale cell and at-
tached 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
DmEB1Pmutant, we recorded sound-evoked compound po-
tentials 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 an-
tennae (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 ?V (Figure 4, B
and C). In DmEB1Pmutants, responses to stimuli of the same
intensity were reduced to an average of 237 ?V (Figure 4, B
and C). Although many of the mutants showed reduced
activity and severe uncoordination, the reduction in re-
sponse amplitude is unlikely to be due to general “sickness,”
because four especially healthy and vigorous mutants gave
similarly reduced responses (average amplitude 220 ?V).
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 ?-tu-
bulin as well as a monoclonal antibody (mAb) 22C10, which
Figure 3.
Normal cell division in DmEB1Pmutant. Central nervous systems of
third instars from wild-type and DmEB1Phomozygotes were dis-
sected and stained with aceto-orcein after being squashed. Average
mitotic indexes (A) and percentages of anaphase among mitotic
cells (B) were shown with standard deviations (bars). Mitotic index
was calculated as the number of mitotic cells per microscope field
containing typically 200–400 cells. (C) All wild-type (w1118) adult
flies hold wings on their backs. (D) All adult DmEB15/DmEB1Pflies
hold their wings downward. (E) Times taken to get up after being
turned over. Each adult fly was turned over on their back by using
a paint brush. Wild-type and DmEB12/DmEB1Pwere represented by
open and shaded bars, respectively.
DmEB1 mutants show neuromuscular defects. (A and B)
EB1 for Chordotonal Sensory Organs
Vol. 16, February 2005895