BACKGROUND
the concerted action of ion-transport mechanisms and gap junctions gives rise to bioelectric phenomena, like ph gradients, voltage gradients and ion fluxes within cells, tissues, organs, and even organisms.
such bioelectric phenomena are known to play regulatory roles in various developmental and regenerative processes, e.g.
proliferation, differentiation, polarisation and migration .
using a variety of approaches, we have gathered considerable information on the electrical properties as well as on the distribution and activity patterns of ion-transport mechanisms and gap junctions in the ovary of drosophila melanogaster .
following this strategy in a model system, we are trying to further clarify the roles that bioelectric phenomena play during development, e.g.
for ph-regulation, osmoregulation, cell-cell communication, cell migration, cell proliferation, cell death, vitellogenesis and growth.
during the course of drosophila oogenesis, characteristic extracellular current patterns  <cit>  as well as membrane-potential changes in germ-line and soma cells have been observed that partly depend on the exchange of protons, potassium ions and sodium ions  <cit> .
moreover, striking stage-specific patterns of membrane potentials and intracellular ph that characterize distinct cell populations have been described recently  <cit> .
these bioelectric patterns were found to be related to the distribution patterns of various membrane channels, namely v-atpases, l-type ca2+-channels, amiloride-sensitive na+-channels and na+,h+-exchangers, as well as innexin-3-containing gap junctions.
therefore, these membrane proteins are likely to be involved in the regulation of membrane potentials and/or intracellular ph  <cit> .
an ovarian follicle of drosophila consists of  <dig> germ-line cells surrounded by a layer of somatic follicle cells  <cit> .
the oocyte and its  <dig> nurse cells form a cytoplasmic continuum via intercellular bridges as well as via gap junctions, and the same holds true for the follicle cells.
on the other hand, the germ-line cells are connected to the soma cells only via gap junctions  <cit> .
by way of the intercellular exchange of microinjected fluorescent tracers, stage-specific communication between germ-line cells and soma cells has been detected, and a variety of treatments was found to either inhibit or stimulate this communication  <cit> .
innexins are considered the main gap-junction proteins of invertebrates , but there is some evidence that additional proteins, like e.g.
ductin, can be part of gap junctions  <cit> .
therefore, it is tempting to analyse whether or not ductin and members of the innexin family are found in common gap-junction plaques.
previously, the localization of ductin  <cit>  as well as the localization of innexins  <dig> to  <dig>  <cit>  have been separately analysed in drosophila ovarian follicles in detail.
in the present study, we try to clarify the relationship of ductin, known as a channel-forming subunit of v-atpases, with innexins  <dig> and  <dig>  two well known components of gap junctions, using double-immunolabeling.
we apply combinations of four antisera directed against  ductin ,  another v-atpase component in the membrane-spanning v <dig> domain ,  innexin  <dig>  and  innexin  <dig>  respectively.
these antisera have previously been shown to specifically recognize cellular antigens in the drosophila ovary  <cit> .
methods
preparation of follicles
drosophila melanogaster wild-type oregon r flies were reared at about 20 °c on standard food with additional fresh yeast.
individual 2– <dig> days old females were killed by crushing the thorax with tweezers without previous anesthetization.
the ovaries were dissected with tweezers in drosophila pbs  <cit> , and single follicles of different stages were isolated by pulling at the anterior tip of an ovariole.
antisera
to localize v-atpase and gap-junction proteins, we used  a rabbit antiserum  raised and affinity-purified against a highly conserved region  of the 16 kda-protein ductin, which forms subunit c of v-atpases and is also supposed to be a part of gap junctions ,  a guinea-pig antiserum  raised against an n-terminal region of subunit a of the manduca sexta v-atpase, kindly provided by b. walz and o. baumann ,  a guinea-pig antiserum  raised against the cytoplasmic loop of innexin  <dig>  and  a guinea-pig antiserum  raised against the c-terminus of innexin  <dig> from drosophila, kindly provided by r. bauer and m. hoch .
a rabbit non-immune serum  was used as control.
immunoblotting
immunoblots were performed as described previously  <cit> .
in short, homogenates of ovaries were sonicated and briefly boiled.
the proteins were separated using 12 % sds-page and transferred to nitrocellulose membranes.
nonspecific binding sites were blocked with 5 % skimmed milk powder/pbs and the blots were incubated in anti-ductin diluted 1: <dig> up to 1: <dig> with 1 % bsa/pbs.
nis diluted 1: <dig> was used as a control  <cit> .
subsequently, biotinylated goat-anti-rabbit igg , streptavidin-peroxidase  and h2o2/4-chloro-1-naphthol  were applied, and photographs were taken using a digital camera.
experiments were performed three times.
indirect immunofluorescence preparations
follicles were fixed for 30 min at 4 °c in 4 % formaldehyde dissolved in pbs, washed in pbs and blocked for 1 h at 20 °c with 2 % bsa/ <dig>  % triton x- <dig> in pbs.
thereafter, the follicles were incubated overnight at 4 °c in pbs containing  <dig>  % bsa/ <dig>  % triton x- <dig> and two of the following antisera for double-labeling: anti-ductin diluted 1: <dig>  anti-v-atpase a diluted 1: <dig>  anti-inx <dig> diluted 1: <dig>  anti-inx <dig> diluted 1: <dig>  controls were performed without antiserum and using nis 1: <dig>  <cit> , respectively.
after washing six times for 10 min, the follicles were treated with goat-anti-rabbit-cy <dig>  and donkey-anti-guinea-pig-fp <dig>  for 1 h in pbs containing  <dig>  % bsa/ <dig>  % triton x- <dig>  washing was repeated six times and the nuclei were stained with  <dig>  μg/ml dapi  in pbs for 3 min.
thereafter, the follicles were mounted in fluoromount g  and viewed, by using x20/ <dig> , x40/ <dig>  or x63/ <dig> oil objectives and the appropriate filter sets, either on a zeiss axiovert  <dig> wide-field fluorescence microscope , equipped with a hamamatsu orca er camera, or they were mounted in glycerine/pbs 1: <dig> and viewed on a leica dmre laser-scanning microscope .
each experiment was repeated at least three times.
RESULTS
immunoblot analysis of anti-ductin
using immunoblots of drosophila ovary preparations, we analysed the binding properties of the commercially available antiserum ab <dig> .
as described earlier for two other anti-ductin sera ), anti-ductin recognized a strong band at 29 kda and weak bands at  <dig> and 45 kda .
no specific binding was obtained with nis .
the 16 kda-band represents the monomer of ductin and the other bands represent the putative dimer and trimer, respectively.
thus, the binding of anti-ductin in indirect immunofluorescence preparations is assumed to be specific for the channel-forming protein ductin in drosophila ovarian follicles.fig.
<dig> ductin is localized stage-specifically in plasma membranes and cytoplasmic vesicles of follicle cells and germ-line cells.
a: on immunoblots of ovary extracts using anti-ductin, a band at 29 kda  and weak bands at about 16 kda  and 45 kda  are recognized.
a’: no specific staining was obtained with nis.
b to g: lsm images; b: ductin is found in plasma membranes and cytoplasmic particles or vesicles, especially in polar cells .
c: ductin becomes increasingly localized to plasma membranes in nurse cells  and follicle cells .
d: ductin is found in plasma membranes of centripetally migrating fc .
e: ductin is localized in lateral and apical fc membranes , in the oolemma  and, most prominently, in pc and stalk cells .
f: ductin is found in plasma membranes of squamous fc and nc  and around nc nuclei .
g: ductin is concentrated in plaques in lateral fc membranes
localization of ductin in indirect immunofluorescence preparations
using anti-ductin and laser-scanning microscopy , we investigated the distribution of ductin during the course of oogenesis .
as shown previously using the two other anti-ductin sera on sections prepared for immuno-fluorescence and immuno-electron microscopy  <cit> , the continuous as well as punctate distribution of ductin was found to change stage-specifically.
in the following, we concentrate on ductin in specialized types of follicle cells  and on some main features of its distribution relevant to the present comparative analysis.
during previtellogenesis , ductin is located in cytoplasmic particles or vesicles as well as in plasma membranes and is enriched in polar cells .
beginning with vitellogenesis, ductin increasingly becomes located in plasma membranes of nurse cells and follicle cells .
during vitellogenesis , ductin is located  in cytoplasmic vesicles,  in plasma membranes of centripetally migrating follicle cells ,  in lateral and apical plasma membranes of columnar follicle cells,  in plasma membranes of squamous follicle cells and nurse cells as well as around nurse-cell nuclei,  in the oolemma, and , most prominently, in polar cells and stalk cells .
predominantly in lateral and apical follicle-cell membranes, ductin is localized in a punctate pattern resembling gap-junction plaques ).
no specific staining was found in control preparations without primary antiserum or using nis .
colocalization of ductin with v-atpase subunit a
since ductin forms subunit c of v-atpases, it is expected to localize, at least in part, together with subunit a in the same cellular regions.
we analysed follicles double-labeled with anti-ductin and anti-v-atpase a using wide-field fluorescence microscopy .
stage-specifically, ductin and subunit a were found colocalized in plasma membranes and in the cytoplasm of follicle cells and germ-line cells, especially in cytoplasmic particles or vesicles.fig.
<dig> ductin and v-atpase subunit a are partly colocalized in plasma membranes and in the cytoplasm of follicle cells and germ-line cells, often in cytoplasmic particles or vesicles.
a to c: ductin ; a’ to c’: v-atpase subunit a; a” to c” and d to f: merged images, ductin , v-atpase subunit a  and colocalization of both , wfm.
nuclei were stained with dapi.
no specific staining was obtained with nis .
in young previtellogenic follicles , ductin and v-atpase subunit a co-occur in anterior and posterior polar cells , in lateral and apical follicle-cell membranes , and in the cytoplasm of fc, nurse cells  and the oocyte .
while cytoplasmic particles or vesicles containing predominantly v-atpase subunit a can be observed , ductin is enriched in stalk cells .
in older previtellogenic follicles , ductin and v-atpase subunit a are enriched in afcm.
while ductin is found in higher amounts in ppc and sc, v-atpase subunit a is found in higher amounts in the cytoplasm of fc, nc and ooc .
in vitellogenic follicles , ductin and v-atpase subunit a are colocalized especially in columnar and squamous fc.
while ductin is enriched in nc membranes , v-atpase subunit a is enriched in border cells .
d: ductin and v-atpase subunit a are colocalized in particles or vesicles  in squamous fc .
e: ductin and v-atpase subunit a are colocalized in lateral fc membranes  as well as in the cytoplasm .
particles or vesicles containing v-atpase subunit a are found in fcn .
f: in older follicles , ductin and v-atpase subunit a are colocalized in particles or vesicles in most of the fc  while, in anterodorsal fc , predominantly v-atpase subunit a is found in the cytoplasm and in the nuclei .
scale bars, 10 μm
during early previtellogenesis, ductin and subunit a co-occur  in polar cells,  in lateral and apical follicle-cell membranes and  in the cytoplasm of follicle cells, nurse cells and the oocyte.
however, while ductin was found to be more prominent in stalk cells, the distinct particles or vesicles in germ-line cells were found to contain predominantly subunit a .
during late previtellogenesis, both ductin and subunit a are enriched in apical follicle-cell membranes.
on the other hand, ductin was found to be more prominent in posterior polar cells and in stalk cells, while subunit a was more prominent in the cytoplasm of follicle cells, nurse cells and the oocyte .
during vitellogenesis, ductin and subunit a co-occur especially in columnar and squamous follicle cells and in border cells.
in columnar follicle cells, the amount is usually higher on one side of the follicle , namely the ventral side .
here, ductin and subunit a are colocalized in the plasma membranes and in the cytoplasm while, in squamous follicle cells, they are are colocalized in vesicles .
on the other hand, ductin is enriched in nurse-cell membranes, while subunit a is enriched in border cells  as well as in nuclear particles or vesicles in follicle cells .
during late vitellogenesis, ductin and subunit a are colocalized in cytoplasmic vesicles in most of the follicle cells, whereas, in the cytoplasm and nuclei of anterodorsal follicle cells, predominantly subunit a was found .
taken together, ductin and subunit a are stage-specifically either colocalized or separately enriched in different cellular regions of germ-line and soma cells.
frequently, ductin is more prominent in plasma membranes, while subunit a is more prominent in cytoplasmic and nuclear particles or vesicles .
colocalization of ductin with innexins
since ductin is known to form intercellular communication channels  <cit> , it might localize, in part, together with members of the channel-forming innexin family in the same cellular regions .
in drosophila ovarian follicles, the localization of innexins  <dig> to  <dig> has been described in detail previously using lsm and wfm  <cit> .
for the present comparative analysis, we concentrate on some main features concerning inx <dig> and inx <dig>  that both showed distribution patterns similar to ductin.
ductin and inx2
we analysed follicles double-labeled with anti-ductin and anti-inx <dig> using wfm .
during previtellogenesis , ductin and inx <dig> co-occur  in lateral and apical follicle-cell membranes and  in the cytoplasm of follicle cells, nurse cells and the oocyte.
however, ductin was found to be enriched  in nurse-cell membranes and  in the oolemma as well as  in polar cells and  in stalk cells, whereas cytoplasmic particles or vesicles containing predominantly inx <dig> were observed in germ-line cells.fig.
<dig> ductin and inx <dig> are partly colocalized in plasma membranes and in the cytoplasm of follicle cells and germ-line cells, but often in different plaques.
a to c: ductin; a’ to c’: inx2; a”: merged image, ductin , inx <dig>  and colocalization of both , wfm.
nuclei were stained with dapi.
no specific staining was obtained with nis .
in previtellogenic follicles , ductin and inx <dig> co-occur in lateral and apical follicle-cell membranes  and in the cytoplasm of fc, nurse cells  and the oocyte .
while prominent cytoplasmic particles or vesicles containing inx <dig> can be observed in germ-line cells , ductin is found to be enriched in nc membranes , in the oolemma , in anterior and posterior polar cells  and in stalk cells .
in vitellogenic follicles , ductin and inx <dig> co-occur in lateral and apical fc membranes , while ductin is found to be enriched in ppc, and inx <dig> in the ool, respectively.
both proteins are concentrated in lateral fcm, but they often localize in different plaques .
scale bars, 10 μm
also during vitellogenesis , ductin and inx <dig> co-occur in lateral and apical follicle-cell membranes.
on the other hand, ductin is still enriched in posterior polar cells, while inx <dig> is now enriched in the oolemma.
in lateral follicle-cell membranes, both proteins are often found concentrated in different plaques that are presumed to represent different gap junctions .
ductin and inx3
follicles double-labeled with anti-ductin and anti-inx <dig> were also analysed using wfm .
in previtellogenic and early vitellogenic stages , ductin and inx <dig> co-occur in follicle-cell membranes.
however, ductin is more prominent  in nurse-cell membranes and  in the oolemma as well as  in polar cells,  in stalk cells and  in the cytoplasm of germ-line cells.
on the other hand, inx <dig> was found to be enriched around nurse-cell nuclei and in lateral plasma membranes of the prospective centripetally migrating follicle cells.fig.
<dig> ductin and inx <dig> are partly colocalized in plasma membranes of follicle cells and germ-line cells, but often in different plaques.
a to c: ductin; a’ to c’: inx3; a” to c”: merged images, ductin , inx <dig>  and colocalization of both , wfm.
nuclei were stained with dapi.
no specific staining was obtained with nis .
in previtellogenic and early vitellogenic follicles , ductin and inx <dig> co-occur in follicle-cell membranes .
while ductin is found to be enriched in nurse-cell membranes , in the oolemma , in polar cells , in stalk cells  and in the cytoplasm of nc and oocyte , inx <dig> is enriched especially in lateral membranes of the prospective centripetally migrating fc  and around nc nuclei .
in later stages , ductin and inx <dig> co-occur in lateral fcm  and in ncm.
while inx <dig> exhibits prominent membrane plaques , ductin is enriched in apical fcm  and in the ool c. both proteins are concentrated in lateral fcm, but they often localize in different plaques .
scale bars, 10 μm
in mid to late vitellogenic stages , ductin and inx <dig> co-occur in lateral follicle-cell membranes and in nurse-cell membranes.
while ductin is enriched in apical follicle-cell membranes and in the oolemma, inx <dig> forms prominent plaques in lateral follicle-cell membranes.
here, ductin and inx <dig> are often found concentrated in different plaques that are presumed to represent different gap junctions .
taken together, ductin and both innexins are stage-specifically either colocalized or separately enriched in different cellular regions of germ-line and soma cells.
frequently, ductin shows a rather continuous membrane distribution, while the distribution of both innexins is more punctate .fig.
<dig> summary of colocalization or local enrichment of ductin, v-atpase subunit a, inx <dig> and inx <dig>  respectively, during previtellogenic stages  and vitellogenic stages .
a, a’: ductin  and v-atpase subunit a; b, b’: ductin and inx2; c, c’: ductin and inx <dig>  ductin and subunit a  are stage-specifically either colocalized in plasma membranes or in the cytoplasm , which is supposed to represent the distribution of v-atpases, or separately enriched in different cellular regions of germ-line and soma cells.
frequently, ductin  is more prominent in plasma membranes, while subunit a  is more prominent in cytoplasmic and nuclear particles or vesicles.
punctate membrane labeling  is presumed to originate from ductin as part of membrane channels or gap-junctional structures.
a: in previtellogenic stages , ductin  is enriched in posterior polar cells  and in stalk cells , while subunit a  is enriched in cytoplasmic particles or vesicles in follicle cells , nurse cells  and the oocyte .
a’: in vitellogenic stages , the dorsoventral gradient of v-atpase distribution in the fc epithelium  is in accordance with the gradual distribution of epithelial membrane potentials  <cit> .
while ductin  is enriched in nc, ppc and sc, subunit a  is enriched in border cells  and nuclear particles or vesicles in fc .
since pc, bc and ventral fc are also characterized through a high activity of l-type ca2+-channels, alterations of membrane potentials through v-atpase activity are supposed to result in cellular responses due to voltage-dependent ca2+-currents  <cit> .
ductin and inx <dig>  as well as ductin and inx <dig>  are stage-specifically either colocalized  or separately enriched  in different cellular regions of germ-line and soma cells.
frequently, ductin  shows a rather continuous membrane distribution, presumably representing v-atpases, while the distribution of both innexins  is more punctate, presumably representing gap-junctional structures.
in lateral fc membranes , both ductin and inx <dig> as well as ductin and inx <dig> are often found concentrated in different plaques  that are presumed to represent different gap-junctional structures .
b: in previtellogenic stages, ductin  is enriched in nc membranes  and in the oolemma  as well as in pc and sc, whereas inx <dig>  is enriched in cytoplasmic particles or vesicles in germ-line cells.
b’: in vitellogenic stages, ductin  is enriched in ppc, while inx <dig>  is enriched in the ool.
antibodies directed against presumed cytoplasmic regions of both ductin or inx <dig> microinjected into the oocyte specifically blocked intercellular communication through ductin- or inx2-containing gap junctions  <cit> .
c: in previtellogenic stages, ductin  is more prominent in ncm and in the ool as well as in pc, in sc and in the cytoplasm of germ-line cells, while inx <dig>  is more prominent around ncn and in lateral plasma membranes of the prospective centripetally migrating fc .
c’: in vitellogenic stages, ductin  is enriched in apical fcm and in the ool as well as in ppc, bc and sc, while inx <dig>  forms prominent plaques in lateral membranes of cfc .
inx3-containing gap junctions are supposed to specifically regulate the distribution of membrane potentials, intracellular ph and ions within cfc and to establish a communication border to neighboring fc  <cit>
discussion
the channel-forming protein ductin has been found in both v-atpases and gap-junctional structures  <cit> .
in v-atpases, it forms the proteolipid component or subunit c, whereof the c- and n-termini as well as loop  <dig> are located either on the vacuolar or on the extracellular side of the membrane.
in gap-junctional structures, where ductin is supposed to form membrane channels, these regions are located on the cytoplasmic side of the membrane  <cit> .
using light and electron microscopy, it has been shown that antibodies against ductin bind to antigens located in the cytoplasm and in plasma membranes of various drosophila tissues, especially of ovarian follicles and embryos .
in part, cytoplasmic labeling might represent storage or transport of ductin.
however, since v-atpases are required for the acidification of organelles , cytoplasmic labeling is likely to also represent secretory vesicles, lysosomes or endosomes in which ligand-receptor complexes become dissociated .
on the other hand, v-atpases coupled to secondary active antiport mechanisms are known to energize transport processes across plasma membranes .
these pumps are expected to exert direct or indirect influence on intracellular ph and membrane potentials.
in the follicle-cell epithelium of drosophila, antibodies against both subunits a and c revealed an asymmetric distribution of v-atpases which is in accordance with the gradual dorsoventral distribution of epithelial membrane potentials  <cit> .
this observation points to a possible involvement of v-atpases in the regulation or maintainance of the spatial coordinates during oogenesis, a phenomenon that has been observed in other systems .
moreover, v-atpases are also known to be involved in the regulation of cell division, migration and differentiation .
labeling of plasma membranes with antibodies against ductin was either continuous or punctate .
continuous labeling is presumed to originate from ductin as part of v-atpases, while punctate labeling might originate from ductin as part of gap-junctional structures.
by microinjection experiments, it has been shown that antibodies directed against presumed cytoplasmic regions of ductin specifically block intercellular communication and exert adverse influence on oogenesis as well as on embryogenesis  <cit> .
interestingly, ductin labeling was prominent in both stalk cells and polar cells that derive from a common cell population in the germarium.
polar cells are known to control stalk formation as well as axis formation, they recruit the border cells and they express various cell-specific markers  <cit> .
evidence amounts that ductin, or subunit c, is a multi-functional channel protein.
it has been observed to form ca2+-inducible membrane pores that are permeable to hydrophilic molecules  <cit> .
it has also been demonstrated that hexamers of subunit c form stacked rings containing an intercellular channel  <cit> .
and, in addition, ductin seems to play a role in the process of vesicle-membrane fusion  <cit> .
the present study lends further support to the notion that ductin might be engaged in several of these functions also during drosophila oogenesis.
although various proteins have been described to be part of drosophila gap junctions , members of the innexin family are assumed to be the main invertebrate gap-junction proteins  <cit> .
in the drosophila ovary, the mrnas of innexins  <dig>   <dig>   <dig>   <dig> and   <dig> have been detected  <cit> .
using various antibodies against innexins , inx <dig> was found to be predominantly located in the baso-lateral domain of follicle cells, whereas inx <dig> is positioned apico-laterally as well as apically between follicle cells and germ-line cells.
inx <dig> was observed laterally in follicle cells and also in nurse cells, and inx <dig> was detected in the oolemma up to stage  <dig> and in nurse-cell membranes up to stage  <dig>  while inx <dig> and inx <dig> are colocalized between somatic cells, inx <dig> and inx <dig> are colocalized between somatic cells and germ-line cells  <cit> .
microinjected antibodies directed against presumed cytoplasmic regions of inx <dig> specifically blocked intercellular communication and lead to inhibitory effects on oogenesis that were similar to those obtained with antibodies directed against presumed cytoplasmic regions of ductin  <cit> .
even during spermatogenesis, inx <dig> was found to be involved in soma-to-germ line intercellular communication  <cit> .
moreover, it has been demonstrated that, within the germarium, inx <dig> supports germ-cell survival, cyst formation and early follicle development  <cit> .
during embryogenesis, inx <dig> was shown to participate in organizing epithelia by interacting with core proteins of adherens and septate junctions, and it has been presumed that inx <dig> exerts its structural functions indirectly through the formation of channels for intercellular communication .
recently, inx <dig> has also been identified as a regulator of eye development  <cit> .
contrary to inx <dig>  inx <dig> shows a striking non-uniform distribution in the follicle-cell epithelium  <cit> : it is enriched in the lateral membranes of centripetally migrating follicle cells.
this indicates distinct coupling conditions between these cells concerning e.g.
regulatory signals, membrane potentials or intracellular ph.
moreover, inx <dig> has been described to be involved in maintaining tissue integrity in response to tension  <cit> , a feature that is particularly important for migrating cells.
the spatial restriction of specialized intercellular channels and the independent regulation of their permeability is suspected to provide mechanisms to generate or maintain boundaries between distinct follicle-cell populations  <cit> .
by the use of various proteins in either homomeric, heteromeric or heterotypic combinations, a wealth of different gap-junction channels might be formed .
compared to neighboring cells, the centripetally migrating follicle cells are specialized in several respect:  they are more acidic and more depolarized  <cit> ,  they possess higher amounts of active voltage-dependent l-type ca2+-channels  <cit>  ,  they contain higher ca2+-concentrations  <cit> ,  they possess higher amounts of na+,k+-atpase  <cit>  , and  they remain coupled via gap junctions to the oocyte when neighboring cells are already uncoupled  <cit> .
the permeability of innexin channels in general has been reported to be sensitive to membrane potential, intracellular ph, k+- and ca2+-concentrations .
gap-junctional communication between oocyte and follicle cells was found to be inhibited by low extracellular ph, by low extracellular k+-concentrations , and by high ca2+-concentrations in the oocyte  <cit> .
taken together, these findings support the notion that intercellular communication in drosophila ovarian follicles is precisely regulated.
similar to ductin and to the pannexins in vertebrates, innexins have also been found to form non-junctional channels  allowing the secretion of small molecules, including atp, into the extracellular space .
innexons were observed to open in response to mechanical stress, depolarization, high intracellular ca2+- and high extracellular k+-concentrations, and to close due to low intracellular ph  <cit> .
CONCLUSIONS
in recent years, the origin as well as the physiological and cellular functions of bioelectric phenomena have attracted growing attention and, thus, their meaning becomes more and more elucidated .
accordingly, the distribution and activity patterns of membrane channels, like ductin and innexins, during the course of drosophila oogenesis are supposed to contribute to developmentally important bioelectric signals  <cit> .
since ductin shows overlapping distribution patterns with both inx <dig> and inx <dig> , these proteins might be, in part, located within the same gap-junctional structures, especially between neighboring follicle cells.
however, the analysed proteins are often found concentrated in different membrane plaques which supports the notion of different gap junctions with a large variety of protein compositions.
particularly, ductin is enriched in polar cells and stalk cells, while inx <dig> is enriched in the oolemma, and inx <dig> is enriched in centripetally migrating follicle cells, but the other proteins are not absent from these regions.
since ductin is part of a proton pump and, like the innexins, can form junctional as well as non-junctional membrane channels, a plethora of cellular functions could be realized by using only the three proteins analysed in the present study.
abbreviations
a, anterior; ad, anterodorsal; bc, border cells; bsa, bovine serum albumine; c, centripetally migrating; dapi, 4’,6-diamidino-2-phenylindole; dmso, dimethyl sulfoxide; fc, follicle cells; inx <dig>  innexin 2; inx <dig>  innexin 3; lsm, laser-scanning microscopy; m, membrane; n, nucleus; nc, nurse cells; nis, non-immune serum; ooc, oocyte; ool, oolemma; p, posterior; pbs, phosphate buffered saline; pc, polar cells; sc, stalk cells; sds-page, sodium dodecyl sulphate polyacrylamide gel-electrophoresis; wfm, wide-field microscopy

 the concerted action of ion-transport mechanisms and gap junctions gives rise to bioelectric phenomena, like ph gradients, voltage gradients and ion fluxes within cells, tissues, organs, and even organisms. such bioelectric phenomena are known to play regulatory roles in various developmental and regenerative processes, e.g. proliferation, differentiation, polarisation and migration. using a variety of approaches, we have gathered considerable information on the electrical properties as well as on the distribution and activity patterns of ion-transport mechanisms and gap junctions in the ovary of drosophila melanogaster. following this strategy in a model system, we are trying to further clarify the roles that bioelectric phenomena play during development, e.g. for ph-regulation, osmoregulation, cell-cell communication, cell migration, cell proliferation, cell death, vitellogenesis and growth. during the course of drosophila oogenesis, characteristic extracellular current patterns as well as membrane-potential changes in germ-line and soma cells have been observed that partly depend on the exchange of protons, potassium ions and sodium ions. moreover, striking stage-specific patterns of membrane potentials and intracellular ph that characterize distinct cell populations have been described recently. these bioelectric patterns were found to be related to the distribution patterns of various membrane channels, namely v-atpases, l-type ca2+-channels, amiloride-sensitive na+-channels and na+,h+-exchangers, as well as innexin-3-containing gap junctions. therefore, these membrane proteins are likely to be involved in the regulation of membrane potentials and/or intracellular ph. an ovarian follicle of drosophila consists of germ-line cells surrounded by a layer of somatic follicle cells. the oocyte and its nurse cells form a cytoplasmic continuum via intercellular bridges as well as via gap junctions, and the same holds true for the follicle cells. on the other hand, the germ-line cells are connected to the soma cells only via gap junctions. by way of the intercellular exchange of microinjected fluorescent tracers, stage-specific communication between germ-line cells and soma cells has been detected, and a variety of treatments was found to either inhibit or stimulate this communication. innexins are considered the main gap-junction proteins of invertebrates, but there is some evidence that additional proteins, like e.g. ductin, can be part of gap junctions. therefore, it is tempting to analyse whether or not ductin and members of the innexin family are found in common gap-junction plaques. previously, the localization of ductin as well as the localization of innexins to have been separately analysed in drosophila ovarian follicles in detail. in the present study, we try to clarify the relationship of ductin, known as a channel-forming subunit of v-atpases, with innexins and two well known components of gap junctions, using double-immunolabeling. we apply combinations of four antisera directed against ductin, another v-atpase component in the membrane-spanning v domain, innexin and innexin respectively. these antisera have previously been shown to specifically recognize cellular antigens in the drosophila ovary. methods preparation of follicles drosophila melanogaster wild-type oregon r flies were reared at about 20 °c on standard food with additional fresh yeast. individual 2– days old females were killed by crushing the thorax with tweezers without previous anesthetization. the ovaries were dissected with tweezers in drosophila pbs, and single follicles of different stages were isolated by pulling at the anterior tip of an ovariole. antisera to localize v-atpase and gap-junction proteins, we used a rabbit antiserum raised and affinity-purified against a highly conserved region of the 16 kda-protein ductin, which forms subunit c of v-atpases and is also supposed to be a part of gap junctions, a guinea-pig antiserum raised against an n-terminal region of subunit a of the manduca sexta v-atpase, kindly provided by b. walz and o. baumann, a guinea-pig antiserum raised against the cytoplasmic loop of innexin and a guinea-pig antiserum raised against the c-terminus of innexin from drosophila, kindly provided by r. bauer and m. hoch. a rabbit non-immune serum was used as control. immunoblotting immunoblots were performed as described previously. in short, homogenates of ovaries were sonicated and briefly boiled. the proteins were separated using 12 % sds-page and transferred to nitrocellulose membranes. nonspecific binding sites were blocked with 5 % skimmed milk powder/pbs and the blots were incubated in anti-ductin diluted 1: up to 1: with 1 % bsa/pbs. nis diluted 1: was used as a control. subsequently, biotinylated goat-anti-rabbit igg, streptavidin-peroxidase and h2o2/4-chloro-1-naphthol were applied, and photographs were taken using a digital camera. experiments were performed three times. indirect immunofluorescence preparations follicles were fixed for 30 min at 4 °c in 4 % formaldehyde dissolved in pbs, washed in pbs and blocked for 1 h at 20 °c with 2 % bsa/  % triton x- in pbs. thereafter, the follicles were incubated overnight at 4 °c in pbs containing  % bsa/  % triton x- and two of the following antisera for double-labeling: anti-ductin diluted 1: anti-v-atpase a diluted 1: anti-inx diluted 1: anti-inx diluted 1: controls were performed without antiserum and using nis 1:, respectively. after washing six times for 10 min, the follicles were treated with goat-anti-rabbit-cy and donkey-anti-guinea-pig-fp for 1 h in pbs containing  % bsa/  % triton x- washing was repeated six times and the nuclei were stained with  μg/ml dapi in pbs for 3 min. thereafter, the follicles were mounted in fluoromount g and viewed, by using x20/, x40/ or x63/ oil objectives and the appropriate filter sets, either on a zeiss axiovert wide-field fluorescence microscope, equipped with a hamamatsu orca er camera, or they were mounted in glycerine/pbs 1: and viewed on a leica dmre laser-scanning microscope. each experiment was repeated at least three times.  immunoblot analysis of anti-ductin using immunoblots of drosophila ovary preparations, we analysed the binding properties of the commercially available antiserum ab. as described earlier for two other anti-ductin sera ), anti-ductin recognized a strong band at 29 kda and weak bands at and 45 kda. no specific binding was obtained with nis. the 16 kda-band represents the monomer of ductin and the other bands represent the putative dimer and trimer, respectively. thus, the binding of anti-ductin in indirect immunofluorescence preparations is assumed to be specific for the channel-forming protein ductin in drosophila ovarian follicles.fig. ductin is localized stage-specifically in plasma membranes and cytoplasmic vesicles of follicle cells and germ-line cells. a: on immunoblots of ovary extracts using anti-ductin, a band at 29 kda and weak bands at about 16 kda and 45 kda are recognized. a’: no specific staining was obtained with nis. b to g: lsm images; b: ductin is found in plasma membranes and cytoplasmic particles or vesicles, especially in polar cells. c: ductin becomes increasingly localized to plasma membranes in nurse cells and follicle cells. d: ductin is found in plasma membranes of centripetally migrating fc. e: ductin is localized in lateral and apical fc membranes, in the oolemma and, most prominently, in pc and stalk cells. f: ductin is found in plasma membranes of squamous fc and nc and around nc nuclei. g: ductin is concentrated in plaques in lateral fc membranes localization of ductin in indirect immunofluorescence preparations using anti-ductin and laser-scanning microscopy, we investigated the distribution of ductin during the course of oogenesis. as shown previously using the two other anti-ductin sera on sections prepared for immuno-fluorescence and immuno-electron microscopy, the continuous as well as punctate distribution of ductin was found to change stage-specifically. in the following, we concentrate on ductin in specialized types of follicle cells and on some main features of its distribution relevant to the present comparative analysis. during previtellogenesis, ductin is located in cytoplasmic particles or vesicles as well as in plasma membranes and is enriched in polar cells. beginning with vitellogenesis, ductin increasingly becomes located in plasma membranes of nurse cells and follicle cells. during vitellogenesis, ductin is located in cytoplasmic vesicles, in plasma membranes of centripetally migrating follicle cells, in lateral and apical plasma membranes of columnar follicle cells, in plasma membranes of squamous follicle cells and nurse cells as well as around nurse-cell nuclei, in the oolemma, and, most prominently, in polar cells and stalk cells. predominantly in lateral and apical follicle-cell membranes, ductin is localized in a punctate pattern resembling gap-junction plaques ). no specific staining was found in control preparations without primary antiserum or using nis. colocalization of ductin with v-atpase subunit a since ductin forms subunit c of v-atpases, it is expected to localize, at least in part, together with subunit a in the same cellular regions. we analysed follicles double-labeled with anti-ductin and anti-v-atpase a using wide-field fluorescence microscopy. stage-specifically, ductin and subunit a were found colocalized in plasma membranes and in the cytoplasm of follicle cells and germ-line cells, especially in cytoplasmic particles or vesicles.fig. ductin and v-atpase subunit a are partly colocalized in plasma membranes and in the cytoplasm of follicle cells and germ-line cells, often in cytoplasmic particles or vesicles. a to c: ductin; a’ to c’: v-atpase subunit a; a” to c” and d to f: merged images, ductin, v-atpase subunit a and colocalization of both, wfm. nuclei were stained with dapi. no specific staining was obtained with nis. in young previtellogenic follicles, ductin and v-atpase subunit a co-occur in anterior and posterior polar cells, in lateral and apical follicle-cell membranes, and in the cytoplasm of fc, nurse cells and the oocyte. while cytoplasmic particles or vesicles containing predominantly v-atpase subunit a can be observed, ductin is enriched in stalk cells. in older previtellogenic follicles, ductin and v-atpase subunit a are enriched in afcm. while ductin is found in higher amounts in ppc and sc, v-atpase subunit a is found in higher amounts in the cytoplasm of fc, nc and ooc. in vitellogenic follicles, ductin and v-atpase subunit a are colocalized especially in columnar and squamous fc. while ductin is enriched in nc membranes, v-atpase subunit a is enriched in border cells. d: ductin and v-atpase subunit a are colocalized in particles or vesicles in squamous fc. e: ductin and v-atpase subunit a are colocalized in lateral fc membranes as well as in the cytoplasm. particles or vesicles containing v-atpase subunit a are found in fcn. f: in older follicles, ductin and v-atpase subunit a are colocalized in particles or vesicles in most of the fc while, in anterodorsal fc, predominantly v-atpase subunit a is found in the cytoplasm and in the nuclei. scale bars, 10 μm during early previtellogenesis, ductin and subunit a co-occur in polar cells, in lateral and apical follicle-cell membranes and in the cytoplasm of follicle cells, nurse cells and the oocyte. however, while ductin was found to be more prominent in stalk cells, the distinct particles or vesicles in germ-line cells were found to contain predominantly subunit a. during late previtellogenesis, both ductin and subunit a are enriched in apical follicle-cell membranes. on the other hand, ductin was found to be more prominent in posterior polar cells and in stalk cells, while subunit a was more prominent in the cytoplasm of follicle cells, nurse cells and the oocyte. during vitellogenesis, ductin and subunit a co-occur especially in columnar and squamous follicle cells and in border cells. in columnar follicle cells, the amount is usually higher on one side of the follicle, namely the ventral side. here, ductin and subunit a are colocalized in the plasma membranes and in the cytoplasm while, in squamous follicle cells, they are are colocalized in vesicles. on the other hand, ductin is enriched in nurse-cell membranes, while subunit a is enriched in border cells as well as in nuclear particles or vesicles in follicle cells. during late vitellogenesis, ductin and subunit a are colocalized in cytoplasmic vesicles in most of the follicle cells, whereas, in the cytoplasm and nuclei of anterodorsal follicle cells, predominantly subunit a was found. taken together, ductin and subunit a are stage-specifically either colocalized or separately enriched in different cellular regions of germ-line and soma cells. frequently, ductin is more prominent in plasma membranes, while subunit a is more prominent in cytoplasmic and nuclear particles or vesicles. colocalization of ductin with innexins since ductin is known to form intercellular communication channels, it might localize, in part, together with members of the channel-forming innexin family in the same cellular regions. in drosophila ovarian follicles, the localization of innexins to has been described in detail previously using lsm and wfm. for the present comparative analysis, we concentrate on some main features concerning inx and inx that both showed distribution patterns similar to ductin. ductin and inx2 we analysed follicles double-labeled with anti-ductin and anti-inx using wfm. during previtellogenesis, ductin and inx co-occur in lateral and apical follicle-cell membranes and in the cytoplasm of follicle cells, nurse cells and the oocyte. however, ductin was found to be enriched in nurse-cell membranes and in the oolemma as well as in polar cells and in stalk cells, whereas cytoplasmic particles or vesicles containing predominantly inx were observed in germ-line cells.fig. ductin and inx are partly colocalized in plasma membranes and in the cytoplasm of follicle cells and germ-line cells, but often in different plaques. a to c: ductin; a’ to c’: inx2; a”: merged image, ductin, inx and colocalization of both, wfm. nuclei were stained with dapi. no specific staining was obtained with nis. in previtellogenic follicles, ductin and inx co-occur in lateral and apical follicle-cell membranes and in the cytoplasm of fc, nurse cells and the oocyte. while prominent cytoplasmic particles or vesicles containing inx can be observed in germ-line cells, ductin is found to be enriched in nc membranes, in the oolemma, in anterior and posterior polar cells and in stalk cells. in vitellogenic follicles, ductin and inx co-occur in lateral and apical fc membranes, while ductin is found to be enriched in ppc, and inx in the ool, respectively. both proteins are concentrated in lateral fcm, but they often localize in different plaques. scale bars, 10 μm also during vitellogenesis, ductin and inx co-occur in lateral and apical follicle-cell membranes. on the other hand, ductin is still enriched in posterior polar cells, while inx is now enriched in the oolemma. in lateral follicle-cell membranes, both proteins are often found concentrated in different plaques that are presumed to represent different gap junctions. ductin and inx3 follicles double-labeled with anti-ductin and anti-inx were also analysed using wfm. in previtellogenic and early vitellogenic stages, ductin and inx co-occur in follicle-cell membranes. however, ductin is more prominent in nurse-cell membranes and in the oolemma as well as in polar cells, in stalk cells and in the cytoplasm of germ-line cells. on the other hand, inx was found to be enriched around nurse-cell nuclei and in lateral plasma membranes of the prospective centripetally migrating follicle cells.fig. ductin and inx are partly colocalized in plasma membranes of follicle cells and germ-line cells, but often in different plaques. a to c: ductin; a’ to c’: inx3; a” to c”: merged images, ductin, inx and colocalization of both, wfm. nuclei were stained with dapi. no specific staining was obtained with nis. in previtellogenic and early vitellogenic follicles, ductin and inx co-occur in follicle-cell membranes. while ductin is found to be enriched in nurse-cell membranes, in the oolemma, in polar cells, in stalk cells and in the cytoplasm of nc and oocyte, inx is enriched especially in lateral membranes of the prospective centripetally migrating fc and around nc nuclei. in later stages, ductin and inx co-occur in lateral fcm and in ncm. while inx exhibits prominent membrane plaques, ductin is enriched in apical fcm and in the ool c. both proteins are concentrated in lateral fcm, but they often localize in different plaques. scale bars, 10 μm in mid to late vitellogenic stages, ductin and inx co-occur in lateral follicle-cell membranes and in nurse-cell membranes. while ductin is enriched in apical follicle-cell membranes and in the oolemma, inx forms prominent plaques in lateral follicle-cell membranes. here, ductin and inx are often found concentrated in different plaques that are presumed to represent different gap junctions. taken together, ductin and both innexins are stage-specifically either colocalized or separately enriched in different cellular regions of germ-line and soma cells. frequently, ductin shows a rather continuous membrane distribution, while the distribution of both innexins is more punctate.fig. summary of colocalization or local enrichment of ductin, v-atpase subunit a, inx and inx respectively, during previtellogenic stages and vitellogenic stages. a, a’: ductin and v-atpase subunit a; b, b’: ductin and inx2; c, c’: ductin and inx ductin and subunit a are stage-specifically either colocalized in plasma membranes or in the cytoplasm, which is supposed to represent the distribution of v-atpases, or separately enriched in different cellular regions of germ-line and soma cells. frequently, ductin is more prominent in plasma membranes, while subunit a is more prominent in cytoplasmic and nuclear particles or vesicles. punctate membrane labeling is presumed to originate from ductin as part of membrane channels or gap-junctional structures. a: in previtellogenic stages, ductin is enriched in posterior polar cells and in stalk cells, while subunit a is enriched in cytoplasmic particles or vesicles in follicle cells, nurse cells and the oocyte. a’: in vitellogenic stages, the dorsoventral gradient of v-atpase distribution in the fc epithelium is in accordance with the gradual distribution of epithelial membrane potentials. while ductin is enriched in nc, ppc and sc, subunit a is enriched in border cells and nuclear particles or vesicles in fc. since pc, bc and ventral fc are also characterized through a high activity of l-type ca2+-channels, alterations of membrane potentials through v-atpase activity are supposed to result in cellular responses due to voltage-dependent ca2+-currents. ductin and inx as well as ductin and inx are stage-specifically either colocalized or separately enriched in different cellular regions of germ-line and soma cells. frequently, ductin shows a rather continuous membrane distribution, presumably representing v-atpases, while the distribution of both innexins is more punctate, presumably representing gap-junctional structures. in lateral fc membranes, both ductin and inx as well as ductin and inx are often found concentrated in different plaques that are presumed to represent different gap-junctional structures. b: in previtellogenic stages, ductin is enriched in nc membranes and in the oolemma as well as in pc and sc, whereas inx is enriched in cytoplasmic particles or vesicles in germ-line cells. b’: in vitellogenic stages, ductin is enriched in ppc, while inx is enriched in the ool. antibodies directed against presumed cytoplasmic regions of both ductin or inx microinjected into the oocyte specifically blocked intercellular communication through ductin- or inx2-containing gap junctions. c: in previtellogenic stages, ductin is more prominent in ncm and in the ool as well as in pc, in sc and in the cytoplasm of germ-line cells, while inx is more prominent around ncn and in lateral plasma membranes of the prospective centripetally migrating fc. c’: in vitellogenic stages, ductin is enriched in apical fcm and in the ool as well as in ppc, bc and sc, while inx forms prominent plaques in lateral membranes of cfc. inx3-containing gap junctions are supposed to specifically regulate the distribution of membrane potentials, intracellular ph and ions within cfc and to establish a communication border to neighboring fc discussion the channel-forming protein ductin has been found in both v-atpases and gap-junctional structures. in v-atpases, it forms the proteolipid component or subunit c, whereof the c- and n-termini as well as loop are located either on the vacuolar or on the extracellular side of the membrane. in gap-junctional structures, where ductin is supposed to form membrane channels, these regions are located on the cytoplasmic side of the membrane. using light and electron microscopy, it has been shown that antibodies against ductin bind to antigens located in the cytoplasm and in plasma membranes of various drosophila tissues, especially of ovarian follicles and embryos. in part, cytoplasmic labeling might represent storage or transport of ductin. however, since v-atpases are required for the acidification of organelles, cytoplasmic labeling is likely to also represent secretory vesicles, lysosomes or endosomes in which ligand-receptor complexes become dissociated. on the other hand, v-atpases coupled to secondary active antiport mechanisms are known to energize transport processes across plasma membranes. these pumps are expected to exert direct or indirect influence on intracellular ph and membrane potentials. in the follicle-cell epithelium of drosophila, antibodies against both subunits a and c revealed an asymmetric distribution of v-atpases which is in accordance with the gradual dorsoventral distribution of epithelial membrane potentials. this observation points to a possible involvement of v-atpases in the regulation or maintainance of the spatial coordinates during oogenesis, a phenomenon that has been observed in other systems. moreover, v-atpases are also known to be involved in the regulation of cell division, migration and differentiation. labeling of plasma membranes with antibodies against ductin was either continuous or punctate. continuous labeling is presumed to originate from ductin as part of v-atpases, while punctate labeling might originate from ductin as part of gap-junctional structures. by microinjection experiments, it has been shown that antibodies directed against presumed cytoplasmic regions of ductin specifically block intercellular communication and exert adverse influence on oogenesis as well as on embryogenesis. interestingly, ductin labeling was prominent in both stalk cells and polar cells that derive from a common cell population in the germarium. polar cells are known to control stalk formation as well as axis formation, they recruit the border cells and they express various cell-specific markers. evidence amounts that ductin, or subunit c, is a multi-functional channel protein. it has been observed to form ca2+-inducible membrane pores that are permeable to hydrophilic molecules. it has also been demonstrated that hexamers of subunit c form stacked rings containing an intercellular channel. and, in addition, ductin seems to play a role in the process of vesicle-membrane fusion. the present study lends further support to the notion that ductin might be engaged in several of these functions also during drosophila oogenesis. although various proteins have been described to be part of drosophila gap junctions, members of the innexin family are assumed to be the main invertebrate gap-junction proteins. in the drosophila ovary, the mrnas of innexins and have been detected. using various antibodies against innexins, inx was found to be predominantly located in the baso-lateral domain of follicle cells, whereas inx is positioned apico-laterally as well as apically between follicle cells and germ-line cells. inx was observed laterally in follicle cells and also in nurse cells, and inx was detected in the oolemma up to stage and in nurse-cell membranes up to stage while inx and inx are colocalized between somatic cells, inx and inx are colocalized between somatic cells and germ-line cells. microinjected antibodies directed against presumed cytoplasmic regions of inx specifically blocked intercellular communication and lead to inhibitory effects on oogenesis that were similar to those obtained with antibodies directed against presumed cytoplasmic regions of ductin. even during spermatogenesis, inx was found to be involved in soma-to-germ line intercellular communication. moreover, it has been demonstrated that, within the germarium, inx supports germ-cell survival, cyst formation and early follicle development. during embryogenesis, inx was shown to participate in organizing epithelia by interacting with core proteins of adherens and septate junctions, and it has been presumed that inx exerts its structural functions indirectly through the formation of channels for intercellular communication. recently, inx has also been identified as a regulator of eye development. contrary to inx inx shows a striking non-uniform distribution in the follicle-cell epithelium: it is enriched in the lateral membranes of centripetally migrating follicle cells. this indicates distinct coupling conditions between these cells concerning e.g. regulatory signals, membrane potentials or intracellular ph. moreover, inx has been described to be involved in maintaining tissue integrity in response to tension, a feature that is particularly important for migrating cells. the spatial restriction of specialized intercellular channels and the independent regulation of their permeability is suspected to provide mechanisms to generate or maintain boundaries between distinct follicle-cell populations. by the use of various proteins in either homomeric, heteromeric or heterotypic combinations, a wealth of different gap-junction channels might be formed. compared to neighboring cells, the centripetally migrating follicle cells are specialized in several respect: they are more acidic and more depolarized, they possess higher amounts of active voltage-dependent l-type ca2+-channels, they contain higher ca2+-concentrations, they possess higher amounts of na+,k+-atpase, and they remain coupled via gap junctions to the oocyte when neighboring cells are already uncoupled. the permeability of innexin channels in general has been reported to be sensitive to membrane potential, intracellular ph, k+- and ca2+-concentrations. gap-junctional communication between oocyte and follicle cells was found to be inhibited by low extracellular ph, by low extracellular k+-concentrations, and by high ca2+-concentrations in the oocyte. taken together, these findings support the notion that intercellular communication in drosophila ovarian follicles is precisely regulated. similar to ductin and to the pannexins in vertebrates, innexins have also been found to form non-junctional channels allowing the secretion of small molecules, including atp, into the extracellular space. innexons were observed to open in response to mechanical stress, depolarization, high intracellular ca2+- and high extracellular k+-concentrations, and to close due to low intracellular ph.  in recent years, the origin as well as the physiological and cellular functions of bioelectric phenomena have attracted growing attention and, thus, their meaning becomes more and more elucidated. accordingly, the distribution and activity patterns of membrane channels, like ductin and innexins, during the course of drosophila oogenesis are supposed to contribute to developmentally important bioelectric signals. since ductin shows overlapping distribution patterns with both inx and inx, these proteins might be, in part, located within the same gap-junctional structures, especially between neighboring follicle cells. however, the analysed proteins are often found concentrated in different membrane plaques which supports the notion of different gap junctions with a large variety of protein compositions. particularly, ductin is enriched in polar cells and stalk cells, while inx is enriched in the oolemma, and inx is enriched in centripetally migrating follicle cells, but the other proteins are not absent from these regions. since ductin is part of a proton pump and, like the innexins, can form junctional as well as non-junctional membrane channels, a plethora of cellular functions could be realized by using only the three proteins analysed in the present study. abbreviations a, anterior; ad, anterodorsal; bc, border cells; bsa, bovine serum albumine; c, centripetally migrating; dapi, 4’,6-diamidino-2-phenylindole; dmso, dimethyl sulfoxide; fc, follicle cells; inx innexin 2; inx innexin 3; lsm, laser-scanning microscopy; m, membrane; n, nucleus; nc, nurse cells; nis, non-immune serum; ooc, oocyte; ool, oolemma; p, posterior; pbs, phosphate buffered saline; pc, polar cells; sc, stalk cells; sds-page, sodium dodecyl sulphate polyacrylamide gel-electrophoresis; wfm, wide-field microscopy 
ion-transport mechanisms and gap junctions are known to cooperate in creating bioelectric phenomena, like ph gradients, voltage gradients and ion fluxes within single cells, tissues, organs, and whole organisms. our results support the notion of a large variety of gap junctions existing in the drosophila ovary. while ductin was often more prominent in plasma membranes, subunit a was more prominent in cytoplasmic and nuclear vesicles. comparably, ductin and both innexins and were either colocalized or separately enriched in different cellular regions. such phenomena have been shown to play regulatory roles in a variety of developmental and regenerative processes. we, therefore, investigated the stage-specific distribution patterns of v-atpase components in relation to gap-junction proteins. while ductin often showed a continuous membrane distribution, the distribution of both innexins was mostly punctate. using drosophila oogenesis as a model system, we aim at characterizing in detail the mechanisms underlying bioelectric phenomena in order to reveal their regulatory functions. these types of follicle cells had previously been shown to exhibit characteristic patterns of membrane channels as well as membrane potential and intracellular ph. moreover, since ductin is the channel-forming part of a proton pump and, like the innexins, is able to form junctional as well as non-junctional membrane channels, a plethora of cellular functions could be realized by using these proteins. the distribution and activity patterns of such membrane channels are expected to contribute to developmentally important bioelectric signals. particularly, ductin was enriched in polar cells, stalk cells, and nurse-cell membranes, whereas subunit a was enriched in the cytoplasm of border cells, columnar follicle cells and germ-line cells. drosophila melanogasteroogenesisbioelectricitycell communicationpattern formationproton pumpgap junctionissue-copyright-statement© the author 2016 we analysed the localization of the v-atpase components ductin and subunit a, and the gap-junction components innexins and especially in polar cells, border cells, stalk cells and centripetally migrating cells. particularly, ductin was enriched in polar cells and stalk cells, whereas innexin was enriched in the oolemma, and innexin in centripetally migrating follicle cells. stage-specifically, ductin and subunit a were found either colocalized or separately enriched in different regions of soma and germ-line cells. in lateral follicle-cell membranes, the three proteins were found colocalized as well as separately concentrated in presumed gap-junction plaques.
 ion-transport mechanisms and gap junctions are known to cooperate in creating bioelectric phenomena, like ph gradients, voltage gradients and ion fluxes within single cells, tissues, organs, and whole organisms. such phenomena have been shown to play regulatory roles in a variety of developmental and regenerative processes. using drosophila oogenesis as a model system, we aim at characterizing in detail the mechanisms underlying bioelectric phenomena in order to reveal their regulatory functions. we, therefore, investigated the stage-specific distribution patterns of v-atpase components in relation to gap-junction proteins.  we analysed the localization of the v-atpase components ductin and subunit a, and the gap-junction components innexins and especially in polar cells, border cells, stalk cells and centripetally migrating cells. these types of follicle cells had previously been shown to exhibit characteristic patterns of membrane channels as well as membrane potential and intracellular ph. stage-specifically, ductin and subunit a were found either colocalized or separately enriched in different regions of soma and germ-line cells. while ductin was often more prominent in plasma membranes, subunit a was more prominent in cytoplasmic and nuclear vesicles. particularly, ductin was enriched in polar cells, stalk cells, and nurse-cell membranes, whereas subunit a was enriched in the cytoplasm of border cells, columnar follicle cells and germ-line cells. comparably, ductin and both innexins and were either colocalized or separately enriched in different cellular regions. while ductin often showed a continuous membrane distribution, the distribution of both innexins was mostly punctate. particularly, ductin was enriched in polar cells and stalk cells, whereas innexin was enriched in the oolemma, and innexin in centripetally migrating follicle cells. in lateral follicle-cell membranes, the three proteins were found colocalized as well as separately concentrated in presumed gap-junction plaques.  our results support the notion of a large variety of gap junctions existing in the drosophila ovary. moreover, since ductin is the channel-forming part of a proton pump and, like the innexins, is able to form junctional as well as non-junctional membrane channels, a plethora of cellular functions could be realized by using these proteins. the distribution and activity patterns of such membrane channels are expected to contribute to developmentally important bioelectric signals. keywords drosophila melanogasteroogenesisbioelectricitycell communicationpattern formationproton pumpgap junctionissue-copyright-statement© the author 2016 