1 Drosophila oocyte polarity and cytoskeleton organization requires ...

1

Drosophila oocyte polarity and cytoskeleton organization requires regulation of 1

Ik2 activity by Spn-F and Javelin-like 2

Simha Amsalem1, Anna Bakrhat

1, Tetsuhisa Otani

2, Shigeo Hayashi

2,3, Bareket 3

Goldstein1, Abdu Uri

1,# 4

1Department of Life Sciences, Ben-Gurion University, Beer-Sheva 84105, Israel. 5

2Laboratory for Morphogenetic Signaling, RIKEN Center for Developmental Biology, 6

2-2-3, Minatojima-Minamimachi, Chuo-ku, Kobe, Hyogo 650-0047, Japan. 7

3Department of Biology, Kobe University Graduate School of Science, Kobe, Hyogo 8

657-8501, Japan 9

10

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Keywords: Cytoskeleton, Drosophila, Gurken, Ik2, Javelin-like, Oskar, Spn-F, 13

Running title: Regulation of Ik2 activity in oocyte polarity. 14

#Correspondence should be addressed to Uri Abdu: [email protected] 15

16

MCB Accepts, published online ahead of print on 9 September 2013Mol. Cell. Biol. doi:10.1128/MCB.00713-13Copyright © 2013, American Society for Microbiology. All Rights Reserved.

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Abstract 18

The Drosophila Spn-F, Ik2 and Javelin-like (Jvl) proteins interact to regulate oocyte 19

mRNA localization and cytoskeleton organization. However, the mechanism by which 20

these proteins interact remains unclear. Using antibodies to activated Ik2, we showed 21

that this protein is found at the region of oocyte and follicle cell where microtubule 22

minus-ends are enriched. We demonstrate that germline Ik2 activation is diminished 23

both in jvl and in spn-F mutant ovaries. Structure-function analysis of Spn-F revealed 24

that the C-terminal end is critical for protein function, since it alone was able to rescue 25

spn-F sterility. On the other hand, germline expression of Spn-F lacking its conserved 26

C-terminal region (Spn-F〉C) phenocopied ik2, leading to production of ventralized 27

eggshell and bicaudal embryos. In Spn-F〉C-expressing oocytes, Gurken protein is 28

mislocalized and oskar mRNA and protein localization is disrupted. Expression of Ik2 29

rescued Spn-F〉C ovarian phenotypes. We found that whereas Spn-F physically 30

interacts with Ik2 and Jvl, Spn-F〉C physically interacts with Ik2 but not with Jvl. 31

Thus, expression of Spn-F〉C, which lacks the Jvl-interacting domain, probably 32

interferes with Ik2 and Jvl interaction. In summary, our results demonstrate that Spn-F 33

mediates the interaction between Ik2 and Jvl to control Ik2 activity. 34

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Introduction 40

During development and cell differentiation, mRNA localization is a crucial step 41

in the regulation of gene expression of many transcripts. Accurate mRNA localization 42

permits precise temporal and spatial regulation of protein production during 43

development in a variety of organisms and cell types. RNA localization has been 44

described in organisms as diverse as yeast and humans and has been observed in many 45

polarized cells, such as oocytes, fibroblasts or neurons. In Drosophila, mRNA 46

localization provides a particularly important mechanism for polar localization of axis-47

determining factors during oogenesis. The asymmetric localization of mRNA within the 48

developing egg chamber relies on both microtubules (MTs) and actin networks, as well 49

as on motor proteins. Although the organization of MTs and actin during mid-oogenesis 50

has been revealed, the mechanism that leads to this complex cytoskeleton organization 51

is still not fully understood. 52

The Drosophila spindle-F (spn-F), IKKi homologue (Ik2), and the novel 53

microtubule (MT)-associated protein, Javelin-like (Jvl), together produce a complex of 54

proteins that affect both oogenesis and bristle development (1-4). We and others have 55

shown that females carrying mutations in these genes produce eggs and embryos with 56

polarity defects that arise due to disruptions in cytoskeleton organization and mRNA 57

localization during oocyte development (1-2, 4). We, moreover, have demonstrated that 58

these three proteins physically interact and that their proper cell localization and 59

function are inter-dependent (3-4). In their physical interaction, Ik2 phosphorylates Spn-60

F, although such phosphorylation does not affect the stability of the protein (4). In 61

addition, ik2 has also been found to be involved in other processes, including spindle 62

organization (5-6), dendrite pruning (7), bristle MT function (8-9), F-actin assembly 63

regulation (10-11) and in the shuttling of recycling endosomes during bristle cell 64

elongation (12). 65

Closer examination of spn-F and ik2 ovarian defects reveals that whereas both 66

mutants share the same defects in terms of cytoskeleton organization, they differ in their 67

effects on mRNA localization. In the mutants, both transport towards the minus-end of 68

the MT and organization of the MTs that surround the oocyte nucleus are strongly 69

affected (1-2). The spn-F and ik2 mutants also present the same defects in terms of grk 70

mRNA and protein localization. However, while over 90% of the embryos produced by 71


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ik2 mutant females are bicaudal (2), this phenotype is only rarely found in spn-F mutant 72

embryos (1). This difference could be attributed to the fact that in ovaries and embryos 73

produced by ik2 mutant females, oskar (osk) mRNA and protein are localized 74

posteriorly and anteriorly, while in spn-F mutant ovaries, osk mRNA and protein 75

localization are not affected. The difference seen in osk mRNA but not in grk mRNA 76

localization defects between spn-F and ik2 mutants raises the question as to which 77

molecular mechanisms control the actions of these proteins. 78

To better understand the function of these genes in mRNA localization and 79

cytoskeleton organization during development, structure-function analysis of Spn-F 80

protein was conducted. We show that the Spn-F protein may act as mediator between 81

Ik2 and Jvl to regulate Ik2 activity. Thus, our results provide a new perspective on the 82

function of these proteins in pattern formation of the Drosophila egg and embryo 83

demonstrating that Spn-F and Jvl act on the core Ik2 function to augment the activity of 84

this complex. 85

86

Materials and Methods 87

Drosophila stocks 88

Oregon-R served as a wild-type control. The following mutants and transgenic flies 89

were used: spn-F1, Df(3R)tll-e, (1), jv1

l, Df(3R)Exel6275 (3), UASp:GFP-Spn-F, 90

UASp:GFP-Ik2 (4); UASp:GFP-Rad9 (13) kinesin く-GAL insertion line KZ503 and the 91

Nod く-GAL insertion line NZ143.2 (14, 15); P{w+, osk-LacZ} (16). Germline 92

expression was performed with P{matg4- GAL-VP16}V37 (subsequently referred to as 93

g-tub Gal4-3), obtained from the Bloomington Stock Center. 94

95

Cloning and transgenic flies 96

The Spn-F-N and Spn-F-C constructs were described in Dubin-Bar et al. (2008). To 97

create the truncated Spn-F〉C, CC2 and CT Spn-F protein, the sequence coding for that 98

region spanning nucleotides 1–840, 573-819 and 810-1092 respectively, of the gene 99

were amplified by PCR and cloned into the pUASp plasmid in fusion with DNA 100

encoding GFP using the XbaI restriction site. To create a non-GFP-tagged Spn-F〉C 101

construct, Spn-F〉C was cloned into plasmind pUASp using the KpnI site. P-element-102

mediated germline transformation of these constructs was carried out according to 103

standard protocols (17). 104


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Eggshell and cuticle preparations 107

Eggs and embryos were allowed to age for 24 h on plates containing a molasses agar 108

medium at 25ºC. For chorion visualization, eggs were collected and prepared as 109

previously described (18). For cuticle preparation, eggs were collected and washed with 110

0.05% Triton X-100 and dechorionated in 50% commercial bleach (3% sodium 111

hypochlorite) for 2-3 min. Following methanol devitellinization, embryos were mounted 112

in Hoyer’s medium (19), and incubated at 65ºC overnight. Eggs and embryos were 113

viewed by phase contrast microscope. 114

115

In situ hybridization 116

RNA in situ hybridization on ovaries and embryos was carried out as described 117

previously (1, 20). 118

119

ß-galactosidase and antibody staining 120

ß-galactosidase staining of ovaries was performed according to Peretz et al. (2007), with 121

the exception that the ovaries were incubated in X-gal stock solution at room 122

temperature. Antibody staining of ovaries was performed as described previously (4). 123

The following primary antibodies were used: mouse anti-Grk (1:10, clone 1D12) (21), 124

rabbit anti-Oskar (1:3000) (22), mouse anti-g-tubulin (1:100, Sigma), and rabbit anti-125

pIKK (10). Goat anti-mouse Cy3 or Cy2, and goat anti-rabbit Cy3 or Cy2 secondary 126

antibodies (Jackson ImmunoResearch) were used at a dilution of 1:100. For g-tubulin 127

staining, ovaries were kept at room temperature after fixation to prevent MT 128

depolymerization. The following dyes were used: Oregon green 488 and Alexa Fluor 129

568 phalloidin (1:250, Molecular Probes). All pictures were imaged on an Olympus 130

FV1000 laser-scanning confocal microscope. 131

132

Western blot analysis 133

Dissected ovaries were ground in Laemmli sample buffer (10 たl per ovary). The protein 134

extracts were boiled for 5 min and loaded onto a 10% polyacrylamide gel. Following 135

electrophoresis, proteins were transferred to nitrocellulose membranes for 1 h at 300 136

mA. The nitrocellulose membranes were blocked by incubation in TTBS (0.2 M Tris-137

HCl, pH 7.5, 1.5 M NaCl, 9 mM Tween 20) containing 2.5% non-fat dry milk for 30 138


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min at room temperature followed by either a 1 h incubation with anti-acetylated tubulin 139

(1:1000, Sigma) or overnight with anti-Ikk (1:20, 10) primary antibodies. The 140

membranes were washed in TTBS and incubated for 30 min with horseradish 141

peroxidase (HRP)-labeled anti-mouse antibodies (1:2000, Amersham). Antibody 142

labeling was visualized in a Fujifilm LAS300 imager using an ECL detection kit 143

(Biological Industries). 144

145

Co-immunoprecipitation and co-localization assay 146

S2 cells expressing constructs as described in the text were treated with lysis buffer and 147

immuno-complexes were recovered using GFP-Trap_A (Chromotek) according to the 148

manufacturer's instruction. To detect interactions between proteins, Western blot with 149

g-mCherry or g-Ikk antibodies was performed. To detect protein localization patterns, 150

mCherry-Jvl, Myc-tagged Ik2, GFP-Spn-F and GFP-Spn-F〉C constructs were used. 151

For Ik2 detection, primary mouse anti-c-Myc antibodies (1:150, Santa Cruz 152

Biotechnology) were used. Goat anti-mouse Alexa Fluor 633 secondary antibodies 153

(Molecular Probe) were used at a dilution of 1:100. 154

155

Yeast two hybrid assay 156

Yeast two hybrid analysis was performed using the Yeast Two Hybrid Phagemid vector 157

kit (Stratagene), following the manufacturer's instructions. The pAD-Spn-F and 158

truncated version plasmids were used as bait while a plasmid encoding full length Jvl 159

protein was used as prey. 160

161

Results 162

Activation of Ik2 phosphorylation is dependent on spn-F and javelin-like genes 163

To study the activation pattern of Ik2 during oogenesis, we used antibodies raised 164

against Ik2 phosphorylated on serine 175 (pIKK�) (12). We found that in egg chambers 165

from stage 5-6, pIKK� is found throughout the oocyte, with higher accumulation seen 166

at the posterior end (Fig. 1A). Later on, after the oocyte nucleus migrates to the dorsal-167

anterior corner of the oocyte, pIKK� is found at the anterior ring, with higher 168

accumulation seen in the vicinity of the nucleus (Fig. 1B) and in a punctate pattern in 169

nurse cells (Fig. 1B). We also noticed that pIKK� is present on the apical side of the 170


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follicle cells (Fig. 1B'-B’’’). Thus, we found that pIKK� accumulates at MT minus 171

end-rich regions both in the oocyte and in follicle cells. Next, we studied the 172

localization pattern of pIKK� in spn-F and in jvl mutant ovaries and found that whereas 173

in spn-F mutants, anti-pIKK� antibody staining of the oocyte and nurse cells was 174

abolished (Fig. 1C-D), in jvl mutants no such pIKK� staining was detected in the 175

oocyte but was still seen in the nurse cells (Fig. 1D). Also, in both the spn-F (Fig. 1 D') 176

and jvl mutants (Fig. 1F'), anti-pIKK� antibody staining was still evident on the apical 177

side of the follicle cells. Thus, our results suggest that both spn-F and jvl are required 178

for Ik2 function in the germline but not in somatic follicle cells. 179

Next, we studied whether the absence of anti-pIKK� antibody staining in the 180

germline of spn-F and jvl mutants could arise due to defects in Ik2 protein stabilization. 181

Using antibodies directed against the Ik2 protein (10), we thus compared the levels of 182

Ik2 protein in ovarian extracts from wild type flies and in spn-F (Fig. 1G) and jvl 183

mutants (Fig. 1H). We found that in the ovarian extracts of both mutants, the level of 184

Ik2 protein was similar to that of the wild type ovarian extract. Thus, our results show 185

that spn-F and jvl are required for Ik2 activation but not for its stabilization in the 186

germline. 187

188

Expression of C-terminally truncated Spn-F affects the antero-posterior and 189

dorso-ventral axes 190

In this study, we sought to understand how Spn-F affects Ik2 activity by conducting 191

structure-function analysis of the Spn-F protein. Our search for conserved regions of 192

Spn-F revealed the presence of two coiled-coil domains, the first extending from amino 193

acid residue 32 to 114 and the second from residue 210 to 243. We had previously 194

shown that the C-terminal end (but not the N-terminal end) of Spn-F is crucial for 195

interaction with Ik2 (4). To investigate the functional importance of these Spn-F 196

domains, we tested the functions of several mutant Spn-F transgenes, deleted of 197

sequences encoding different domains of the protein. Examination of multi-species spn-198

F protein sequences aligned by ClustalX showed that the two coiled-coil domains are 199

conserved in all species considered. Using this alignment, an additional C-terminal 200

conserved region spanning from amino acid 285 to the end of the protein sequence was 201

found (data not shown). Three deletion constructs were thus generated in plasmid 202

pUASp to yield truncated versions of Spn-F N-terminally tagged with GFP. The first 203

variant encodes the N-terminal region of Spn-F (residues 1-162, hereafter termed Spn-204


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F-N), encompassing the first coiled-coil domain. The second construct encodes the C-205

terminal domain of Spn-F (residues 165-364, hereafter termed Spn-F-C) that includes 206

the second coiled-coil domain of the protein. Finally, the third construct encodes a 207

version of Spn-F that includes the two coiled-coil domains but lacks the 84 C-terminal 208

residues (i.e. residues 1-280, hereafter termed Spn-F〉C) (Fig. 2A). Transgenic flies 209

expressing each of the truncated proteins under the control of the UAS/Gal4 system 210

were created. Three independent transgenic lines were tested for the expression of each 211

of the constructs. All lines showed the same pattern, as described below. To drive the 212

expression of the different Spn-F-encoding constructs, we used mat gTub-GAL4-VP16, 213

a GAL4-VP16 fusion protein expressed under the control of the alphaTub67C promoter 214

(Bloomington stock # 7062 or 7063). These Gal4 drivers lead to higher protein 215

expression when starting from stage 5-6 egg chambers. 216

First, we tested the ability of each of the constructs to rescue spn-F mutant 217

female sterility. We found that neither spn-F-N nor spn-F〉C germline expression 218

rescued spn-F female sterility. On the other hand, 95% of the eggs (n=236) laid by 219

females expressing spn-F-C in spn-F mutants background hatched, demonstrating that 220

the C-terminal half of Spn-F is sufficient for Spn-F function. Interestingly, we noticed 221

that whereas germline expression of spn-F-N and spn-F-C in a wild type background 222

had no effect on female fertility, expression of spn-F〉C led to complete female sterility. 223

Closer examination revealed that 92% (n=1036) of eggs laid by females expressing spn-224

F〉C in the germline produced ventralized eggshells (Figure 2 B-E). Furthermore, we 225

found that expression of spn-F〉C severely affected the anterior-posterior axis of the 226

embryos. 98% (n=126) of the embryos produced by females expressing spn-F〉C 227

showed a strong bicaudal phenotype, with several abdominal segments appearing in 228

mirror image symmetry, and Filzkoerper and telson at both ends (Fig. 2G). The same 229

results were obtained with non-tagged constructs, demonstrating that the defects seen 230

with spn-F〉C flies are not due to fusion of the GFP tag to the protein. 231

232

Expression of C-terminally truncated Spn-F affects Gurken (Grk) protein 233

localization 234

Dorsal-ventral polarity defects can be attributed to disruptions in the grk-Egfr signaling 235

pathway. Since specific expression of C-terminally truncated Spn-F protein in the 236

germline led to ventralized egg production, we examined the localization and expression 237

of grk RNA and Grk protein in these ovaries. In situ hybridization analysis with a grk 238


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probe was performed in female flies expressing Spn-F〉C and wild type ovaries. We 239

found that similar to wild type ovaries (arrow in Fig. 3A, 23-25), ovaries expressing 240

spn-F〉C showed no effect on grk mRNA localization, with grk mRNA being detected 241

as a cap around the oocyte nucleus in the anterior-dorsal corner of the oocyte (arrow in 242

Fig. 3B). Next, we tested the localization pattern of Grk protein and found that 243

expression of spn-F〉C in the germline led to a profound effect on Grk localization 244

throughout oocyte development. In wild type ovaries from stage 7 onward, Grk is 245

restricted to the anterior-dorsal corner of the oocyte (arrow in Fig. 3C), much as seen in 246

terms of grk mRNA localization. In stage 7 (data not shown) to stage 9 (arrow in Fig. 247

3D) egg chambers from Spn-F〉C-expressing females, Grk protein was localized to 248

abnormally large puncta close to the oocyte. These results indicate that the dorsal-249

ventral defects in Spn-F〉C-expressing eggs are due to mislocalization of Grk protein. 250

251

Secreted and microfilament-related proteins are localized to ectopic actin cages in 252

oocytes from flies expressing spn-F〉C. 253

The integrity of both the microtubule and actin networks is essential for correct mRNA 254

localization in the oocyte. To examine the organization of the actin cytoskeleton, 255

ovaries were stained with rhodamine-conjugated phalloidin (Fig. 3). We found that 256

expression of Spn-F〉C affected oocyte actin organization. Whereas in a stage 8-9 wild 257

type egg chamber actin is evenly distributed at the cortical surface (Fig. 3C), the ectopic 258

F-actin network is juxtaposed to the oocyte nucleus (arrow in Fig. 3E). 259

Ectopic actin clumps were also observed in trailer hitch (tral) and BicC mutants, 260

where it was shown that also other microfilament-related and secreted proteins associate 261

with the ectopic actin cages (26-27). We saw that in flies expressing Spn-F〉C, the 262

secreted protein Grk is co-localized with the abnormal actin clumps in oocytes (arrow in 263

Fig. 3F). Moreover, we found that Spectrin, a microfilament-related protein, is also 264

associated with the actin clumps (Fig. 3G-J). Thus, our results show that both secreted 265

proteins, such as Grk, and the microfilament-related protein, Spectrin, co-localized to 266

the ectopic actin network in oocytes from flies expressing Spn-F〉C. 267

268

Expression of C-terminally truncated Spn-F affects osk mRNA and protein 269

localization 270

Establishment of the anterior-posterior axis of the embryo depends on the localization 271

of bcd and osk mRNA to the anterior and posterior poles of the oocyte, respectively (28-272


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30). Indeed, most of the bicaudal phenotypes reported arise from either mis-localization 273

of osk mRNA to the anterior of the oocyte (29, 31) or from premature translation of osk 274

mRNA (32-34)0 As described above, 98% of the eggs from females expressing Spn-275

F〉C produced bicaudal embryos. We initially examined bcd mRNA localization and 276

found that in Spn-F〉C-expressing egg chambers, bcd mRNA was localized to the 277

anterior ring of the oocyte, similarly to wild type flies (data not shown). Next, we 278

examined the localization pattern of osk mRNA and protein in ovaries and eggs from 279

Spn-F〉C-expressing females. In wild type ovaries, osk mRNA was tightly localized to 280

the posterior pole of the oocyte (Fig. 4A). However, in 47% of stage 9 or 10 egg 281

chambers (n=36) from Spn-F〉C-expressing females, osk mRNA was found both at the 282

posterior and anterior poles and/or abnormally accumulated in the middle of the oocyte 283

(arrows in Fig. 4B). Analysis of Osk protein localization revealed that in comparison to 284

wild type oocytes, where Osk protein is found as a tight crescent at the oocyte posterior 285

end (Fig. 4C), in Spn-F〉C-expressing stage 10 oocytes, Osk protein was distributed 286

more diffusely (Fig. 4D). 287

To characterize the localization pattern of Osk protein in oocytes at later stages 288

of oogenesis, we used an osk-LacZ construct as a reporter of osk mRNA translation 289

since most antibodies fail to penetrate at these stages (16). We found that in mature eggs 290

produced by wild type females, Osk-LacZ protein localized exclusively to the posterior 291

pole (Fig. 4E). However, in eggs from Spn-F〉C-expressing females, Osk-LacZ protein 292

accumulated at the anterior end (arrow in Fig. 4F) and was seen at high levels 293

throughout the egg, in addition to its normal localization to the posterior pole, 294

suggesting that anteriorly-localized osk mRNA in Spn-F〉C-expressing egg chambers is 295

only translated in the mature egg and does not undergo premature translation during 296

oogenesis. Next, we analyzed osk mRNA localization in early embryos and found that 297

in wild type embryos, osk transcripts are concentrated at the posterior of the embryo, as 298

well as being expressed at low levels throughout the embryo (Fig. 4H). In 90% of the 299

embryos (n=65) produced by Spn-F〉C-expressing females, osk mRNA levels were 300

found at the posterior pole and all over the embryo, as compared to wild type, and also 301

slightly accumulated at the anterior end of the embryo (arrow in Fig. 4I). 302

303

Microtubule polarity defects in oocytes from flies expressing Spn-F〉C 304


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We next examined the integrity of the microtubule network, as revealed by g–tubulin 305

antibody staining, and found that expression of Spn-F〉C affects MT organization in 306

mid-stage egg chambers. In stage 9 egg chambers, oocyte MTs are organized in a 307

gradient, with higher accumulation observed at the anterior cortex of the oocyte (arrow 308

in Fig. 5A). In oocytes from females expressing Spn-F〉C, a significant reduction in g-309

tubulin levels along the anterior cortex was observed (arrow in Fig. 5B). 310

To investigate the polarity and functionality of the MT network, we tested 311

transport to MT plus and minus ends, using the kin:く-gal and Nod:く-gal markers, 312

respectively (14-15). In wild type stage 8 egg chambers, Nod:く-gal is localized to the 313

anterior cortex of the oocyte and is enriched in the dorsoanterior corner, near the oocyte 314

nucleus (arrows in Fig. 5C). However, in egg chambers from females expressing Spn-315

〉C, Nod:く-gal could not be detected (Fig. 5D). In analyzing transport to the MT plus 316

end, we found that whereas in wild type stage 9-10 egg cambers, kin:く-gal is tightly 317

concentrated at the oocyte posterior (arrow in Fig. 5E), in Spn-F〉C-expressing egg 318

chambers, kin:く-gal is abnormally localized. In 58% of the egg chambers examined 319

(n=64), the protein was not detected (Fig. 5F), while in 12% of the egg chambers, the 320

protein was found at the posterior end and in the middle of the oocyte (arrows in Fig. 321

5G). In 27% of cases, the protein accumulated only at the center of the oocyte (arrow 322

Fig. 5H). These results suggest that transport towards the MT minus and plus ends is 323

severely disrupted in Spn-F〉C-expressing ovaries. 324

325

Expression of Ik2 rescues the sterility of females expressing Spn-F〉C 326

Since expression of Spn-F〉C phenocopied ik2 but not spn-F loss-of-function, we tested 327

whether Spn-F〉C ovarian phenotypes are due to inactivation of Ik2 activity. We saw 328

that expression of Ik2 significantly rescued Spn-F〉C ovarian phenotypes. Whereas 329

expression of Spn-F〉C (co-expressed with UASp-Rad9, which serves as a negative 330

control) led to complete female sterility (n=1036), in females co-expressing Spn-F〉C 331

and Ik2, 61% (n= 228) of the eggs hatched. Moreover, in Spn-F〉C-expressing females, 332

94% of the eggs (n=321) laid showed a ventralized pattern (a detailed description of 333

these eggshell patterns is found in Fig. 2 C-F), 17% of them were weakly ventralized, 334

74% were ventralized, and 3% were strongly ventralized eggshell (Fig. 6A). Following 335

the addition of Ik2 protein, these ventralized eggshell pattern was reduced to 26% 336

(n=293), where 19% were weakly ventralized, 4% were ventralized, and 3% were 337

strongly ventralized eggshell (Fig. 6A). Also, expression of Ik2 rescued the effect of 338


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Spn-F〉C expression on the anterior-posterior pattern of the embryo, reducing the 339

bicaudal phenotypes from 98% to 39% (n=144). 340

341

Expression of C-terminally truncated Spn-F protein affects Ik2 activation 342

Next, we studied the localization pattern of pIKK� in ovaries expressing C-terminally 343

truncated Spn-F. We found that expression of Spn-F protein lacking its C-terminal 344

completely abolished pIKK� in the germline (Fig. 6 A-C) but did not affect Ik2 protein 345

stability (Fig. 6E). The use of the mat-GAL4 promoter, which drives high expression 346

from stage 6-7, allows for demonstration of the dramatic effects of Spn-F〉C expression 347

on pIKK�. In stage 5 egg chambers, where GFP:Spn-F〉C expression is low 348

(arrowhead in Fig. 6A), anti-pIKK� immune-staining in the oocyte is similar to that 349

seen in the wild type (arrow head in Fig. 6B). However in stage 8 egg chambers, where 350

GFP-tagged Spn-F〉C is expressed at high levels (arrow in Fig. 6A), anti-pIKK� 351

antibody staining was abolished (arrow head in Fig. 6B). Anti-pIKK� immuno-352

staining was also evident in follicle cells (Fig. 6B), since Spn-F〉C was expressed 353

exclusively in the germline. 354

Since we found that expression of Ik2 rescues the sterility of females expressing 355

Spn-F〉C, we tested whether Ik2 expression also restores Ik2 activity. We saw that 356

expression of Ik2 is sufficient to also restore Ik2 activity in flies expressing the C-357

terminally truncated Spn-F protein, as revealed by the restoration of anti-pIKK� 358

immunostaining in the ovaries of these flies (arrows in Fig. 6D). 359

360

Spn-F〉C protein can bind Ik2 but not Jvl 361

To better understand the mechanism by which Spn-F〉C over-expression affects 362

Ik2 activity, we studied the physical interaction between Spn-F〉C and Ik2 or Jvl. Our 363

previous results showed that the C-terminal but not the N-terminal end of the Spn-F 364

protein binds the Ik2 protein (4). The truncated version of Spn-F employed here 365

includes the second coiled-coil domain and the conserved region at the C-terminal 366

region of the protein (spanning from residue 285 to the end of the protein). Since the 367

expression of Spn-F〉C, which includes the second coiled-coil domain but lacks the C-368

terminal conserved region, affects Ik2 activity, we asked whether Spn-F〉C binds Ik2. 369

Accordingly, we co-expressed myc-Ik2 with either GFP-Spn-F or GFP-Spn-F〉C in S2 370

cells and then performed immunoprecipitation with anti-GFP antibodies. We found that 371

myc-Ik2 was co-precipitated with both GFP-Spn-F constructs, demonstrating that Spn-F 372


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〉C is able bind the Ik2 protein (Fig 7B). These results suggest that the second coiled-373

coil domain within Spn-F is required for Ik2 binding. Indeed, the second coiled-coil 374

domain alone is able to co-precipitate with Ik2 (Shigeo Hayashi, personal 375

communication). 376

Next, we studied the ability of Jvl to interact with Spn-F〉C in a yeast two 377

hybrid assay. We found that whereas Spn-F physically interacts with Jvl (Fig. 7C and 378

Dubin-Bar et al., 2011), no interaction between Spn-F〉C and Jvl was detected (Fig. 379

7C). Most importantly, we were able to show that the C-terminal region of Spn-F is able 380

to bind Jvl (Fig. 7C). To verify these results, we considered the ability of Jvl to 381

physically interact with Spn-F protein and its truncated forms in immunoprecipitation 382

assays in S2 cells. Similar to the results of the yeast two hybrid assay, we found that full 383

length Spn-F protein and the C-terminal region of Spn-F but not Spn-F〉C were able to 384

bind Jvl (Fig 7D). 385

386

Spn-F but not Spn-F〉C protein co-localized with Ik2 and Jvl 387

The results showing that different domains within Spn-F bind Jvl and Ik2 suggest that 388

Spn-F, Jvl and Ik2 act as complex of proteins, where Spn-F acts as mediator between 389

Ik2 and Jvl. Thus, deleting Jvl-binding domain in Spn-F will affect the formation of the 390

complex. To test this prediction, we studied the localization pattern of all three proteins 391

in S2 cells. Expressions of each of this protein alone revealed that mCherry-Jvl is 392

localized to a filamentous structure (Fig. 8A), shown to be MT network (3). Myc-tagged 393

Ik2 (Fig. 8B) and GFP-Spn-F (Fig. 8C) were found in a punctate pattern, while GFP-394

Spn-F〉C (Fig 8D) was found mainly throughout the cytoplasm. We found that when 395

mCherry-Jvl, Myc-tagged Ik2 and GFP-Spn-F were co-expressed, all three protein co-396

localized in a filamentous structure which resembles the localization of mCherry-Jvl 397

alone (Fig. 8E-F). One the other hand, when GFP-Spn-F〉C was co-expressed with 398

mCherry-Jvl and Myc-tagged Ik2, both Myc-tagged Ik2 and GFP-Spn-F〉C were no 399

longer associated with mCherry–Jvl (Fig. 8 H-J). 400

401

Discussion 402

Previously, it was shown that Ik2 is activated locally at the tip of bristles (12). 403

In this study, we have demonstrated that during oogenesis, Ik2 is also locally activated 404

at MT minus end regions in the oocyte and follicle cells, as well as in the nurse cells, 405


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where Ik2 presents a punctate pattern. We found that in the germline, jvl and spn-F are 406

required for activation of Ik2. To better understand the mechanism by which Spn-F 407

affects Ik2 activation, we performed structure-function analysis of the Spn-F protein. 408

Using this approach, we were able to demonstrate that the C-terminal end of Spn-F is 409

sufficient for protein function. Following the expression of several truncated Spn-F-410

encoding constructs in the germline, we noticed that expression of Spn-F lacking 84 411

amino acids from the C-terminal end produced similar defects in both the dorsal-ventral 412

and anterior-posterior axes as found in ik2 loss of function ovaries (2). A high 413

percentage of ventralized eggs and bicaudal embryos are produced by both the ik2 414

mutant (2) and Spn-F〉C-expressing females. Most importantly, the fact that expression 415

of Ik2 was able to significantly rescue defects in eggshell and embryo development, as 416

detected by Spn-F〉C expression, suggests that the C-terminal end of Spn-F regulates 417

Ik2 protein function. 418

The results of the current study demonstrated that in terms of mRNA 419

localization, expression of Spn-F〉C protein produced, similarly to ik2 mutants, high 420

percentages of bicaudal embryos due to defects in osk mRNA localization. Previously, 421

we showed that females mutant for spn-F produce low percentage of bicaudal embryos 422

ranging from embryos with the typical reduced head skeleton to rare symmetrical 423

bicaudals (1). Thus, we believe that Ik2 function in oocyte anterior-posterior patterning 424

has two components, one that depends on Spn-F and the other that does not. 425

One can thus ask how Ik2 activity affects osk mRNA localization. The role of 426

the cytoskeleton in transporting osk mRNA to its final destination required cooperation 427

between MTs and between MTs and actin motor proteins. Initially, osk mRNAs are 428

transcribed in nurse cells and transported into the MT minus end at the anterior of the 429

oocyte by dynein, along with the accessory factors BicD and Egalitarian (35-36). 430

Within the oocyte, it was shown that the localization of osk to the posterior end required 431

MTs, Khc and myosin V (37-39). Several models explaining how osk transcripts are 432

transported toward the posterior of the oocyte have been proposed, including active 433

transport to the posterior (38), diffusion and trapping (40), or exclusion from the 434

anterior and lateral cortex (41). One recent model suggested that osk mRNA is actively 435

transported along microtubules in all directions, with a slight bias toward the posterior 436

(42). As to the role of Khc in osk mRNA transport, it was suggested that Khc is required 437

either directly (38, 41) or indirectly (43-45). In the present study, we have demonstrated 438

that over-expression of Spn-F〉C, which eliminates Ik2 activity, affects Khc-LacZ 439


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function and the posterior localization of osk mRNA. Based on our results, it is possible 440

that regulation of Ik2 by the Spn-F C-terminal region affects MT-biased polarity 441

towards the oocyte posterior, thus indirectly affecting Khc and osk mRNA localization. 442

Alternatively, the effect on osk mRNA localization could be due to a direct effect on the 443

regulation of Khc motor protein activity. 444

We have demonstrated that the defects in dorso-ventral axis in flies expressing 445

Spn-F〉C are due to the Grk protein but not mRNA mislocalization. Moreover, we 446

showed that Grk protein, a secreted protein, as well as Spectrin, a microfilament-related 447

protein, is localized to ectopic actin clumps in the oocyte. The localization of Grk to 448

ectopic actin clumps was reported for several mutants, including Bic-C (26-27), trailer 449

hitch (tral) (26-27), spn-F (1) and ik2 (2). In all of these mutants, grk mRNA was 450

mislocalized but in a different pattern than was Grk protein, suggesting that defects in 451

grk mRNA localization cannot account for defects in Grk protein localization. It was 452

suggested that Bic-C and tral are part of the same pathway that regulate efficient Grk 453

secretion (26-27). Accumulation of ectopic Grk protein in the oocyte was also found in 454

Khc and Dhc mutants, and it was suggested that both genes are also required for Grk 455

protein exocytosis (44). Thus, we suggest that the aberrant localization of Grk protein in 456

ik2 mutants and in flies expressing Spn-F〉C revealed a role of these proteins in 457

regulating Grk protein secretion. 458

To better understand the mechanism by which over-expression of Spn-F〉C 459

affects Ik2 activity, we studied interactions between Spn-F〉C and the Ik2 and Jvl 460

proteins. Previously, we had shown that Spn-F is able to directly bind Ik2 and Jvl. In 461

this study, we demonstrated that whereas Ik2 physically interacts with Spn-F〉C, Jvl 462

was not able to bind Spn-F〉C. We also found that Ik2 binds to the second coiled-coil 463

domain of Spn-F (Shigeo Hayashi, personal communication), while Jvl interacts with 464

the conserved C-terminal region of this protein. Moreover, we were able to show that 465

Spn-F but not Spn-F〉C protein forms a complex with Ik2 and Jvl. We believe that 466

expression of Spn-F〉C, which able to bind Ik2 but not Jvl, interfered with the 467

interaction between Ik2 and Jvl. These results suggest that specific interference with the 468

interaction between Ik2 and Jvl, as revealed upon Spn-F〉C expression, is critical for 469

Ik2 core functions during oogenesis. The fact that Spn-F mediates Ik2 interaction with 470

Jvl, a MT-associated protein, and the finding that Ik2 is activated at the MT-minus-end, 471

together with the specific effects of ik2 on oocyte MT organization (2), suggest that Ik2 472

plays a crucial role in MT organization and/or function during oogenesis. 473


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474

475

476

Acknowledgements 477

We thank Anna Ephrussi and the Bloomington stock center for generously providing fly 478

strains and reagents. This research was supported by Israel Science Foundation grant 479

968/10 (to U.A.). 480

481

482

483

484


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485

486

Figure legends: 487

Figure 1: spn-F and javelin-like (jvl) are required for Ik2 activation. (A-F) 488

Confocal images of egg chambers stained with antibodies against phosphorylated Ik2, 489

pIKK (red), and actin (green). In all figures, the egg chamber posterior is to the right. 490

(A-B) Wild type, (C-D) spn-F mutant, (E-F) jvl hemizygous. (A, C, and E) Stage 6 egg 491

chambers, (B, D and F) stage 7-8 egg chambers. (A) pIKK shows posterior 492

accumulation in the oocyte. (B) pIKK is localized to the anterior end of the oocyte and 493

close to the oocyte nucleus. In nurse cells, pIKK is found in a punctuate pattern 494

(arrows). (B'-B'') pIKK is also found at the apical side of follicle cells. (C-D) pIKK is 495

not detected in the spn-F germline but is found in follicle cells (arrows in D'). (E-F) 496

pIKK is not detected in jvl mutants oocytes but is found in nurse cells (arrows in F) and 497

follicle cells (arrows in F'). (G-H) Western blot analysis of Ik2 levels in ovaries. Ik2 498

levels in ovarian extracts from wild type and mutant flies were detected using antibodies 499

against this protein. The level of acetylated tubulin served as a loading control. 500

Mutations in spn-F and jvl do not affect the level of Ik2 protein. Df- Deficiency. 501

502

Figure 2: Expression of Spn-F〉C leads to the appearance of ventralized eggshell 503

and bicaudal embryos. (A) Schematic presentation of Spn-F protein domains and 504

deletion constructs used to make transgenic flies. CC1, coiled-coil 1, CC2, coiled- coil 505

2, CT, conserved C-terminal. (B) Western blot analysis of ovarian extract from flies 506

expressing either GFP- Spn-F〉C, GFP-Spn-F-C or GFP- Spn-F-N. The level of actin 507

served as a loading control. (C-F) Eggshells from flies expressing Spn-F〉C. (B) 8% of 508

the eggshells had a wild type appearance, (C) 13% had a weakly ventralized eggshell 509

with fused appendage, (D) 76% had a ventralized eggshell with one appendage, and (E) 510

3% had a strongly ventralized eggshell with no dorsal appendages. (G) Wild type 511

embryo, (H) 98% of embryos from flies expressing Spn-F〉C had a bicaudal phenotype. 512

In all figures, the Gal4 that was used was P{matg4- GAL-VP16}V37. 513

514

Figure 3: Secreted and microfilament-related proteins are localized to ectopic actin 515

cages in oocytes from flies expressing spn-F〉C. (A-B) In situ grk mRNA localization 516

in stage 9 egg chambers. (A) Wild type, (B) Spn-F〉C, grk mRNA is found at anterior-517


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dorsal corner of the oocyte (arrows in A and B). (C-F) Confocal images of egg 518

chambers stained with antibodies against Grk (white) and actin (red). (C) Stage 9 wild 519

type egg chamber, (D-F) stage 9 egg chamber from flies expressing Spn-F〉C. In the 520

wild type, Grk protein is localized to the dorsal-anterior corner of the oocyte (arrow in 521

C), however, in egg chambers from flies expressing Spn-F〉C, Grk protein is localized 522

to ectopic actin clumps (arrow in D, E and in F). (G-J) Confocal images of egg 523

chambers stained with antibodies against Spectrin (white) and actin (red). (G) Stage 9 524

wild type egg chamber, (H-J) stage 9 egg chamber from flies expressing Spn-F〉C. 525

Spectrin protein is also localized to ectopic actin clumps (arrows in H-J). 526

527

Figure 4: Defects in osk mRNA and protein localization in flies expressing Spn-528

F〉C. (A-B) In situ osk mRNA localization in stage 10 egg chambers. A) Wild type, (B) 529

Spn-F〉C. (C-D) Confocal images of egg chambers stained with antibodies against Osk 530

(red). (C) Wild type, (D) Spn-F〉C. (E-F) く-galactosidase staining of Osk-く-GAL in egg 531

chambers from (E) wild type, (F) Spn-F〉C flies. (H-I) osk mRNA in embryos from (H) 532

wild type, (I) Spn-F〉C flies. Both osk mRNA (arrows in B and I) and Osk protein 533

(arrow in F) are mislocalized upon Spn-F〉C expression. 534

535

Figure 5: Microtubule organization and function is affected upon Spn-F〉C 536

expression. (A-B) Confocal images of egg chambers stained with antibodies against 537

tubulin (red). Stage 9 egg chambers from (A) wild type, (B) flies expressing Spn-F〉C. 538

The anterior to posterior tubulin gradient detected in the wild type (arrow in A) is 539

abolished upon Spn-F〉C expression (arrow in B). (C-H) く-galactosidase staining of 540

Nod く-GAL (C-D) and kinesin く-GAL (E-H). Wild type egg chamber (C and E) and 541

Spn-F〉C egg chambers (D, F-H). In flies expressing Spn-F〉C, wild type anterior Nod 542

く-GAL staining (arrows in C) is abolished (D). In wild type flies and in 3% of egg 543

chambers from flies expressing Spn-F〉C, kin:く-gal is accumulated at the oocyte 544

posterior (arrow in E). Kin:く-gal in flies expressing Spn-F〉C was either not detected 545

(F) or was found at the posterior end and in the middle of the oocyte (arrows in G) or 546

accumulated only at the center of the oocyte (arrow in H). 547

548

Figure 6: Expression of Spn-F〉C affects Ik2 activation. (A) Expression of Ik2 549

rescues the dorsal-ventral defects of Spn-F〉C expression. Whereas 94% (n=321) of 550

eggs laid by females expressing spn-F〉C produced a range of ventralized eggshells 551


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(detailed description of these eggshells pattern is found in the legends to Fig. 2 C-F), 552

expression of Ik2 significantly suppressed these dorsal-ventral defects. (B-E) Confocal 553

images of egg chambers stained with antibodies against pIKK (red). (B-D) Egg 554

chambers from flies co-expressing GFP::Spn-F〉C and Flag-tagged Rad9. (E) Egg 555

chamber from flies co-expressing GFP::Spn-F〉C and GFP::Ik2. Whereas high 556

expression of Spn-F〉C during mid-oogenesis (arrows in B) eliminates Ik2 activation 557

(arrow in C), the lower expression levels seen in early oogenesis (arrows head in B) had 558

no effect on Ik2 activation (arrow head in C). (F) Western blot analysis of Ik2 levels in 559

ovaries. Ik2 levels in ovarian extracts from wild type and Spn-F〉C-expressing flies 560

were detected using antibodies against Ik2. The level of acetylated tubulin served as a 561

loading control. Spn-F〉C expression did not affect the level of Ik2 protein. 562

563

Figure 7: Spn-F〉C physically interacts with Ik2 but not with Jvl. (A) Schematic 564

presentation of Spn-F protein domains and deletion constructs used in this study. (B) 565

Co-immunoprecipitation of GFP-Spn-F constructs with Myc-Ik2 from S2 cells. GFP-566

Spn-F was precipitated using a Protein Trap kit and Myc-Ik2 was detected by western 567

blotting using anti-Ikk antibodies. Ik2 binds to the Spn-F〉C. (C) A yeast two hybrid 568

assay was used to detect the interaction between Spn-F and Jvl. Jvl binds to the 569

conserved C-terminal domain of Spn-F. (D) Co-immunoprecipitation of GFP-Jvl with 570

mCherry-Spn-F constructs from S2 cells. GFP-Jvl was precipitated using a Protein Trap 571

kit and mCherry-Spn-F was detected by western blotting using g–mCherry antibodies. 572

Jvl binds to the conserved C-terminal domain of Spn-F. 573

574

Figure 8: Spn-F〉C do not co-localized with Ik2 and Jvl. (A-D) Confocal images of 575

Schneider cell expressing mCherry-Jvl (A), Myc-tagged Ik2 (B, white pseudo color), 576

GFP-Spn-F (C) and GFP-Spn-F〉C (D). Confocal images of Schneider cells co-577

expressing either mCherry-Jvl, Myc-tagged Ik2 and GFP-Spn-F (E-G) or mCherry-Jvl, 578

Myc-tagged Ik2 and GFP-Spn-F〉C (H-J). Co-expression of mCherry-Jvl, Myc-tagged 579

Ik2 and GFP-Spn-F led to co-localization of all three proteins in a filamentous structure. 580

However, when GFP-Spn-F〉C was co-expressed with mCherry-Jvl and Myc-tagged 581

Ik2, the three proteins no longer co-localized. 582

583

584


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585

586

587

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1 Drosophila oocyte polarity and cytoskeleton organization requires ...

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