Dysferlin is essential for endocytosis in the sea star oocyte

Nathalie Oulhen, Thomas M. Onorato 1, Isabela Ramos 2, Gary M. Wessel n

Department of Molecular and Cell Biology and Biochemistry, Brown University, Providence RI 02912, USA

a r t i c l e i n f o

Article history:Received 4 October 2013Received in revised form5 December 2013Accepted 11 December 2013

Keywords:DysferlinSea starPlasma membraneOocytesEndocytosisGastrulation

a b s t r a c t

Dysferlin is a calcium-binding transmembrane protein involved in membrane fusion and membranerepair. In humans, mutations in the dysferlin gene are associated with muscular dystrophy. In this study,we isolated plasma membrane-enriched fractions from full-grown immature oocytes of the sea star, andidentified dysferlin by mass spectrometry analysis. The full-length dysferlin sequence is highly conservedbetween human and the sea star. We learned that in the sea star Patiria miniata, dysferlin RNA andprotein are expressed from oogenesis to gastrulation. Interestingly, the protein is highly enriched in theplasma membrane of oocytes. Injection of a morpholino against dysferlin leads to a decrease ofendocytosis in oocytes, and to a developmental arrest during gastrulation. These results suggest thatdysferlin is critical for normal endocytosis during oogenesis and for embryogenesis in the sea star andthat this animal may be a useful model for studying the relationship of dysferlin structure as it relates toits function.

& 2014 Elsevier Inc. All rights reserved.

Introduction

Dysferlin is a transmembrane protein associated with rareforms of muscular dystrophies, such as limb-girdle musculardystrophy type 2B (Bashir et al., 1998), Miyoshi myopathy (Liuet al., 1998), and distal myopathy (Illa et al., 2001). Loss of dysferlinfrom the plasma membrane of muscle fibers leads to abnormalitiesin vesicular trafficking and membrane repair (Kobayashi et al.,2012). Moreover, freshly isolated monocytes of dysferlino-pathy patients show deregulated expression of fibronectin andfibronectin-binding integrins which is recapitulated by transientknockdown of dysferlin in the human monocytic cell line, THP1.Dysferlin regulates cellular interactions by forming a complex withintegrins at the cell membrane (de Morree et al., 2013). In humans,the dysferlin transcript is highly expressed in skeletal muscle,heart, placenta, and more weakly in liver, lung, kidney andpancreas (Bashir et al., 1998). More recently, it was also found incultured vascular endothelial cells (Leung et al., 2011; Sharmaet al., 2010). Dysferlin is a member of the ferlin family of proteins,based on the similarity to the C. elegans protein Fer-1 (Fertilizationdefective-1). In C. elegans, the full-length dysferlin homolog Fer-1(230 kDa) is present early in spermatogenesis and becomes lessabundant in spermatids, perhaps because it is proteolytically

processed into smaller isoforms (195 and 180 kDa) (Washingtonand Ward, 2006). Fer-1 is required for the fusion of specializedvesicles, called membranous organelles, with the plasma mem-brane during spermatogenesis (Achanzar and Ward, 1997;Washington and Ward, 2006).

Two other ferlin proteins have been identified in humans,myoferlin and otoferlin. Myoferlin was first considered as a musclespecific protein and its deletion results in impaired mouse myo-blast fusion into mature skeletal myotubes (Doherty et al., 2005).However, myoferlin has been shown to be critical for endocytosisin endothelial cells (Bernatchez et al., 2009). Myoferlin is alsoabundantly expressed in invasive breast tumor cells and remark-ably, its depletion stalls the invasion of these cells (Li et al., 2012).Otoferlin is essential for hearing and pathogenic mutations areassociated with nonsynchronic autosomal recessive deafness(Yasunaga et al., 1999). Otoferlin is required in the priming andfusion of synaptic vesicles during sound encoding, which occurs atsynapses between cochlear inner hair cells and the auditory nerve(Pangrsic et al., 2012).

Ferlin proteins function in various developmental and repro-ductive processes in several organisms. While dysferlin andmyoferlin are co-expressed in the human placenta, only dysferlinexpression is positively correlated to cell fusion in trophoblasticcells (Robinson et al., 2009). In Drosophila melanogaster, only oneferlin gene, called misfire (mfr), has been found. The transcript isexpressed in the testis and ovaries of adult flies. In males, mfrexpression is required for efficient breakdown of the spermplasma membrane and completion of sperm activation duringfertilization. In females, it is required for egg patterning by limitingthe spread of proteins such as Gurken. Mutations in misfire delayembryogenesis (Ohsako et al., 2003; Smith and Wakimoto, 2007).

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Developmental Biology

0012-1606/$ - see front matter & 2014 Elsevier Inc. All rights reserved.http://dx.doi.org/10.1016/j.ydbio.2013.12.018

n Correspondence to: 185 Meeting Street, Box G, Brown University, Providence,RI 02912, USA.

E-mail address: rhet@brown.edu (G.M. Wessel).1 Current address: Department of Natural Sciences, LaGuardia Community

College, Long Island City, NY 11101.2 Current address: Instituto de Bioquímica Médica, Universidade Federal do Rio

de Janeiro, RJ 21940-590, Brazil.

Please cite this article as: Oulhen, N., et al., Dysferlin is essential for endocytosis in the sea star oocyte. Dev. Biol. (2014), http://dx.doi.org/10.1016/j.ydbio.2013.12.018i

Developmental Biology ∎ (∎∎∎∎) ∎∎∎–∎∎∎

In zebrafish, the dysferlin transcript is first detected at 11 h post-fertilization, remains detectable until 96 h, and is expressed indifferent tissues including muscle, brain and eye (Kawahara et al.,2011). Reduction of dysferlin expression in this animal by mor-pholino injection causes abnormal formation of muscle structures(Kawahara et al., 2011). The dysferlin gene is also present in the seaurchin genome (Sodergren et al., 2006) and this animal was usedto test the role of dysferlin in membrane healing (Covian-Nareset al., 2010). Laser wounding of one cell in a 4-cell stage embryotriggered calcium spikes in neighboring cells through voltage-gated calcium channels whereas embryos injected with a dysferlinmorpholino lost their calcium spike activity in neighboring cellsafter wounding. The authors conclude that the dysferlin-depletedembryos have lost an ability to receive a signal, possibly anucleotide, released by the wounded cell.

Here, we found that dysferlin is enriched in the plasma mem-branes of oocytes and embryos from the sea star, an animal withoocytes and embryos tractable for studying cellular dynamics(Fresques et al., in press; Hinman et al., 2003a; Wessel et al., 2010).Sea stars are invertebrate deuterostomes, a sister group to chordates.Oocytes, eggs and embryos of the species used here, Patiria miniata,are optically clear, enabling excellent observations of development.Developing oocytes are plentiful, manipulatable, are arrested inprophase of first meiotic division and meiotic reinitiation is inducedby the hormone 1-methyladenine (Kanatani et al., 1969). Afterfertilization, embryos develop quickly, reach the blastula stage inone day, and gastrulate in two days. In this study, we show that in thestarfish Patiria miniata (Pm), knockdown of the dysferlin proteinresults in reduced endocytosis during oogenesis and significantdevelopmental defects in embryos.

Materials and methods

Animals

P. miniata and A. forbesi were housed in aquaria with artificialseawater (ASW) at 16 1C (Coral Life Scientific Grade Marine Salt;Carson, CA). Gametes were acquired by opening up the animals.Oocytes were collected in filtered seawater and sperm was collecteddry. To obtain mature oocytes, the full-grown immature oocyteswere incubated for an hour in filtered sea water containing 2 mM 1-methyladenine. After addition of sperm, fertilized eggs were culturedin filtered seawater at 16 1C (Foltz et al., 2004; Wessel et al., 2010).

Isolation of plasma membrane-enriched fractions

Full-grown immature or mature oocytes were isolated inCalcium-Free Sea Water (CFSW), resuspended in buffer A (Sucrose0.36 M, 50 mM Tris pH8, 5 mM EDTA) and homogenized on ice byfive up and down strokes of a loose-fitting glass pestle in a 40 mLglass dounce tissue grinder. Following a 15-min incubation on ice,the sample was homogenized with an additional five strokes andthe homogenate was then incubated at 4 1C overnight to settle theplasma membrane-enriched fraction (PMEF) by gravity. This frac-tion was washed three times with buffer A and the membraneswere collected for analysis, or pelleted in a microfuge at 14 000 gfor 5 min. For SDS-PAGE, the pellet was resuspended in LaemmliSample Buffer (LSB) containing 5 mM DTT, and the samples wereboiled for 5 min at 100 1C.

Rhodamine-B-Isothiocynate (RITC) labeling of oocyte surface proteins

Full-grown immature oocytes were isolated in CFSW and alllabeling steps were performed in CFSW. Oocytes were pelleted bygentle centrifugation and rotated at 16 1C for about 90 min in a

15 ml conical centrifuge tube containing RITC at 0.5 µg/ml inCFSW. Oocytes were then rinsed with filtered SW from the MarineBiological Laboratories (MBL; Woods Hole, MA) three times. Analiquot (200 μl) of RITC-labeled whole oocytes was pelleted andsuspended in 200 μl of 2� LSB containing 5 mM DTT for SDS-PAGE analysis. PMEFs were isolated from these labeled oocytesusing the protocol described above. Images were taken with afluorescence dissecting stereoscope (Olympus SZX16) connectedto an Olympus X cite 120 lamp and to a camera (Nikon D90),managed by Pro 2 software. Samples were electrophoresedthrough a 4%–12% SDS-PAGE, stained with Coomassie BrilliantBlue for 1 h and the RITC signal was detected using a TyphoonImager (GE Healthcare, Typhoon 9410).

Mass spectrometry and protein analysis

PMEF was loaded on a SDS-PAGE for Coomassie Brilliant Bluestaining. The two main high molecular weight proteins wereobtained and processed for in gel digestion using the In gel trypticdigestion kit (Pierce). Proteins were digested overnight at 37 1C inpresence of 10 ng/ml trypsin. Samples were analyzed using aThermo-Finnigan LTQ linear ion trap mass spectrometer. Trypticpeptides were fractionated on a reversed phase column andintroduced directly onto an LTQ mass spectrometer via electrosprayionization. Pm-dysferlin was identified via database matching withthe program SEQUEST (Eng et al., 1994), using the Pm ovarytranscriptome (Adrian Reich and Gary Wessel, unpublished data)which has been entered at NCBI (KJ081282).

EMBOSS Needle was used to obtain protein identities andsimilarities between the species (EMBL-EBI). SOSUI was used toidentify the transmembrane domains (Mitaku et al., 2002). Con-served Domain Database (CDD) was used to identify the proteindomains (Marchler-Bauer et al., 2011). The DysF domains of thehuman dysferlin protein were also defined according to a previousanalysis (Lek et al., 2010). YinOYang (Gupta and Brunak, 2002),NetPhos (Blom et al., 1999), Sulfinator (Monigatti et al., 2002), andProP (Duckert et al., 2004) were used to analyze the potential post-translational modifications of the proteins.

Antibody production

Rabbit polyclonal antibody against the peptide sequenceALPRPKFSDSTGKI of dysferlin from P. miniata was custom devel-oped and affinity purified by GenScript (Piscataway, NJ). Thispeptide, specific to dysferlin, is located in the DysFN domain(amino-acids 889–902).

Western blot

Western blot analysis was performed following electrophoretictransfer of proteins from SDS-PAGE onto 0.22 μm nitrocellulosemembranes (Towbin et al., 1979). Membranes were incubated withantibodies directed against Pm-dysferlin (1/5000) in 20 mM Tris–HCl (pH7.6), 3% BSA, and 0.1% Tween-20, overnight at 4 oC. Theantigen–antibody complex was measured by chemiluminescenceusing horseradish peroxidase-coupled secondary antibodiesaccording to the manufacturer0s instruction (ECL; GE HealthcareBiosciences, Pittsburgh, PA, USA).

Real-time quantitative PCR (QPCR)

RNA was extracted from the different developmental stages usingthe RNeasy Micro Kit (Qiagen; Valencia, CA). cDNAwas prepared usingthe TaqMans Reverse Transcription Reagents kit (Applied Biosystems;Foster City, CA). QPCR was performed on a 7300 Real-Time PCR system(Applied Biosystems; Foster City, CA) with the FastStart Universal

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SYBR Green Master Kit (Roche). Pm-dysferlin was amplified usingthe following primers: F (5-AACGAAGCCCTGGAAGAGAT-30) and R(50-GCATGGAGACCGGAGTAGAG-30). Experiments were run in tripli-cate, and the data were normalized to ubiquitin RNA levels (Hinmanand Davidson, 2003; Hinman et al., 2003b). Pm-ubiquitin was ampli-fied using the primers F (50-TTCGGTGAAAGCCAAGATTC-30) and R(50-CCCACCTCTCATGGCTAGAA-30).

Whole mount RNA in situ hybridization (WMISH)

The sequence used to make antisense WMISH probes for Pm-dysferlin was amplified from Pm ovary cDNA and cloned intopGEM T-Easy (Promega). Pm-dysferlin was amplified using thefollowing primers: F (50-ATACGCGTGTGGGTTGCCTGG-30) and R(50-AGCGCGCTACACGCTGATCG-30). The pGEM T-Easy plasmids werelinearized using NdeI (T7 transcription) (Promega; Madison, WI).Antisense, DIG-labeled RNA probes were constructed using a DIGRNA labeling kit (Roche; Indianapolis, IN). WMISH experiments wereperformed as described previously (Minokawa et al., 2004), and thealkaline phosphatase reaction was carried out for 1 h. A non-specificDIG-labeled RNA probe complementary to neomycin resistance gene,obtained from the pSport 18 (Roche; Indianapolis, IN) was used as anegative control. Samples were imaged on a Zeiss Axiovert 200 Mmicroscope equipped with a Zeiss color AxioCam MRc5 camera(Carl Zeiss, Inc.; Thornwood, NY).

Immunofluorescence

Oocytes and embryos were cultured as described above, andsamples were collected at the indicated stages for whole-mountantibody labeling. The fertilization envelope was manually removedin the early stages. Cells were fixed in 4% paraformaldehyde in ASWand washed with PBS before being treated with 0.1% Triton X-100 for20 min. After 3 washes with PBS-Tween 20, oocytes and embryoswere blocked for an hour at room temperature in blocking buffer: PBS-0.05% Tween 20-3% BSA (Sigma; St. Louis, MO). For labeling, the cellswere incubated overnight at 4 1C with either the anti Pm-dysferlinpurified antibody diluted by 1:500 (final concentration: 2 μg/ml) inblocking buffer, or this antibody pre-incubated for 2 h at roomtemperature with the corresponding blocking peptide used at thefinal concentration of 40 μg/ml. The cells were washed three timeswith PBS-Tween 20, and then incubated with an anti-rabbit AlexaFluor 488 (green) (Life technologies, Grand Island, NY), diluted by1:500 in blocking buffer for two hours at room temperature. Oocytesand embryos were then washed 3 times with PBS-Tween 20. Imageswere captured using a LSM 510 laser scanning confocal microscope(Carl Zeiss, Inc.; Thornwood, NY).

Morpholino injection

Oocytes were injected with the dysferlin morpholino 50- TCGACA-CAATCACCATCAGCGACAT-30 and incubated for 36 h before in vitromaturation. An irrelevant morpholino (the sea urchin, Strongylocen-trotus purpuratus, SCP2) was used as a control: 50-GGACATACTTGT-CAGGTCGTGCCAA-30. The injection solution contained 1mM ofmorpholino. Each young and full-grown oocyte received approxi-mately 1.4 and 0.5 nM of morpholino respectively. Embryos wereimaged on a Zeiss Axiovert 200M microscope equipped with a Zeisscolor AxioCam MRc5 camera (Carl Zeiss, Inc.; Thornwood, NY).

In vitro endocytosis assay

Morpholinos injected oocytes were incubated in FSW for36–48 h at 16 1C and then transferred to filtered sea watercontaining 15 μg/ml of FM1-43 (Molecular Probes, Grand Island,NY) and incubated for additional 3–4 h at 16 1C. Oocytes were then

washed in FSW and observed at the confocal microscope formonitoring the presence of endocytic vesicles.

Results

Dysferlin is enriched in the plasma membrane of oocytes

To identify the proteins expressed in the plasma membrane of thefull-grown immature oocytes, the surface was labeled with RITC andthe labeled membranes were isolated (Fig. 1A). Proteins obtained fromwhole oocytes or plasma membrane-enriched fractions (PMEFs) wereelectrophoresed in a 4%–20% SDS-PAGE and visualized by CoomassieBlue staining and by RITC fluorescence imaging (Fig. 1B). Two bands ofhigh molecular mass, flanking the 182 kDa marker (n) were repro-ducibly observed in the plasma membrane fraction. These wereprepared for mass spectrometry analysis and the resulting peptidesequences were identified by a SEQUEST search against the P. miniataovary transcriptome (Adrian Reich and Gary Wessel, unpublisheddata). Dysferlin was identified with two dysferlin peptides from bothbands (Fig. S1). The largest dysferlin transcript (Pm 1104) found in thePm transcriptome contains an open reading frame of 6240 nucleotidesthat encodes for a 2080 aa protein, with an expected size of 233 kDa.An affinity-purified rabbit polyclonal antibody was generated againstPm-dysferlin and immunoblot analysis on the PMEF led to a specificsignal surrounding the 182 kDmarker (Fig.1B), supporting the identityof the isolated protein. PMEFs were also isolated from a second seastar species, Asterias forbesi (Af), and a main band, also present at182 kDa, was observed (Fig. S2). An Af-dysferlin transcript was identi-fied from the Af ovary transcriptome (Adrian Reich and Gary Wessel,unpublished data), and similar to the Pm-dysferlin, it contained anopen reading frame of 6231 nucleotides, encoding a cognate protein of2077 amino acids in length.

Dysferlin is highly conserved between human and sea star

The alignment of amino acid sequences indicates that Pm-dysferlin is highly conserved with the human dysferlin: Pm and Af-dysferlin are 85.6% identical and 92.4% similar. Human dysferlinshares 46.9% of identity and 63.2% of similarity with Pm, and 46.9%of identity and 63.7% of similarity with Af. Moreover, the samedomains are found in the dysferlins of these three species (Fig. S3).In both human and sea star, dysferlin contains six main C2domains. The C2 domain (approximately 130 residues) was firstidentified in the Ca2þ-dependent protein kinase C (PKC) and isinvolved in Ca2þ-dependent membrane binding. It is also found inpresynaptic proteins such as synaptotagmins (Cho and Stahelin,2006). Dysferlin also contains two highly conserved DysF domainsthat are tandemly repeated. The presence or absence of thisdomain in ferlin family members is used to define two subgroups:dysferlin and myoferlin (presence) and otoferlin (absence)(Lek et al., 2012; Lek et al., 2010), although the role of this domainis not fully understood. However, the DysF domain appears moresusceptible to retain mutations and is involved in dysferlinopathy(Patel et al., 2008). FerA and FerB, which lie in the long stretchbetween C2C and C2D, are also conserved between human and seastar and both domains are unique to ferlin proteins. FerB isconserved in all ferlins, while FerA is only found in DysF contain-ing ferlins (Type 1), suggesting FerA and DysF may have comple-mentary or additive function (Lek et al., 2010). The FerI domain,which lies between the C2B and C2C domains, is conserved in Hs,Pm and Af. This domain was also identified in ferlin-like proteins ofprotozoan Apicomplexan parasites Plasmodium (malaria), Thei-leria (East Coast fever), Babesia (tick fever), and Toxoplasma(toxoplasmosis), that account for significant worldwide mortalityand morbidity amongst humans and livestock (Lek et al., 2010).

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Finally, human and sea star dysferlin contain a transmembranedomain similarly placed near the C-terminus of the protein (Bansaland Campbell, 2004).

The dysferlin transcript is expressed throughout oogenesis andembryogenesis in sea star

To determine the temporal expression of the dysferlin transcriptin Pm, an RNA probe was synthesized for in situ hybridization and

a probe against the neomycin-resistance gene was used as anegative control (Fig. 2A). Pm-dysferlin mRNA accumulates uni-formly in the stages tested, from young oocytes (100 μm diameter)to gastrula. Quantitative PCR was used to measure the relative RNAlevel in young oocytes, full-grown immature oocytes (4160 μm),mature oocytes, fertilized eggs, two-cell stage, blastula and gas-trula (Fig. 2B). All values were normalized against Pm-ubiquitinmRNA. Although differences were seen in qPCR signals, these werenot significantly different (p40.05) between the 100 μm oocytes

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Fig. 1. Dysferlin is expressed in the cortex of sea star oocytes. (A) Proteins present at the surface of Pm oocytes were labeled with RITC. Images were taken on intact oocytes(b), and after purification of the plasma membranes (d). The corresponding bright field images are respectively shown in a and c. (B) RITC labeled proteins from wholeoocytes (WO) or from the plasma membrane-enriched fraction (PMEF) were loaded on SDS-PAGE and visualized by either Coomassie staining (lanes 1 and 2) or RITCfluorescence (lanes 3 and 4). Two bands (n) near the 182 kDa marker were reproducibly found enriched in the plasma membrane fraction and identified as Pm-dysferlin bymass spectrometry. A signal at the similar molecular weight was obtained by Western blot using the affinity-purified antibody against Pm-dysferlin on the PMEF (lane 5).

GastrulaBlastulaMatureImmature Fertilized 2 cell100 μm

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Fig. 2. Pm-dysferlin mRNA is uniformly expressed throughout oogenesis and embryogenesis. (A) Whole mount in situ hybridization in the sea star P. miniata, using probesagainst Pm-dysferlin in 100 μm oocytes (a), immature full-grown oocytes (b), mature oocytes (c), 30 min after fertilization (d), at the two-cell stage (e), in blastula (f), and ingastrula (g). Neomycin resistance gene is used as a negative control (h–n). (B) qPCR was used to measure the RNA levels of Pm-dysferlin at the indicated developmental stages:100 μm oocytes, full-grown immature oocytes, mature oocytes, 30 min after fertilization, two-cell stage, at blastula, and gastrula. All values were normalized against thePm-ubiquitin mRNA and represented as a fold-change relative the amount of RNA present in the 100 μm oocytes. Significance was assessed using One-Way ANOVA test.No significant differences were obtained at p¼0.05.

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and each of the other stages. These results indicate that thetranscript level of dysferlin in Pm uniformly accumulates through-out oogenesis and early development.

The dysferlin protein is enriched in the plasma membranes of oocytesand embryos

An affinity-purified rabbit polyclonal antibody was generatedagainst Pm-dysferlin and used to determine dysferlin localizationby immunofluorescence (Fig. 3). Pm-dysferlin is detected at thecortex of the oocytes during oogenesis. Interestingly, dysferlin ishighly expressed in young oocytes (Fig. 3a) and decreases in full-grown immature oocytes (Fig. 3b). Pm-dysferlin is barely detectablein mature oocytes (Fig. 3c) but is readily detectable at the plasmamembrane right after fertilization (Fig. 3d) and at the two-cell stage(Fig. 3e). The protein is also detected in blastula (Fig. 3f) and gastrulastages (Fig. 3g). No immunofluorescence signal was detected underthe same conditions when the cells were exposed to the Pm-dysferlinantibody preincubated with the corresponding blocking peptide(Fig. 3o–u). Moreover, the expression of Pm-dysferlin protein inoocytes was further tested by Western blot analysis after enrichmentof the plasma membranes (Fig. 4). A similar level of Pm-dysferlin wasobtained in the fractions from immature and mature oocytes,suggesting that in mature oocytes, even though the protein cannotbe detected by immunofluorescence, Pm-dysferlin is present andassociated with the plasma membrane.

Endocytosis is reduced in Pm-dysferlin knockdown oocytes

To test if dysferlin plays a role in endocytosis during oogenesisin the sea star, a morpholino was injected into young oocytes toblock dysferlin translation (Fig. 5). Although these dysferlin-

depleted oocytes remained viable, endocytosis was decreased bymore than 40% in the dysferlin injected oocytes, compared tooocytes injected with a control morpholino (Fig. 5A and B). Theefficiency of the morpholino was tested by immunofluorescenceusing the antibody against Pm-dysferlin, and quantificationrevealed that the morpholino reduced the protein expression by35% (Fig. 5C and D), suggesting that not only was dysferlin activelybeing synthesized in the oocyte, it was also being activelydegraded. A strong correlation was seen between the decrease inendocytosis and the decrease in dysferlin expression.

Pm-dysferlin knockdown induces a developmental arrest atgastrula stage

To determine the function of dysferlin during embryonicdevelopment in the sea star, the same morpholino was injectedinto full-grown immature oocytes. Injection of dysferlin morpho-linos did not affect oocyte maturation, fertilization, or earlydevelopment in Pm when compared to the control (data notshown). Interestingly, dysferlin morpholino-injected embryoswere smaller and arrested at gastrula (Fig. 6A). The dysferlinknockdown led to a disorganization of the mesenchyme cellsand the archenteron. The efficiency of the morpholino was testedby immunofluorescence using the antibody against Pm-dysferlin(Fig. 6B); the morpholino decreased the expression of the proteinsignificantly, on average by 30% during this developmental win-dow (Fig. 6C).

Discussion

Dysferlin is a transmembrane protein involved in membranefusion and membrane repair but a paucity of information limits

Fig. 3. The Pm-dysferlin protein is expressed during oogenesis and embryogenesis. Immunofluorescence using the antibody against Pm-dysferlin (a–g) on 100 μm diameteroocytes (a), full-grown immature oocytes (b), mature oocytes (c), fertilized eggs (d), two-cell stage (e), blastula (f), and gastrula (g). The corresponding bright field images arerespectively shown in a0–g0 . To test the specificity of the Pm-dysferlin antibody, a control of immunofluorescence was performed by pre-incubating this antibody with itscorresponding blocking peptide (o–u). The corresponding bright field images are respectively shown in o0–u0 . Images were taken using the same microscope settings (laserintensity, pinhole opening).

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our understanding of its function during oogenesis and embry-ogenesis. Only one dysferlin transcript was found in the Pm ovarytranscriptome (Pm_1104), but since the complete Pm genome hasnot been annotated yet, additional dysferlin isoforms could beexpressed at a lower level and not detected in the transcriptome.However, three transcripts were found for Pm-otoferlin (Pm_10061,Pm_54312, Pm_21293) although we did not follow-up on theirfunction in these cells. We found that dysferlin is highly enrichedin the cortex of oocytes (Fig. 3) though it may also be presentbelow detectable thresholds in other cellular compartments. Inbovine aortic endothelial cells (BAEC), a GFP-tagged dysferlin wasalso found in the nucleus and in the Golgi apparatus (Sharma et al.,2010). Ferlin domains also have similarity to several nucleolarprotein domains (Staub et al., 2004). The nucleolus is the center ofribosome biogenesis (Tschochner and Hurt, 2003) but is also ableto immobilize proteins as a post translational regulatory mechan-ism (Audas et al., 2012).

We learned that dysferlin could be knocked down in young seastar oocytes by morpholinos (Fig. 5). This result is important sinceit shows that the oocyte is constantly undergoing protein turnoverand synthesis of dysferlin, and by extension likely other proteinsas well. Even with dysferlin being a transmembrane protein,turnover is robust in these cells, opening the opportunity to

IM M

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Fig. 4. The Pm-dysferlin protein is expressed in the plasma membrane of bothimmature and mature oocytes. A signal above 182 kDa was obtained by Westernblot using the affinity-purified antibody against Pm-dysferlin on PMEFs fromimmature (IM) or mature (M) oocytes.

Fig. 5. Endocytosis is reduced in dysferlin knockdown oocytes. (A) Young oocytes were injected by either a control (a) or the dysferlin morpholino (b) and incubated withFM1-43 to test endocytosis. The corresponding bright field images are respectively shown in (c) and (d). (B) The fluorescence obtained in (A) was quantified usingMetamorph. Twenty four (with an average size of 137.878.6 μm) and twenty five (with an average size of 139.179.4 μm) injected oocytes were quantified respectively forthe control and the dysferlin morpholino. Significant difference (n) was observed between the control and the dysferlin morpholino using Student0s t-test, Po0.05. (C) Theexpression of Pm-dysferlin decreases in oocytes after morpholino injection. Immunofluorescence on young oocytes injected with either a control (a) or the dysferlinmorpholino (b). The corresponding bright field images are respectively shown in (c) and (d). (D) The fluorescence obtained in (C) was quantified using Metamorph. Twenty(with an average size of 13877.2 μm) and twenty two (with an average size of 140.778.2 μm) injected oocytes were used respectively for the control and for dysferlinmorpholino. Significant difference (n) was observed between the control morpholino and the dysferlin morpholino using Student0s t-test, Po0.05.

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knockdown and test other oocyte-associated factors for function-ality in for example, maturation, fertilization, and early develop-ment. Dysferlin could be required for the fusion of specific vesicleswith the plasma membrane to add extra membrane at the fusionsite during oocyte growth, as described in C. elegans duringspermiogenesis (Washington and Ward, 2006) but how thismechanism might work is not well understood. We could envisiona feedback system whereby if exocytosis is reduced by thedysferlin knockdown, then endocytosis would be reduced to retaintotal plasma membrane surface area. Endocytosis is a criticalprocess utilized by growing oocytes to import yolk proteinprecursors (Brooks and Wessel, 2004; Opresko and Wiley, 1987).In C. elegans, endocytosis of cell surface proteins was also found tobe critical for the regulation of meiotic maturation in oocytes(Cheng et al., 2008). Studies in both Xenopus (El-Jouni et al., 2007)and mouse (Lowther et al., 2011) reveal that endocytosis occurs inoocytes, and contributes to cAMP signaling to regulate meioticarrest. The physiological function of endocytosis in the sea staroocyte is currently under investigations.

Interestingly, we found by Western blot that the dysferlinprotein is expressed at the plasma membrane in mature oocytes,even though it is undetectable by immunofluorescence in situ atthis, but not other stages. The antibody is directed against a singlepeptide (amino acids 889–902) contained in the DysFN domain(Fig. S3) and one possible explanation for this paradox is that inmature oocytes, the protein might have either changed its con-formation or bound a new partner, preventing the interaction withthe antibody. The DysF domains have maintained high sequence

conservation during evolution (Lek et al., 2010) and in humans,mutations of this domain leads to dysferlinopathy, almost cer-tainly due to degradation of the unfolded protein (Patel et al.,2008). The solution structure of the inner DysF domain of thedysferlin paralogue myoferlin has been solved. It consists of twolong β-strand (residues 929–939 and 1018–1028) oriented anti-parallel with each other. The intervening 77 residues form a longloop which packs against both sides of the central β-hairpin;several regions of this loop form short stretches of β-strand. Theregion annotated by SMART as DysFN supplies the first strand andaround two-thirds of the loop and the region annotated as DysFCprovides a third of the loop and the second strand (Patel et al.,2008). In Pm mature oocytes, the protein also could be post-translationally modified near the immune peptide site, resulting ina change of its conformation. A YinOYang prediction analysissuggests that serine 896 included in the targeted peptide can bemodified by phosphorylation and by addition of O-linked β-N-acetylglucosamine. An additional NetPhos analysis predicts thatserines 896 and 898 can be phosphorylated. These specificmodifications might have been lost during the plasma membraneenrichment, leading to the recognition of the protein by immuno-blot but not in situ. Moreover, additional modifications or cleavageof the protein, outside of the peptide sequence, could also changethe conformation of the entire protein. Sulfinator predicts thatfour tyrosines in the protein can be sulfated, and ProP predictsthree arginine or lysines that could be used as cleavage sites. Inmouse, dysferlin has been found to interact with the histonedeacetylase 6 via its C2D domain, and with the alpha tubulin

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Fig. 6. Knockdown of Pm-dysferlin leads to a developmental arrest at gastrula stage. (A) Immature oocytes were injected by either a control (a) or the dysferlin morpholino(b). After maturation and fertilization, the embryos were cultured until the gastrula stage, 72 h after fertilization (72h AF). (B) The expression of Pm-dysferlin decreases inembryos after morpholino injection. Immunofluorescence on embryos injected with either a control (a) or the dysferlin morpholino (b). The corresponding bright fieldimages are respectively shown in (c) and (d). (C) The fluorescence obtained in (B) was quantified using Metamorph. Nine and eight injected embryos were used respectivelyfor the control and for dysferlin morpholino. Significant difference (n) was obtained between the control morpholino and the dysferlin morpholino using Student0s test,Po0.05.

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Please cite this article as: Oulhen, N., et al., Dysferlin is essential for endocytosis in the sea star oocyte. Dev. Biol. (2014), http://dx.doi.org/10.1016/j.ydbio.2013.12.018i

through its C2A and C2B domain during myogenesis (Azakir et al.,2010; Di Fulvio et al., 2011). Dysferlin can also form a dimerin vitro, and in living adult skeletal muscle fibers isolated frommice. Calcium insensitive C2 domains mediate a high affinity self-association of dysferlin in a parallel homodimer, leaving the Ca-sensitive C2A domain free to interact with membranes (Xu et al.,2011). In Pm mature oocytes, dysferlin could be interacting withnew partners and/or forming dimers, reducing its accessibilityto the antibody and thereby making this reagent a functionalreporter.

Dysferlin is also expressed during early development afterfertilization, and through blastula and gastrula stages (Fig. 3).Reduction of its expression did not lead to any developmentaldefects during the early developmental periods. This may be aresult of low knockdown efficiency, but it could also suggest thatdysferlin is not essential right after fertilization, during normaldevelopment. Taking into account the role of dysferlin in mem-brane wounding in sea urchin 4-cell stage embryos (Covian-Nareset al., 2010), the main function of this protein, right after fertiliza-tion, could be in membrane sealing during the rapid cellulardivisions, but is expendable, and perhaps compensated by othermembranous proteins. Interestingly, this reduction of dysferlinexpression leads to a developmental arrest during gastrulation, asshown by the morpholino injection experiments (Fig. 6). Injectionof the dysferlin morpholino in sea star seems to disorganize themesenchyme cells and the archenteron. These data suggest thatdysferlin could be required for the cellular rearrangements neededfor the elongation of the archenteron, and/or for cellular signalingthat may be important for stimulating this process. Sea starsappear to be a good model to study the function of dysferlin andthe role of each domain within a context of oogenesis andembryogenesis.

Acknowledgments

We thank Jim Clifton, Proteomics Facility Manager, for his helpwith mass spectrometry and Adrian Reich for sharing his tran-scriptome sequences and expertise in their screening. TMO was aVisiting Professor sponsored by the American Society for CellBiology Minorities Affairs Committee from 2010–2012 (fundingprovided from NIH grant #GM008622). We appreciate K. Ookie forsharing with us the biotintylation protocol. We gratefully acknowl-edge NSF grant IOS-1120972 and NIH grant 2R01HD028152 toGMW. This research is based in part upon work conducted usingthe Rhode Island NSF/EPSCoR Proteomics Share Resource Facility,which is supported in part by the National Science FoundationEPSCoR Grant no. 1004057, National Institutes of Health Grant no.1S10RR020923, S10RR027027 (Orbitrap XL ETD Mass Spectro-meter), a Rhode Island Science and Technology Advisory Councilgrant, and the Division of Biology and Medicine, Brown University.

Appendix A. Supplementary materials

Supplementary data associated with this article can be found inthe online version at http://dx.doi.org/10.1016/j.ydbio.2013.12.018.

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