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BOR - Papers in Press, published online ahead of print April 14, 2004.
Biol Reprod 2004, 10.1095/biolreprod.104.027730
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BIOLOGY OF REPRODUCTION 71, 705–713 (2004)
DOI: 10.1095/biolreprod.104.027730
© 2004 by the Society for the Study of Reproduction, Inc.

Gamete Biology

The Major Yolk Protein of Sea Urchins Is Endocytosed by a Dynamin-Dependent Mechanism1

Jacqueline M. Brooks, and Gary M. Wessel2

Department of Molecular Biology, Cell Biology and Biochemistry, Brown University, Providence, Rhode Island 02912


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Sea urchin oocytes grow to 10 times their original size during oogenesis by both synthesizing and importing a specific repertoire of proteins to drive fertilization and early embryogenesis. During the vitellogenic growth period, the major yolk protein (MYP), a transferrin-like protein, is synthesized in the gut, transported into the ovary, and actively endocytosed by the oocytes. Here, we begin to dissect this mechanism by first testing the hypothesis that MYP endocytosis is dynamin-dependent. We have identified a sea urchin dynamin cDNA that is highly similar in amino acid sequence, structure, and size to mammalian dynamin I: it contains an N-terminal GTPase domain, a pleckstrin-homology domain, and a C-terminal proline-rich domain. Sea urchin dynamin is enriched at the cortex of oocytes and colocalizes to MYP endocytic vesicles at the oocyte periphery. To test for a functional relationship between MYP endocytosis and dynamin, we used a dominant-negative human dynamin I mutant protein containing an alteration within the GTPase domain (hDynK44A) to specifically compete for dynamin function. Using a fluorescent MYP construct to follow its endocytosis solely, as well as a general endocytosis marker, we demonstrate that the disruption of dynamin function significantly reduces MYP uptake but does not affect fluid-phase endocytosis. Using this specific biochemical approach, we are able to separate distinct pathways of endocytosis during oogenesis and learn that dynamin-mediated endocytosis is responsible for MYP endocytosis but not fluid-phase uptake.

gamete biology, gametogenesis, oocyte development


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Yolk uptake by growing oocytes is a dramatic example of the receptor-mediated endocytosis pathway. This is especially prominent in the yolk-laden oocytes of many vertebrates, e.g., frog, fish and chicken, and most invertebrates, e.g., Drosophila and Caenorhabditis [1]. In these organisms, the major yolk constituents are either vitellogenins (vertebrates and many invertebrates) or yolk proteins (as in dipteran insects such as Drosophila). These two classes of yolk proteins are transported from an external site of synthesis (liver, fat body, or gut) via the circulatory system and are targeted to the ovary, where they are endocytosed into membrane-bound clathrin-coated vesicles of oocytes via receptors of the low-density lipoprotein (LDL)-receptor superfamily [1].

The yolk of sea urchin oocytes is distinct from vertebrate vitellogenins and insect yolk proteins [2]. It is comprised of two main components, each of different origin: YP30, a 30-kDa yolk-platelet protein that is synthesized by the oocyte and packaged into yolk platelets [3], and the major yolk protein, MYP. The MYP is synthesized in the gut and is secreted into the coelomic fluid of the body cavity. It is transferred across the ovarian capsule of epithelial cells and is finally taken up into vesicles within the growing oocyte. There it is packaged into the YP30 yolk platelets, where it accumulates to 10–15% of the total egg protein [4, 5]. MYP belongs to the transferrin superfamily [6, 7], although its receptor has yet to be identified.

Endocytic mechanisms are diverse between cell types and between the different molecules destined for uptake. Endocytosis in a cell may include macropinocytosis, clathrin-, or caveolin-mediated endocytosis, and clathrin- or caveolin-independent mechanisms. Early studies indicated that a large GTP-hydrolyzing protein, dynamin, is required for the release of clathrin-coated pits from the cell surface [8, 9]. It is now appreciated that dynamin serves as a master regulator of cell surface, membrane trafficking events [10]. Mammals exhibit tissue-specific expression of three closely related (>80% identical) dynamin isoforms [11] while, Drosophila and Caenorhabditis elegans appear to contain only a single isoform of dynamin [12–14]. Mutations in the GTPase domain common to all isoforms of these dynamin homologues disrupt endocytosis but not other membrane trafficking events [10].

Genetic evidence for a direct role of dynamin in endocytosis originally came from phenotypic analysis of the temperature-sensitive shibire mutant of Drosophila. The shibire gene product, Drosophila dynamin (dDyn), encodes a protein that is 70% identical to mammalian dynamins [12, 15]. The shibirets flies have a dynamin GTPase domain mutation that renders the protein defective in both GTP binding and hydrolysis. When flies are shifted to the nonpermissive temperature, endocytosis is blocked and coated pits accumulate at the plasma membrane in all tissues examined [16–19]. The phenotypes of temperature-sensitive alleles in the shibire locus in both garland cells (nephrocyte) and oocytes showed reduced uptake of the fluid-phase tracer, horseradish peroxidase [17, 19, 20]. In oocytes, this included the disappearance of endosomal compartments consisting of tubules and small yolk spheres [19, 20]. These studies suggested that dDyn is required for all pathways of endocytosis in the cell types examined, suggesting that the shibire protein is required for all pinocytotic events as well as yolk endocytosis in oocytes.

Endocytic mechanisms in mammalian cells have been dissected using dynamin GTPase mutants, such as K44A-dynamin, defective in GTP binding and hydrolysis [21]. In mammalian cells overexpressing dynamin-1 mutants, receptor-mediated endocytosis of transferrin is blocked, but in contrast with shibire cells, fluid-phase endocytosis proceeds normally [21, 22], indicating that the pinocytotic pathway is dynamin independent. Both the shibire and K44A mutation are in the GTPase domain of dynamin but at different amino acid residues. To determine if the dramatic differences in phenotype between the two mutations is the result of different sites of mutation in the GTPase domain, Damke et al. [23], generated a mammalian HeLa-shibire cell line expressing a human dynamin mutant (G273D) that is homologous to the shibirets allele in Drosophila. HeLa cell transformants expressing this protein, like their shibire counterparts, exhibit a rapid, reversible, and temperature-sensitive block in receptor-mediated endocytosis of transferrin. This recapitulates the results seen for the previously characterized HeLa cell line expressing the dynK44A mutant. However, unlike the shibire phenotype, fluid-phase endocytosis was only partially inhibited and, after prolonged incubation at the nonpermissive temperature (30 min), fluid-phase uptake returned to normal levels, independent of dynamin mutations. The conclusion from this work was that some clathrin- and dynamin-independent pathways, such as pinocytosis, are upregulated in cells that lack functional dynamin [23]. Given these results, it appears that the relative contributions of dynamin-dependent and dynamin-independent pinocytosis varies between different organisms.

Because endocytosis is a mechanism by which cells interface with their environment, important aspects of this process are likely to differ between not only different organisms but also between the different cell types that comprise them. Here, we describe the use of fluorescently labeled major yolk protein to visualize yolk endocytosis in live sea urchin oocytes. We have previously established a methodology to reproduce MYP uptake in vitro that recapitulates the biology of endogenous MYP, as ascertained by both immunofluorescence and immuno-electron microscopy studies [24] and can introduce macromolecules into the oocyte by microinjection to study the biochemical function independent of genetic manipulations and compensation by the cell. We used this assay to probe the function of dynamin in specific MYP endocytosis as well as for endocytosis in general. We document that dynamin in oocytes functions in a strict subset of endocytic processes, including yolk endocytosis, but not fluid-phase uptake, in this animal. These results enable a further dissection of dynamin-dependent and dynamin-independent mechanisms by use of a cellular assay in a specialized cell type—the oocyte.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals

Adult Lytechinus variegatus were obtained from Dr. James R. Garey (Tampa, FL) and the Duke University Marine Lab (Beaufort, NC) and kept at 20°C in artificial seawater (ASW; Coral Life Scientific Grade Marine Salt; Energy Savers Unlimited, Inc., Carson, CA) until needed. Females were shed by KCl (0.5 M) injection and ovaries were then removed and minced with a scalpel in artificial seawater. Dissociated oocytes were isolated by pipette and cultured at 22°C in ASW supplemented with 100 µg/ml ampicillin (Sigma, St. Louis, MO). To minimize damage to the surface of the cells, all glassware that came into contact with the oocytes was treated with SafetyCoat Nontoxic Coating (J.T. Baker, Philippsburg, NJ). In addition, the microinjection needles were siliconized with Sigmacote (Sigma).

Cloning of the cDNA

A L. variegatus {lambda}ZAP ovary cDNA library (gift of Dr. David R. McClay, Duke University, Durham, NC [25]) was screened using degenerate primers designed according to regions of conserved primary structure based on the amino acid sequences of dynamin 1 in human [26], C. elegans [13], and Drosophila [12]. The primers used were as follows: sense, 5'-ATG GAY GAR GGI ACI GAY G-3,' corresponding to amino acids 210–216 of human dynamin 1; antisense. 5'-AAI GCC ATR TCI GGI GTR AA-3,' corresponding to amino acids 403–410 of human dynamin 1.

PCR products were obtained with Taq DNA polymerase (Life Technologies Inc., Gaithersburg, MD) with the following PCR conditions: after initial denaturation for 5 min at 94°C, the reaction was subjected to 25 cycles of denaturation (94°C, 1 min), primer annealing (43°C, 45 sec), and extension (72°C, 1 min). The amplification products were cloned into pGEM-T easy (Promega Corp., Madison, WI). To obtain full-length cDNA encoding dynamin, plaque-lift hybridizations were carried out using L. variegatus {lambda}ZAP cDNA libraries from ovary and prism, using radiolabeled PCR clones. Positive plaques were purified by repeated plating and hybridization and the recombinant cDNA of each phage was excised with helper phage R408 and recovered as a Bluescript plasmid ([27]; Stratagene, La Jolla, CA). Products from both of these screens were sequenced either by the Sanger chain termination method [28] using [35S] dATP (DuPont, Boston, MA) and detected with BioMax MR film (Kodak, Rochester, NY) or by the macromolecular sequencing facility at Brown University, using an ABI 77 prism automated DNA sequencer (Perkin Elmer, Foster City, CA). Sequence data were assembled and analyzed using the University of Wisconsin Genetic Computer Group sequence analysis package [29]. The sequence of the overlapping clones was entered in GenBank (Accession No. AY562992).

Reverse Transcription-Polymerase Chain Reaction Analysis

Reverse transcription-polymerase chain reaction (RT-PCR) was carried out according to the manufacturer's directions using the Access RT-PCR kit (Promega), with the addition of 1% DMSO and 1 M betaine to increase amplification efficiency. The RT template was 0.1 µg of total ovary RNA and the reaction was performed for 45 min at 48°C; after denaturation for 5 min at 94°C, PCR amplification was performed for 50 cycles of denaturation for 1 min at 94°C, annealing for 1 min at 50°C, and extension for 1 min at 68°C. The sequence of the primers used at 35 pmol per reaction were 5'-CGTGAGCGTGAGTCCAAGAC-3' and 5'-CAAGCCATCAAGATTCATC-3.' The amplification products were cloned into the pGEM-T Easy vector (Promega), and DNA sequencing was performed by the macromolecular sequencing facility at Brown University.

Antibody Generation

Dynamin peptide (with N-terminally added cysteine) synthesis, BSA coupling, and generation of rabbit polyclonal serum against the peptides were performed by Sigma Genosys (St. Louis, MO) using two rabbits for each peptide immunogen. The N-terminal dynamin peptide corresponds to amino acids 48–62 [VLENFVGRDFLPRGS] and lies within the GTPase domain of sea urchin dynamin while the C-terminal peptide corresponds to amino acids 809–823 [GQPNIPGRPDMPNRP] and lies within the proline-rich domain (PRD) domain of sea urchin dynamin.

Electrophoresis and Immunoblot Analysis

For immunoblot analysis, egg proteins (300 µg total protein) were subjected to SDS-PAGE and immunoblotted essentially as described [30]. Samples for analysis were pelleted, resuspended in 2x sample buffer containing 10 mM dithiothreitol, and denatured for 5 min at 100°C. The proteins were then resolved by SDS-PAGE on a 4–20% gradient gel (Gradipore, Hawthorne, NY) and either stained with Coomassie blue or transferred to nitrocellulose for immunolabeling. Blots were preblocked for 30 min by incubation in blotto (50 mM Tris-Cl, pH 7.5, 0.18 M NaCl, 0.05% Tween 20, 3% nonfat dry milk) and then incubated for 1 h in blotto containing anti-dynamin peptide pAbs diluted 1:100. The blots were then washed three more times over 1 h and incubated for 1 h with goat anti-rabbit antibodies conjugated with alkaline phosphatase diluted 1:30 000. Blots were washed in blotto three more times and then washed in blotto minus milk two times. Each of the above washes were at least 10 min in duration. Detection of immunolabel signals was carried out with BCIP/ NBT colorimetric development according to Harlow and Lane [31] (Promega Corp). The specificity of the antibodies was checked by comparing the two different antibodies with the N-terminal peptide and the two different antibodies to the C-terminal peptide separately, all of which recognized the same single dynamin band of predicted size. In combination, the antibodies also recognized the same single band. We also performed peptide competition experiments to check for specificity and use of preimmune serum or secondary antibody alone (data not shown), and these experiments resulted in no detectable signal.

Immunolocalizations

Immunofluorescent localization was performed in whole mounts that were fixed in 4% formaldehyde in ASW and processed as previously described [32]. Polyclonal antibodies against the N- and C-terminal dynamin peptides were diluted 1:200 in tris-buffered saline Tween-20 (TBST). The secondary antibody, CY-3-conjugated affinity-purified goat anti-rabbit IgG (Jackson Research Laboratories, West Grove, PA), was diluted 1:300 in TBST. For dynamin colocalization to Tr-MYP in vitro labeled oocytes [24], the secondary antibody used was Alexa-conjugated affinity-purified goat anti-rabbit IgG (Molecular Probes, Eugene, OR), at a 1:500 dilution in TBST. Signals were visualized and recorded by laser-scanning microscopy with a Zeiss LSM 410 confocal microscope (Zeiss Inc., Thorwood, NY).

Membrane Topology and Endocytosis

Coelomic fluid enriched with MYP was fluorescently labeled with the Texas-red-X protein labeling kit (Molecular Probes) as previously described [24]. FM1-43 (Molecular Probes) was resuspended in methanol at 1 mg/ml, then diluted in ASW to give a working concentration of 1 µg/ ml. To evaluate endocytosis, eggs and oocytes were transferred to either the Tr-MYP in ASW or FM1-43 in ASW. To quantitate endocytosis, confocal sections of live cells were acquired with a Zeiss LSM 410 laser-scanning microscope and analyzed using Metamorph software (version 4.6r5; Universal Imaging Corp., Downingtown, PA). The relative intensity of each fluorochrome was calculated per area of each cell by using the region measurement function. The intensity of fluorescence was divided by the area of each cell to obtain a standardized value. These values were averaged and compared using Excel (Microsoft Corporation, Seattle, WA). The values were subjected to ANOVA followed by a Student t-test.

Preparation of Proteins and Microinjections

Oocytes were placed in a Kiehart chamber [33] in ASW and injected with either human dynamin I as control or human dynamin I K44A, a GTPase defective mutant [21]. An oil droplet of dimethypolysiloxane (Sigma) or a 0.1-mg/ml solution of fluorescent dextran (labeled with rhodamine or Oregon green; Molecular Probes) was coinjected into cells as a marker. Both control and test reagents were injected into the same batch of cells, which were then incubated at room temperature for at least 90 min. The oocytes were then removed from the chamber via mouth pipette and placed in a Pyrex spot well containing ASW and Texas-red labeled MYP [24] or ASW and FM1-43. The plate was placed in a humid chamber to avoid evaporation and endocytosis was allowed to occur at room temperature for 15 min. The oocytes were then rinsed with ice-cold ASW and mounted on slides for immediate visualization.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Sea Urchin Dynamin Is a Conserved Member of the Dynamin GTPase Protein Superfamily

A PCR screen of L. variegatus ovary and prism cDNA libraries, using degenerate primers designed from the human dynamin 1 sequence, resulted in overlapping cDNAs, the sequence of which resembled dynamin. The full-length L. variegatus dynamin was obtained by subsequent PCR screens using gene-specific primers and by plaque hybridization of both an ovary and a prism cDNA library. We did not observe significant variations in sequence identity of the clones obtained from either screen, nor did we find other dynamin types. Our complete cDNA sequence encompasses 3590 nucleotides with a 74-base pair (bp) 5' untranslated region (UTR) and (TAG)-stop codon at position 2715 followed by at least 876 bp of 3' UTR sequence. The sequence contains a potential 2640-bp open reading frame, coding for an 880-amino acid polypeptide. The predicted molecular mass of the sea urchin dynamin full-length protein is 98.8 kDa, which is in close agreement with the 97.4 kDa predicted for the full-length human dynamin I open reading frame of 864 amino acids.

When the full-length deduced amino acid sequence of L. variegatus dynamin (Fig. 1) was compared with other proteins in the NCBI database, it was most like dynamins. When sea urchin and human dynamin I were aligned to one another (Lalign; [34]), 70% identity was seen with a high degree of sequence conservation that extends throughout their lengths (Fig. 1). The dynamin family members are characterized by their common structural motifs and by conserved sequences in the GTP-binding domain. The N-terminal GTPase, which is approximately 300 amino acids, is always followed by a middle domain (of unknown function) of approximately 150 amino acids and an assembly domain of approximately 100 amino acids, named the GTPase effector domain or GED. Dynamins have a pleckstrin homology (PH) domain that binds phosphoinositides between the middle and the GED domain and have a PRD at their C-terminus. None of the other large GTPase family members have a PH domain or a PRD [14]. The sea urchin dynamin contains all five of the dynamin signature motifs and most closely resembles a dynamin I family member.



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  		FIG. 1. Sea urchin dynamin shares sequence identity with members of the dynamin family as shown by this alignment. The complete predicted open reading frame of Lytechinus variegatus dynamin is aligned to the human dynamin I consensus sequence. Amino acids are numbered from the first predicted methionine. Asterisks denote identities (70%), colons denote conserved substitutions, and periods denote semiconserved substitutions. The lysine residue that corresponds to the human dynK44A mutant is highlighted in red and the peptides used to generate our polyclonal antibodies are in bold and underlined. Note that the sea urchin dynamin sequence contains the five dynamin signature motifs including the N-terminal GTPase, middle domain, pleckstrin homology (PH) domain, GTPase effector domain (GED), and proline-rich domain (PRD) at the C-terminus

Sea Urchin Dynamin Is Expressed in Oocytes and Localized to the Plasma Membrane

Independent of the ovary cDNA screen, we tested for the presence of the dynamin transcript in ovaries by RT-PCR using dynamin-specific primers designed from the dynamin cDNA sequence. The ovary RNA screen resulted in a single, and expected, 313-bp product (Fig. 2A) that was subsequently cloned, sequenced, and confirmed to represent the dynamin transcript. When reverse transcriptase was omitted from the RT-PCR reaction, no product was detectable.



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  		FIG. 2. Dynamin mRNA is present in the ovary and the protein is enriched at the cortex of oocytes and eggs. A) RT-PCR indicates that the dynamin transcript is present in ovaries. Shown is a representative ethidium bromide-stained gel with the expected 313-bp product in the ovary RNA lane and an absence of amplified product in the control lane, where reverse transcriptase was omitted from the reaction. B) Immunoblot analysis of dynamin indicates that this protein is present in eggs. Lane 1: total egg protein, 300 µg/lane, probed with dynamin preimmune sera; lane 2: total egg protein, 300 µg/lane, probed with dynamin immune sera. C) In situ immunolocalization shows that dynamin protein is present at the surface (white arrow) of both oocytes (top panel) and unfertilized eggs (bottom panel) of L. variegatus. Note that dynamin is also associated with cytoplasmic vesicles in oocytes (top panel). Images were visualized with a Zeiss LSM 410. Brightfield images are on the left and the corresponding fluorescent images are on the right. Bar = 25 µm; gv = germinal vesicle

Polyclonal antisera were raised separately against sea urchin dynamin peptides to a portion of the N-terminal GTPase domain (amino acids 48–62) and to a portion of the C-terminal PRD domain (amino acids 809–823). Both sera selectively detect a single immunoblot protein product near 100 kDa in total egg protein (Fig. 2B) that is in close agreement with the predicted molecular mass of our full-length dynamin sequence (98.8 kDa) and human dynamin 1 (97.4 kDa). Next, we used these antibodies to do whole-mount immunolocalizations on both oocytes and eggs that revealed a substantial amount of dynamin at the plasma membrane in both of these cells (Fig. 2C). In oocytes, the dynamin peptide antibodies also labeled prominent punctate foci, enriched near the cell surface and scattered throughout the cytoplasm. Sea urchin oocytes are endocytically active throughout oogenesis and this punctate staining is reminiscent of endocytic vesicles [24]. We see a rapid drop in endocytic activity when oocytes mature into eggs and the disappearance of dynamin-positive vesicles in eggs is consistent with this cessation. The dynamin remaining at the cortex of the egg may be poised for the rapid reactivation of endocytosis that occurs at fertilization [35, 36].

Dynamin Is a Component of MYP Endocytic Vesicles

To initially define the localization and function of dynamin in MYP endocytosis, the dynamin antibodies were used in whole-mount immunolocalizations. Oocytes were first incubated with Tr-MYP for 15 min at room temperature to allow for a pulse of yolk endocytosis. The oocytes were then washed in ice-cold seawater, fixed, and processed for dynamin immunolocalization. Confocal scans of these oocytes reveals a striking colocalization of dynamin with MYP endocytic vesicles at the cell surface (Fig. 3) that is very similar to the clathrin pattern of immunostaining (data not shown).



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  		FIG. 3. In situ immunolocalization shows that dynamin is a component of MYP endocytic vesicles (arrows) at the periphery of the oocyte. A) Anti-dynamin serum. B) Texas-red MYP endocytic vesicles. C) overlay of A and B. This image shows a magnified surface scan of the oocyte cortex as visualized with laser-scanning microscopy with a Zeiss LSM 410. Bar = 25 µm

Dynamin Functions in MYP Endocytosis in Oocytes

To directly test the role of dynamin in MYP endocytosis, we used the dominant-negative human dynamin I K44A (hDynK44A) protein [21]. This dynamin mutant has a K44A (lysine -> arginine) point mutation introduced within the nucleotide-binding region that substantially reduces its affinity for GTP binding and hydrolysis [21, 37]. This mutant is often used to block receptor-mediated endocytosis in the most specific manner and is proposed to exert its dominant-negative effect by blocking endocytic vesicle inception at an intermediate stage of internalization before final constriction of the vesicle neck [26, 38, 39]. BLAST searches indicate that sea urchin dynamin is homologous to human dynamin I, and local sequence alignment across the GTPase domain shows 86.2% identity between these two proteins. Most important, like all of the other members of the dynamin family of proteins, the sea urchin sequence shares the same conserved lysine residue as human dynamin's position 44 (Fig. 1).

We injected oocytes with either the recombinant hDynK44A or wild-type human dynamin I (hDynI). The injected cells were allowed to equilibrate for 90 min and were then incubated for 15 min in the continuous presence of Tr-MYP to load endosomes. The cells were then washed in ice-cold seawater and kept on ice until confocal imaging (Fig. 4, A–C). First, we demonstrate that the injected cells do not display any gross morphological defects compared with control injected or uninjected cells. Based on dynamin's colocalization to MYP endocytic vesicles, we anticipated that a dominant negative-mediated inhibition of dynamin function could have profound effects on MYP internalization. Initially, we imaged live cells that were either uninjected or injected with hDynI controls and found the classic punctate periplasmic staining pattern, characteristic of the endosomal MYP internalization pathway (Fig. 4, A and B). In contrast, cells injected with the mutant dynamin exhibited a profound inhibitory effect on MYP uptake and internalization appears nearly ablated (Fig. 4C). Quantitation of MYP uptake shows that disruption of dynamin function significantly reduces the endocytic uptake of yolk (Fig. 4D). We conducted an ANOVA test on these data and found that the variance in MYP endocytosis among the three groups (uninjected, hDynI control injected, and hDynK44A mutant injected) was significant (P < 0.02). We then proceeded to do a pairwise comparison of the three groups using the t-test and found that the uptake of MYP for both the uninjected and control injected oocytes was statistically significant when compared with the hDynK44A-injected (uninjected vs. mutant hDynK44A, P < 0.01; control hDynI vs. mutant hDynK44A, P < 0.01). From these results, we conclude that disruption of dynamin function significantly reduces MYP uptake in sea urchin oocytes.



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  		FIG. 4. Dynamin functions in MYP endocytosis in oocytes. A) Uninjected oocyte. B) Human dynamin I (hDynI) control injected oocyte. C) Human dynamin I K44a mutant (hDynK44A)-injected oocyte. The hDynI control injected oocytes appear to undergo normal MYP endocytosis when compared with the uninjected oocytes. In contrast, oocytes injected with the hDynK44A mutant show a dramatic decrease in MYP internalization. A–C) Images of live cells were recorded with laser-scanning microscopy with a Zeiss LSM 410. The brightfield image is shown on the left, with the corresponding fluorescent image on the right. nc, Nucleolus; gv, germinal vesicle; oil, marks the oil droplets of injected cells; bar = 25 µM. D) The maximum values of endocytic activity for the control uninjected oocytes were set to 100%, and the remaining values were scaled accordingly. The difference in MYP endocytosis in the hDynIK44A-injected oocytes is statistically different than uninjected oocytes and hDynI control injected oocytes (both P < 0.01) as determined by a two-sample t-test following ANOVA (P < 0.02) analysis

Dynamin Does Not Function in Fluid-Phase Uptake in Oocytes

We used the lipophilic dye FM1-43 to look at the relative total endocytic activity of oocytes. FM1-43 is a nontoxic, water-soluble dye that is membrane-impermeable and virtually nonfluorescent in aqueous media. Upon insertion in the plasma membrane, it becomes intensely fluorescent and is thus used to view plasma membrane dynamics, including the internalization of cell membranes during endocytosis in sea urchins [24, 35, 36, 40]. We isolated oocytes from ovaries and cleared them of endogenous ligands by culture at room temperature in artificial seawater for several hours before our experiments. We then injected oocytes with either the recombinant hDynK44A or wild-type hDynI protein. The injected oocytes were allowed to recover for 90 min and were then incubated for 15 min in the continuous presence of FM1-43 to mark plasma membrane dynamics. The cells were then washed in ice-cold seawater and kept on ice until confocal imaging (Fig. 5, A–D).



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  		FIG. 5. Dynamin does not function in fluid-phase uptake in oocytes. A) Uninjected egg. B) Uninjected oocyte. C) Human dynamin I (hDynI) control injected oocyte. D) Human dynamin I K44a mutant (hDynK44A)-injected oocyte. Both the hDynI control and hDynK44A mutant-injected oocytes appear to undergo normal FM1-43 endocytosis when compared with uninjected oocytes. Images of live cells were recorded with laser-scanning microscopy with a Zeiss LSM 410. The brightfield image is shown on the left, with the corresponding fluorescent image on the right. pn, Pronocleus; nc, nucleolus; gv, germinal vesicle; oil, marks the oil droplets of injected cells; bar = 25 µm (D). E) The maximum values of endocytic activity for the control uninjected oocytes were set to 100%, and the remaining values were scaled accordingly. The difference in FM1-43 endocytosis in the hDynIK44A-injected oocytes is not statistically different as determined by ANOVA analysis

We imaged these live oocytes under confocal microscopy and found that they had uniform FM1-43 surface labeling along their plasma membrane. In addition, all of the oocytes appeared to be indistinguishable in their endocytic activity as discerned by the punctate periplasmic staining pattern, characteristic of a wave of internalization (Fig. 5, B–D). For comparison, we included mature eggs in these experiments to illustrate the downregulation of endocytosis following oocyte maturation. We conducted an ANOVA test on these data and found that uninjected, control hDyn-injected, and mutant hDynK44A-injected oocytes were invariant in their uptake of FM1-43 (Fig. 5E). From these results, we conclude that fluid-phase endocytosis in sea urchin oocytes is not dynamin dependent. In addition, these results support the specific role that dynamin is playing in yolk endocytosis and indicate that the inhibitory effect of dynK44A on MYP uptake is not due to a defect in oocyte integrity nor an indirect consequence of decreased membrane uptake.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Oocytes are capable of endocytosing massive amounts of extracellular material during oogenesis, including proteins, lipids, and carbohydrates. Oviparous animals store much of this material in specialized membrane-enclosed vesicles called yolk platelets. The repertoire of proteins deposited in these organelles support early development and vary for different species of embryos, depending on the external environmental demands. The yolk proteins in sea urchin are distinct from vertebrates and insects because they are synthesized both by the oocytes themselves as well as by other adult tissues and are then imported into the oocyte [2]. The MYP in the sea urchin is predominately made within the adult gut and is transported to the gonad during gametogenesis. Yolk endocytosis systems in many organisms have been compared with the best-studied endocytosis pathways, such as those of mammalian LDLs and transferrin [41]. MYP belongs to the transferrin superfamily, and its packaging into yolk may serve as an iron reserve during embryogenesis [6, 7]. MYP's receptor has yet to be identified, but endocytic trafficking of this protein is thought to proceed through pathways similar to those used by transferrin in somatic cells [1, 41].

Dynamin was originally characterized in the mechanism of receptor-mediated, clathrin-dependent endocytosis and more recently has been shown to be required for other membrane trafficking events at the cell surface, including phagocytosis, caveolar endocytosis, and some clathrin- and caveolae-independent endocytic pathways [10, 42, 43]. A number of recent studies in different cell types of both mammals and Drosophila suggest that different cells may exhibit dynamin-dependent and dynamin-independent pathways of endocytosis for both membrane and fluid-phase markers. For example, D2 dopamine receptors [44] and glycosylphosphatidylinosital-anchored proteins (GPI-Aps; [45]) are internalized from the surface of mammalian cells by a dynamin-dependent mechanism. Interestingly, GPI-Aps are endocytosed in a clathrin- and dynamin-independent pinocytotic manner into distinct compartments that contain a majority of the internalized fluid phase [45]. Primary cultures of Drosophila larval hemocytes and embryonic macrophages have also been shown to have dynamin-independent GPI-AP endocytosis via the fluid-phase pathway ascertained using dextran as a marker for bulk phase uptake [46]. This result is contrary to the earlier studies conducted in Drosophila larval garland cells [47] and oocytes [19, 20] with the fluid-phase tracer horseradish peroxidase (HRP). Guha et al. [46] bring up the possibility that HRP may be taken up by multiple means, including receptor-mediated pathways, and therefore may not be restricted to the fluid phase. It appears that the relative contributions of dynamin-dependent and dynamin-independent pinocytosis varies between not only different organisms but also among different cell types within the organism. This is a likely scenario because the environmental interface of a cell can be highly variable depending on the cell type.

We have focused our own studies on vitellogenesis in the sea urchin oocyte. We had previously documented that oocytes at all stages of oogenesis are very active in endocytosis compared with eggs [24, 39]. Regardless of access to the yolk precursor, oocytes are only endocytically active for MYP during their vitellogenic growth phase, which is when they reach about 50 µm in diameter. Both MYP endocytosis [24] and FM1-43 uptake [40] are microfilament mediated, precluding the possibility that the onset of the vitellogenic program is due to a global absence or presence of microfilaments. We looked at another possible component of the oocyte's endocytic machinery, the GTPase dynamin, to address the specificity of yolk vesicle uptake. Recent work has identified proteins that bridge the GTPase dynamin to the actin cytoskeleton, including profilin, cortactin, and the F-actin-binding protein Abp1 [48, 49]. Dynamin is enriched at the plasma membrane in sea urchin oocytes coincident with the thick microfilament meshwork position at the cortex of these cells [40, 50]. Here, we demonstrate that MYP-containing endocytic vesicles carry endogenous dynamin. Using a dominant-negative approach, we find that dynamin depletion significantly reduces MYP uptake. Together, these results demonstrate a structural and functional association between yolk uptake in the oocyte and the GTPase dynamin. Dynamin may provide the linkage of MYP endosomes to microfilaments and its GTPase function may drive this internalization.

Endocytosis assays in C. elegans oocytes using a yolk YP170-GFP fusion protein show that the trafficking pathway components used in this cell are remarkably similar to those used in mammalian cells. When dynamin function is perturbed by RNAi, they report a strongly reduced accumulation of YP170-GFP within oocytes that is one third of that of wild type [51]. The participation of dynamin in fluid-phase endocytosis was not tested in these cells. The significant but incomplete depletion of yolk inception in these oocytes is consistent with what we report here for sea urchin oocytes. This incomplete cessation of yolk endocytosis may be due to the upregulation of a dynamin-independent pathway that is stimulated when MYP is present and dynamin function is perturbed. Proper targeting and sequestering of MYP within the yolk platelet is not only important for its role in embryogenesis but also for tight safeguarding against the deleterious effects that free iron could have on the cell.


   ACKNOWLEDGMENTS
 
We are especially grateful to Emma Green for her technical expertise in the cloning of sea urchin dynamin. We thank Dr. Sean Conner for initial piloting of the microinjections and insightful comments throughout this work. Additional thanks are extended to the Schmid Lab for their generous contribution of the recombinant human dynamin proteins, Ekaterina Voronina for her continued interactions with Metamorph statistical analysis, and Jia Song for microinjection Sigmacote protocols.


   FOOTNOTES
 
1 This work was supported by grants from the NIH and the NSF. Back

2 Correspondence: Gary M. Wessel, Department of Molecular Biology, Cell Biology and Biochemistry, Box G, 69 Brown Street, Brown University, Providence, RI 02912. FAX: 401 863 1182; e-mail: rhet@brown.edu Back

Received: 22 January 2004.

First decision: 16 February 2004.

Accepted: 12 March 2004.


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TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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