The mammalian homologue of Nuclear fallout, Arfo2 physically associates and colocalizes with Rab11, a key component of the RE (Hickson, 2003). Rab11 is required for the integrity of the RE, and is believed to mediate transport of vesicles from the RE to the TGN, early endosome, and plasma membrane via a "slow" recycling route (Ullrich, 1996; Ren, 1998). Dollar (2002) characterized the pattern of Rab11 localization in the developing Drosophila oocyte. Rab11 was shown to localize at the posterior pole and is necessary for proper microtubule organization and Oskar mRNA localization. The pattern of Rab11 localization during the cortical divisions was examined in the early Drosophila embryo. During interphase, Rab11 exhibits a diffuse punctate localization that concentrates around the nuclei. As the embryos progress into prophase, Rab11 maintains its punctate morphology, but exhibits significantly increased concentration at the centrosomes. During metaphase, the centrosomal concentration of Rab11 decreases and there is a concomitant dispersal of Rab11 throughout the cytoplasm encompassing each chromosomespindle complex. This trend continues as the nuclei enter anaphase. Even though the nuclear envelope is substantially broken down during metaphase and anaphase, Rab11 does not enter the interior nuclear space. During telophase, Rab11 puncta concentrate around the newly formed nuclear envelope. There is a slight increase in the concentration of Rab11 puncta at the centrosomes. Cellularization occurs during the prolonged interphase of nuclear cycle 14. At this time, Rab11 is highly concentrated around the pair of apically located sister centrosomes (Riggs, 2003).
The pericentriolar concentration of Rab11 in Drosophila embryos is equivalent to Rab11 localization observed in mammalian cells. In CHO cells, Rab11 is primarily localized to a discrete pericentriolar region with a lower concentration of puncta distributed throughout the cell (Ullrich, 1996). Colocalization experiments with internalized transferrin indicated that Rab11 localizes to the pericentriolar RE (Ullrich, 1996; Sheff, 2002). GFP-Rab11 also exhibits a pericentriolar localization and colocalizes with the transferrin receptor (Sonnichsen, 2000). Given the equivalent staining patterns, it is concluded that Rab11 also localizes to the RE in syncytial and cellularized Drosophila embryos (Riggs, 2003).
Immunofluorescent analyses was performed using anti-Nuf and anti-Rab11 antibody. During prophase, when both antigens are highly concentrated in the pericentriolar region, areas of maximal Nuf localization correspond to areas of maximal Rab11 localization. Almost without exception, Nuf colocalizes with Rab11. However, the converse is not true, and in regions more distal from the centrosome, Rab11, but not Nuf, is present. During cellularization at interphase of nuclear cycle 14, Nuf and Rab11 exhibit high pericentriolar concentrations and extensive colocalization. As observed for prophase of the cortical divisions, Nuf always colocalizes with Rab11, but there are regions of Rab11 localization in which Nuf is not present. Given that Rab11 is an excellent marker of the RE (Ullrich, 1996; Ren, 1998), these results support the notion that Nuf localizes to the RE during cortical syncytial divisions and during cellularization at interphase of nuclear cycle 14 (Riggs, 2003).
To gain insight into the role of Rab11 in the generation of oocyte polarity, its subcellular distribution was determined in developing oocytes by staining fixed ovaries with Rab11 antisera. The distribution of green fluorescent protein (GFP)-tagged Rab11 was examined in living tissues. Both approaches revealed a similar Rab11 expression pattern (Dollar, 2003).
Rab11 is abundant in stage 1-10 oocytes. Detection of the protein in later stage oocytes is problematic due to the deposition of the chorion and extrachorionic membranes. The protein is also expressed in follicle cells and nurse cells, but at reduced levels compared with oocytes. In stage 1-7 oocytes, Rab11 accumulates in a distinct perinuclear compartment and is abundantly distributed in a thick crescent along the lateral and posterior cortexes. In stage 8-10 oocytes, Rab11 continues to accumulate in the perinuclear compartment, but the thick cortical crescent is gradually replaced by a small cap of protein at the extreme posterior pole of the oocyte. Double label experiments showed near perfect colocalization of Rab11 with Osk in stage 9 and 10 oocytes (Dollar, 2003).
In situ hybridization for Rab11 mRNA revealed no specific accumulation of the transcript at the posterior pole of the oocyte. Rather, the mRNA is uniformly dispersed throughout the oocyte through at least stage 9. Thus, in contrast to Osk, Rab11 localization would appear to be mediated by a protein-based localization machinery (Dollar, 2003).
Rab11 mediates post-Golgi trafficking of rhodopsin to the photosensitive apical membrane of Drosophila photoreceptors
To evaluate the role of Rab11 in Rh1 transport, Rh1 transport was investigated in Rab11 mutants. Since animals lacking Rab11 die as embryos (Dollar, 2002; Jankovics, 2001), mosaic animals, with eyes containing a mixture of normal photoreceptors and photoreceptors with severely reduced Rab11, were made. Comparatively few Rab11 mutant photoreceptors were observed in mosaic eyes, probably reflecting a cell-essential role for the protein. Rab11-reduced photoreceptors fail to transport Rh1 to the rhabdomere. Mutant rhabdomeres are reduced in size and a profusion of vesicles fills the photoreceptor cytoplasm. These cells lack normal globular MVBs, but contain infrequent, irregular vesicular organelles resembling defective MVBs. Other vesicular compartments, including ER and Golgi, retain normal appearance (Satoh, 2005).
Rab11 reduction via dsRNA expression in developing eyes similarly blocks Rh1 delivery to the rhabdomere and disperses it throughout photoreceptor cytoplasm. Electron microscopy shows MVBs are lost and irregular vesicles fill the cytoplasm. Rab11 activity was also reduced via expression of a dominant-negative Rab11, Rab11N124I, a GTP-binding mutant (Duman, 1999). Rab11N124I expression during the time when TRP is normally delivered to the rhabdomere blocks TRP delivery with a concomitant accumulation of TRP in photoreceptor cytoplasm. Expression during the Rh1 delivery period recapitulates genetic Rab11 reduction: Rh1 fails to reach the rhabdomere and instead accumulates in vesicles dispersed throughout the cytoplasm. Confocal and electron microscope examination of Rab11N124I photoreceptors shows a parallel loss of RLVs and MVBs (Satoh, 2005).
These results above show that loss of Rab11 activity results in the accumulation of Rh1-containing vesicles in the cytoplasm and the absence of MVBs. The possibility is considered that the Rh1 vesicles may originate from a defective endocytic pathway, such that the cytoplasm accumulates early endocytic vesicles unable to consolidate into MVBs. To address this possibility, the effect was examined of a dominant-negative form of Rab5, Rab5N142I, on the process of rhodopsin transport and MVB accumulation. This inhibits endocytosis and prevents MVB formation (Shimizu, 2003). Indeed, MVBs containing both TRP and Rh1 are lost in flies expressing Rab5N142I. The same images show that Rab5N142I does not inhibit TRP and rhodopsin delivery to the rhabdomere, confirming that MVBs are not required for transport to the rhabdomere (Satoh, 2005).
Simultaneous expression of Rab11N124I and Rab5N142I allowed a determination of whether the Rab11N124I phenotype is the result of absence of Rab11 activity prior to TRP and Rh1 delivery to the rhabdomere, or after endocytic removal of these two proteins from the rhabdomere. If the TRP and Rh1 vesicles accumulating in cytoplasm upon Rab11N124I expression are endocytosed from the rhabdomere, Rab5N142I expression should inhibit their biogenesis. This is not the case; numerous cytoplasmic TRP- or Rh1-bearing vesicles accumulate and rhabdomeres do not stain for TRP and Rh1 (Satoh, 2005).
The interpretation of these results could be complicated by consideration that guanine-nucleotide-deficient small GTPase dominant negatives sequester activating GEF proteins, and crosstalk may exist among RabGEF signaling pathways. However, the fidelity with which Rab11N124I recapitulates genetic and RNAi Rab11 loss, the observation that Rab5N142I mutant shows the expected endocytic defect, and the marked contrast in the Rab11N124I and Rab5N142I individual phenotypes, suggest these dominant negatives do generate a specific loss of function for each of these genes. From this perspective, the failure of Rab5N142I expression to impact the Rab11N124I phenotype argues Rab11 is required upstream of Rab5 and prior to the initial delivery of TRP and Rh1 to the rhabdomere (Satoh, 2005).
It was also observed that Golgi morphology visualized by CFP-galactosyl transferase in confocal microscopy is unaffected in Rab11N124I photoreceptors. These data, in agreement with the localization studies, suggest Rab11 is required for a post-Golgi step in rhodopsin and TRP movement to the rhabdomere (Satoh, 2005).
The proposed role of Rab11 in delivery of membrane proteins to the rhabdomere does not account for the loss of MVBs in the Rab11 mutant photoreceptors. It is possible that loss of Rab11 activity depletes the rhabdomere of Rh1 and other membrane proteins, and the lack of protein in these membranes limits the rate of endocytosis and MVB formation. To investigate this possibility more directly, the effect was examined of Rab11N124I on uptake of an endocytic tracer, Texas Red-conjugated avidin (TR-avidin), by larval Garland cells, large and easily accessible endocytic specialists (the Garland cell is a nephrocyte which takes up waste products from the haemolymph). In normal cells, internalized TR-avidin could be seen in peripheral, vesicular structures 10 minutes after exposure to TR-avidin. In Garland cells expressing Rab11N124I, however, TR-avidin is not internalized (Satoh, 2005).
Electron microscopic observations provided insight into this defect. In wild-type Garland cells, numerous labyrinthine channels invaginate deeply from plasma membrane. These channels are the sites of active endocytosis; clathrin-coated buds mark the tips of the channels and the channels elongate when endocytosis is inhibited in the dynamin mutant shibirets. The labyrinthine channels are absent in Garland cells expressing Rab11N124I. Thus, the labyrinthine channels do not form correctly in the absence of Rab11 activity. These results are consistent with the view that membrane components essential to sustaining vigorous endocytosis are lost when Rab11-dependent apical delivery is compromised (Satoh, 2005).
Embryonic cleavage leads to the formation of an epithelial layer during development. In Drosophila, the process is specialized and called cellularization. The trafficking pathways that underlie this process and that are responsible for the mobilization of membrane pools, however, remain poorly understood. Functional evidence is provided for the role of endocytic trafficking through Rab11 endosomes in remobilizing vesicular membrane pools to ensure lateral membrane growth. Part of the membrane stems from endocytosed apical material. Mutants in the endocytic regulators rab5 and shibire/dynamin inhibit basal-lateral membrane growth, and apical endocytosis is blocked in shibire mutants. In addition, shibire controls vesicular trafficking through Rab11-positive endosomes. In shibire mutants, the transmembrane protein Neurotactin follows the secretory pathway normally but is not properly inserted in the plasma membrane and accumulates instead in Rab11 subapical endosomes. Consistent with a direct role of shibire in vesicular trafficking through Rab11 endosomes, Shibire is enriched in this compartment. Moreover, electron microscopy demonstrates the large accumulation of intracellular coated pits on subapical endocytic structures in shibire mutants. Finally, Rab11 is shown to be essential for membrane growth and invagination during cellularization. Together, the data show that endocytic trafficking is required for basal-lateral membrane growth during cellularization. Rab11 endosomes are key trafficking intermediates that control vesicle exocytosis and membrane growth during cellularization. This pathway may be required in other morphogenetic processes characterized by the growth of a membrane domain (Pelissier, 2003).
Epithelia separate different environments within an organism. This property relies on the existence of different membrane domains, the apical surface that forms the lumen of most organs, and the basal-lateral surface. Epithelia can form via two cellular pathways: the mesenchyme-to-epithelium transition as studied in Madin Darby canine kidney (MDCK) cells is the most common pathway, and the other is embryonic cleavage, which leads to the formation of primary epithelial tissues in a variety of organisms. Although the processes are different, the columnerization of flat squamous epithelial cells and embryonic cleavage are both accompanied by the growth of a basal lateral membrane domain. The extent of membrane growth varies between tissues. The cellular mechanisms underlying polarized membrane growth remain poorly understood (Pelissier, 2003).
In MDCK cells, the process depends mostly on vesicular sorting along the secretory pathway from the trans Golgi network (TGN) and on vesicular insertion at adherens junctions via the recruitment of the multiprotein complex called exocyst. In other cases, such as cultured hepatocytes, however, the process relies on vesicular sorting through endocytic trafficking pathways. In this case, called transcytosis, all proteins are first targeted to the future apical surface and endocytic targeting from the apical domain to another membrane domain ensures the formation of the basal-lateral membrane surface. The contribution of membrane trafficking pathways to the growth and polarization of the cell surface during embryonic cleavage has not been thoroughly addressed yet (Pelissier, 2003).
Drosophila cellularization, a specialized form of embryonic cleavage, is a very good system for addressing this problem, although the process may in part rely on specific mechanisms that may not apply to other cases of epithelial morphogenesis. In the early syncytial embryo, the nuclei undergo 13 synchronous division cycles in a common cytoplasm. Upon entry into cycle 14, the cell surface increases, and the plasma membrane invaginates between the nuclei. This invagination results in a columnar epithelium. Cellularization proceeds in two distinct phases, slow phase and fast phase. The process is characterized by the polarized growth of the cell surface as revealed by surface-labeling experiments in living embryos. Distinct membrane behaviors are observed in slow phase and fast phase. When the plasma membrane is labeled at the onset of cellularization, the surface marker gradually disappears during slow phase from the apical surface rich in microvilli and accumulates instead along the growing basal-lateral surface. If the surface is labeled instead at the onset of fast phase, a patch of unlabeled membrane appears in fast phase in the apical-lateral membrane. Two different mechanisms could underlie the disappearance of apical surface marker specific to the slow phase and its basal-lateral accumulation. The process could depend on the direct apical insertion of intracellular unlabeled membrane and a sorting between the newly inserted and recipient membranes within the plane of the epithelium. Conversely, the process could stem from an endocytic-based membrane transfer from the apical to the basal-lateral surface in a manner akin to transcytosis (Pelissier, 2003).
The transport of vesicles along the endocytic pathway proceeds via a series of organelles defined in part by the transport of cargo molecules (e.g., the transferrin receptor) and in part by the specific localization of regulatory molecules. Internalized vesicles at the plasma membrane are transported to early endosomes, also called sorting endosomes. This step is dependent on the activity of the small GTPase Rab5 as well as on Dynamin. Dynamin is encoded by shibire in Drosophila and controls vesicle budding and internalization in a large number of tissues. Vesicles are then transported back to the plasma membrane, destined for the late endosome and lysosome degradation pathway, or transported to the pericentriolar recycling endosome. The recycling endosome is involved in the recycling of vesicles back to the plasma membrane. In polarized epithelial cells, the apical recycling endosome is required for cell polarization as well as for transcytosis. Trafficking through recycling endosomes is dependent on the small GTPase Rab11 in mammals as well as in Drosophila. Rab11 localizes to this organelle in both vertebrates and invertebrate. Dynamin was also shown to control vesicle budding from recycling endosomes in mammalian cells.In order to address the implication of endocytic trafficking in plasma membrane growth during cellularization, the roles of key effectors of these pathways were tested. The requirements for Dynamin, Rab5, and Rab11 were tested (Pelissier, 2003).
The overexpression of Rab5S43N, a dominant-negative variant of Rab5, has been shown to block endocytosis in a number of Drosophila tissues. Because cellularization coincides with the zygotic induction of gene expression, high enough levels of the UAS-Rab5S43N transgene cannot be expressed. To circumvent this problem, GST+Rab5S43N was prepared in vitro and the proteins were injected into embryos during cycle 13, 10-15 min before cellularization. Injection of GST+Rab5S43N reduces the speed of membrane invagination near and opposite to the site of injection during slow phase:this is in contrast to the injection of GST alone and of GST+Rab5WT. The delay in membrane invagination can be seen via a comparison of the relative positions of the membrane front and of the nuclei at the end of slow phase (+26'), and in fast phase (+29' and +32'). Confocal images show the different positions of the membrane front and of the plasma membrane within the injected and the control area. Similar results are obtained with another dominant-negative variant of Rab5, Rab5N142I. However, no invagination defect was observed after injection of a dominant-negative form of Rab1, Rab1N124I, which is involved in ER-to-Golgi transport. These observations support the fact that the injection of Rab5S43N has a specific effect during cellularization (Pelissier, 2003).
The role of endocytosis from the plasma membrane can be examined by using the temperature-sensitive allele of shibire/dynamin. The timing of gene inactivation can be precisely controlled by shifting embryos to the restrictive temperature at 32°C. In shibire-ts mutant embryos this causes the total inhibition of membrane invagination in slow phase, unlike in control embryos, where cellularization proceeds normally. In contrast, both control and shibire-ts mutant embryos cellularize when the embryos are shifted to the restrictive temperature at the beginning of fast phase, albeit at a slightly slower rate in shibire-ts mutants. The specific requirement for shibire during slow phase is remarkable in light of the fact that, in wild-type embryos, apical membrane-labeled material is cleared from the plasma membrane specifically in slow phase. In agreement with the known role of shibire/dynamin in the internalization of plasma membrane vesicles, this study shows that apically labeled membrane is no longer endocytosed in a shibire mutant, as opposed to wild-type control embryos. Together, the data so far suggest that endocytosis from the apical plasma membrane is required throughout slow phase in order for the basal-lateral surface to grow (Pelissier, 2003).
Although the results are consistent with the known role of shibire in plasma membrane endocytosis, Dynamin has also been implicated in vesicle budding and trafficking from the TGN and from recycling endosomes. The possibility that other trafficking defects might account for the role of shibire during cellularization was also examined. Neurotactin is a transmembrane protein synthesized de novo during slow phase. In control embryos, the protein traffics through the secretory pathway and, at steady state, Nrt is predominantly localized at the plasma membrane. Small vesicular staining is also detected in the Golgi. shibire mutant embryos at the restrictive temperature were shifted at the onset of cellularization (e.g., when Nrt is not yet synthesized), incubated for 20 min during slow phase, and fixed to monitor the localization of Nrt. The striking result is that Nrt is not properly inserted in the plasma membrane and accumulates instead in a large subapical compartment. This compartment is not the Golgi apparatus, which can be detected with the middle-trans Golgi marker p120. In control and shibire mutant embryos, p120 is indeed mostly concentrated in the basal cytoplasm in the form of small (<2microm) vesicular structures. The very different distribution of these two proteins suggests that Nrt follows the secretory pathway normally but fails to be inserted in the plasma membrane apically. Note that Toll, a maternally provided transmembrane protein that is already present at the plasma membrane at the onset of cellularization, remains in the plasma membrane, unlike Nrt in a shibire mutant. The trafficking defects observed with Nrt, a newly synthesized protein en route to the plasma membrane, suggest that shibire controls another step in vesicular trafficking (Pelissier, 2003).
Rab11 was found in the search for markers of intracellular compartments in which Nrt accumulates. In Drosophila, during slow phase, Rab11 accumulates mostly in the subapical cytoplasm in two large pericentriolar endocytic compartments. An additional staining is seen in small puncta (<1microm) concentrated mostly in the basal cytoplasm and, in some cases, in the apical cytoplasm. These small puncta colocalize with Golgi markers, unlike the large apical staining. At steady state, Nrt is virtually absent from the large apical Rab11 endosomes, although some colocalization is occasionally detected in the small Rab11-positive Golgi puncta. However, in a shibire mutant, the subapical accumulation of Nrt predominantly colocalizes with Rab11 in the large subapical pericentriolar endosomes (Pelissier, 2003).
The sequestration of Nrt in Rab11 apical endosomes is unlikely to be the indirect effect of perturbing globally endocytic traffic in a shibire mutant. Indeed, an antibody to Shibire was used to detect the protein and a clear enrichment of Shibire was found in Rab11 endosomes, although the colocalization is not total. Shibire may mark a subdomain of recycling endosomes, as reported for other endosomal proteins. Moreover, the localization of Rab11 and of Shibire follows a similar developmental regulation. The proteins colocalize in subapical endosomes during slow phase but not in fast phase, when shibire is in fact no longer required for cellularization (Pelissier, 2003).
These results argue that shibire is also required for vesicular budding from Rab11 recycling endosomes during cellularization. In support of this conclusion, very large amounts of intracellular subapical coated pits were found in shibire mutant embryos, by using transmission electron microscopy. Large subapical endocytic structures, all of which contain one or more dark uncleaved vesicle, were found. In wild-type embryos, however, no such vesicular structures were detected in the subapical cytoplasm, suggesting that these structures may form very rapidly and transiently in the wild-type. Given the role of Dynamin/Shibire in severing vesicles, these observations support the role of shibire in vesicular traffic from subapical endosomes in addition to its role in plasma membrane endocytosis (Pelissier, 2003).
The role of trafficking through recycling endosomes was further tested by using a dominant-negative form of Rab11, Rab11S25N, which is an effector of this process. Injection of GST+Rab11S25N during cycle 12-13 causes a striking inhibition of membrane invagination during slow phase. Such an effect is not detected when either GST or GST+Rab11WT are injected in embryos at the same stages (Pelissier, 2003).
In vertebrates, Rab11 interacts with members of the Arfophilin family of proteins. Drosophila nuclear-fallout (nuf) encodes a centrosomal protein that is a member of the Arfophilin family, suggesting that, in Drosophila, rab11 and nuf may function in a similar functional pathway. In support of this proposal, nuf is required for the formation of membrane furrows prior to cellularization as well as during slow phase. In addition, the apical Rab11 endosomes are assembled at the centrosomes, as revealed by the centrosomal protein gamma-Tubulin. Interestingly, on top of the cellularization defects, Rab11S25N-injected embryos show a distinct 'nuclear fallout' phenotype reminiscent of nuf mutant embryos. Instead of being regularly aligned at the cortex, the nuclei are disorganized and occasionally 'fall' inside the embryo (Pelissier, 2003).
Two classes of phenotypes, distinguished by their severity, are described. In both cases, the phenotype is concentrated in the area of the embryo that was injected, at the injection site, and opposite to it. Class 1 (weak) represents 50% of the injected embryos (n = 56) and shows weak nuclear defects and membrane invagination defects. Of the injected embryos, 37.5% fall in class 2 (strong) and have strong nuclear and membrane invagination defects in slow phase. These data further support the role of trafficking through Rab11 endosomes to ensure membrane growth during cellularization (Pelissier, 2003).
The identification of Rab11 endosomes as a key intermediate in the trafficking of vesicles necessary for lateral membrane growth raises two questions. Where does the membrane come from? Where is it eventually inserted? The facts that shibire and rab5 are both required for basal-lateral membrane growth and that in a shibire mutant apically labeled membrane is not internalized suggests that the plasma membrane present in the villous projections accounts in part for the growth of the lateral surface. The plasma membrane is indeed a huge reservoir whose capacity largely exceeds the membrane needed for lateral membrane growth during slow phase. Trafficking through Rab11 endosomes appears to also be essential for cellularization, as assayed by the injection of a dominant-negative form of Rab11 and as revealed by the role of shibire in vesicular trafficking through this compartment. In a shibire mutant, the transmembrane protein Nrt, which is de novo synthesized and traffics normally through the Golgi, accumulates in part in Rab11 endosomes. This suggests that secretory material might also contribute to the growth of the basal-lateral surface. This is supported by the fact that the injection of the drug Brefeldin A, an inhibitor of Endoplasmic Reticulum (ER) to Golgi transport, blocks cellularization and that the Golgi structural protein Lava-Lamp is required for cellularization. Note that the failure to detect any invagination defect after injection of dominant-negative Rab1 could stem from the fact that Rab2 may compensate for ER-to-Golgi vesicular transport. Together, the data suggest that Rab11 endosomes constitute a point of integration of vesicles from the secretory and endocytic pathways necessary for the rapid exocytosis of vesicles required for the growth of the lateral membrane. Note that the ER has been suggested to be a membrane reservoir required in part for the formation of pseudopods, another example of rapid membrane growth during phagocytosis. In this process, an early step called 'focal exocytosis' is controlled in part by Dynamin-2 (Pelissier, 2003).
After trafficking through Rab11 endosomes, where might the vesicles be finally inserted? The sites of membrane insertion are likely to dictate how polarity arises. In light of the published data, the most likely proposal would state that vesicles are inserted at the basal junctions, a transient adherens junction that forms between the membrane front called 'furrow canal' and the lateral surface. The adherens junction is indirectly required for lateral membrane growth and basal-lateral targeting in polarizing MDCK cells through the recruitment of Sec6 and Sec8, key components of the exocyst that localize in part at adherens junctions and control vesicle insertion, presumably from the Golgi apparatus. Consistent with a role of junctions in regulating vesicular insertion, when basal adherens junctions fail to form, as in slam mutant embryos, membrane invagination is blocked and Nrt is less efficiently inserted in the plasma membrane (Pelissier, 2003).
Why does cellularization involve such a pathway to ensure the growth and invagination of the plasma membrane? The comparison with other examples of cells in which the plasma membrane is rapidly remodeled provides insight into this question. Endocytic trafficking is usually much more efficient than secretory trafficking at remobilizing membrane pools. Antigen-presenting cells and migrating Dictyostelium ameobae can, for instance, recycle their whole surface within a few minutes. In a situation where the membrane grows rapidly, remobilizing preexisting membrane pools at the plasma membrane and in the secretory pathway through recycling endosomes might be a very efficient way to transfer membrane 'en masse' toward a defined site of the plasma membrane. Note that endocytic recycling is indeed involved in the formation of membrane protrusions, ruffles, and lamellipodesa but also in other cases in which invaginations form, for example during cytokinesis. rab11 is required for late stages of cytokinesis in the C. elegans embryo (Skop, 2001). Endobrevin/VAMP8, a regulator of endosome trafficking, is also required for cytokinesis in mammalian cells, together with Syntaxin 2. Trafficking through recycling endosomes may also provide an efficient way to couple surface polarization to membrane growth. The current findings are consistent with the similarities between cellularization and cytokinesis. Through the identification of a trafficking pathway involved in the growth of the lateral surface during cellularization, this work may also provide insight into the formation of an epithelial sheet during embryonic cleavage (Pelissier, 2003).
Abdomen and germ cell development of the Drosophila embryo requires proper localization of Oskar mRNA to the posterior pole of the developing oocyte. Oskar mRNA localization depends on complex cell biological events like cell-cell communication, dynamic rearrangement of the microtubule network, and function of the actin cytoskeleton of the oocyte. To investigate the cellular mechanisms involved, a novel interaction type of genetic screen was developed by which 14 dominant enhancers were isolated of a sensitized genetic background composed of mutations in oskar and in TropomyosinII, an actin binding protein. The detailed analysis of two allelic modifiers is described that identify Drosophila Rab11, a gene encoding small monomeric GTPase. Mutation of the Rab11 gene, involved in various vesicle transport processes, results in ectopic localization of Oskar mRNA, whereas localization of Gurken and Bicoid mRNAs and signaling between the oocyte and the somatic follicle cells are unaffected. The ectopic oskar mRNA localization in the Rab11 mutants is a consequence of an abnormally polarized oocyte microtubule cytoskeleton. These results indicate that the internal membranous structures play an important role in the microtubule organization in the Drosophila oocyte and, thus, in Oskar RNA localization (Jankovics, 2001).
Whatever the role of the Drosophila Rab11 gene in the Osk localization pathway, it must be indirect and act in the reorientation of the oocyte microtubule network, as seen by the Tau:GFP microtubule visualization and by mislocalization of the Kinesin:ß-galactosidase fusion protein to the center in the Rab11 mutants. Central mislocalization of the Kinesin:ß-galactosidase protein has been observed in mutants that impair any step of the reciprocal signaling events between the oocyte and the posterior follicle cells. Given that the best-characterized role of the Rab11 proteins is the targeting of recycling endosomes or trans-Golgi vesicles to the plasma membrane, it seemed to be plausible that Rab11 mutants would exert their phenotypes by blocking signaling events between the oocyte and the follicular cells. However, two types of evidence suggest that both the oocyte-to-follicle cells and the follicle cells-to-oocyte signals are functional in the Rab 11 mutants: (1) absence of expression of an enhancer trap in follicle cells at the posterior cap indicates that the posterior polar follicle cell fate is properly adopted; (2) a focus of microtubules at the posterior pole was never observed by Tau:GFP labeling, indicating that posterior MTOC disassembles. Consistently, mislocalization of the Bcd mRNA to the posterior pole was never observed, indicating again that the back signaling from the posterior polar follicle cells is received, and the MTOC and the minus ends of the microtubules disappear from the posterior pole. However, working with hypomorphic allele combinations, the possibility that mislocalization of Bcd mRNA was not detected because of its relative insensitivity to the microtubule reorientation cannot be excluded. The analysis of the hypomorphic phenotypes also supports this interpretation. An intermediate Osk mislocalization phenotype occurs in Rab11 mutants, when Osk mRNA is detected in the center and simultaneously at the normal posterior position in the same oocyte. This indicates that in such mutant oocytes the MTOC does indeed disappear and the minus ends of the microtubules are replaced by plus ends at the posterior. Rab11 phenotypes are reminiscent of that of par1. In par1 mutant oocytes, the posterior MTOC also disappears but central osk mRNA localization is observed. It is therefore concluded that even though the posterior MTOC normally disassembles, the reorientation of the oocyte microtubule network is incomplete in Rab11 mutants. It is proposed that instead of having reverse polarity when the microtubules are nucleated predominantly from the anterior, in Rab11 mutants only a subset of microtubules, which are nucleated over the entire cortex of the oocyte, is intact driving Kinesin:ß-galactosidase motor protein and Osk mRNA to the center of the oocyte. In Rab11 mutants, Osk mRNA mislocalization phenotype is not fully penetrant and transient, and by stage 10-11, egg chambers exhibit wild-type Osk mRNA localization. It is suggested that the Rab11-dependent localization pathway for Osk RNA itself is redundant and the recovery observed in later stages is due to an alternative, Rab11-independent Osk localization mechanism when cytoplasmic streaming, which begins at stage 10, directs the Osk mRNA to the posterior pole. These results indicate that posterior MTOC breakdown may not be sufficient for reorientation of the microtubule network during stages 6-8 of the Drosophila oocyte; rather, the reorientation process depends on other factors too, like internal membrane functions. The precise mechanism by which Rab11 contributes to the microtubule reorientation is still unclear. A similar phenotype, characterized by Osk mislocalization to the center of the oocyte, is observed in Ter94 mutant ovaries. Ter94 encodes an AAA type ATPase that is also responsible for internal membrane trafficking, namely, for homotypic fusion of endoplasmic reticulum vesicles. Rab11 and Ter94 phenotypes reveal that the internal membranous structures and the cytoskeleton of the Drosophila oocyte have a functional connection to conduct cytoplasmic mRNA localization (Jankovics, 2001).
The Drosophila embryonic body plan is specified by asymmetries that arise in the oocyte during oogenesis. These asymmetries are apparent in the subcellular distribution of key mRNAs and proteins and in the organization of the microtubule cytoskeleton. Evidence suggests that the Drosophila oocyte also contains important asymmetries in its membrane trafficking pathways. Specifically, alpha-adaptin and Rab11, which function critically in the endocytic pathways of animal cells, are localized to neighboring compartments at the posterior pole of stage 8-10 oocytes. Rab11 and alpha-adaptin localization occurs in the absence of a polarized microtubule cytoskeleton, i.e. in grk null mutants, but is later reinforced and/or refined by Osk, the localization of which is microtubule dependent. Analyses of germline clones of a rab11 partial loss-of-function mutation reveal a requirement for Rab11 in endocytic recycling and in the organization of posterior membrane compartments. Such analyses also reveal a requirement for Rab11 in the organization of microtubule plus ends and osk mRNA localization and translation. It is proposed that microtubule plus ends and, possibly, translation factors for osk mRNA are anchored to posterior membrane compartments that are defined by Rab11-mediated trafficking and reinforced by Rab11-Osk interactions (Dollar, 2002).
The best evidence that the plasma membrane of the oocyte and membrane trafficking pathways are polarized in the Drosophila oocyte comes from the expression pattern of human transferrin receptor (Htr) in transgenic flies. During stages 8-10, the posterior pole of the oocyte becomes enriched with Htr, both along the plasma membrane and in vesicles. Htr rapidly disappears from vesicles upon inhibition of endocytosis, indicating that Htr is actively internalized and recycled in Drosophila oocytes as it is in many other examined cells. Together with the observation that Htr is restricted to the posterior plasma membrane, its active internalization and recycling strongly suggests that the membrane recycling pathway of the oocyte is polarized towards the posterior pole (Dollar, 2002 and references therein).
To investigate the role of Rab11 in the organization of the posterior pole of the oocyte, the distribution of osk mRNA and protein was examined in rab11P2148 germ-line clones (GLCs). In wild-type oocytes, osk mRNA is transported to the posterior pole during stages 8 and 9, coincident with the polarization of the microtubule cytoskeleton. During transport, and in the initial hours following transport, osk mRNA is seen as a large ball. By the end of stage 9, the ball resolves into a thin cap along the posterior cortex, which persists through the end of oogenesis. The nature of the transition from the ball to the cap is not clear, but coincides with the activation of osk translation. The cap structure is much more resistant to disruption with colchicine than is the ball and appears, then, to represent the binding of the mRNA to an anchor, which might be Osk. rab11P2148 germ-line clones (GLCs) are defective in the transport of osk mRNA to the posterior pole, and in its subsequent translation and anchoring. The transport defect is temporal in nature. Thus, while most osk transcripts reach the posterior pole of wild-type oocytes during stage 8, only a small fraction of transcripts reach the posterior pole of rab11 oocytes during stage 8. Typically, the lagging transcripts are aggregated into a mass near the center of the cell, possibly representing stalled transport at an intermediate step. Although most osk transcripts eventually reach the posterior pole of rab11 oocytes, two observations suggest that they are never anchored: (1) no Osk protein was ever detected in rab11 oocytes; (2) the osk transcripts of rab11P2148 GLCs never formed the characteristic cap at the posterior pole, but instead remained as a ball. Moreover, during late stages of oogenesis (e.g. when microtubules are bundled along the entire egg cortex), the ball of osk mRNA appears to drift away from the posterior pole and is often fragmented into several smaller balls. It is concluded that Rab11 is required for the efficient transport of osk mRNA to the posterior pole of the oocyte and for its subsequent translation and anchoring (Dollar, 2002).
Rab11 and alpha-adaptin are localized to the posterior pole of mid-stage oocytes and such localization does not require a polarized microtubule cytoskeleton or grk signaling. A reduction of rab11 activity in the oocyte alters the subcellular distribution of Rab11, alpha-adaptin and internalized transferrin. These alterations indicate that Rab11 is required for endocytic recycling and to organize posterior membrane compartments. A reduction of rab11 activity in the oocyte also causes defects in Kin:ß-gal localization, osk mRNA transport, and osk mRNA translation and anchoring. These latter defects suggest that the posterior membrane compartments established by Rab11 organize microtubule plus ends and, possibly, the translation factors and/or anchors for osk mRNA (Dollar, 2002).
The expression pattern of Htr in transgenic flies shows clearly that the oocyte establishes a posterior plasma membrane domain (PMD). Further evidence for such a domain comes from the finding that Rab11 localizes to the posterior pole of wild-type oocytes. Interestingly, the PMD is established independently of microtubule polarity or grk signaling, since Rab11 is localized normally in grk null mutants. Given the rapid rate at which Htr is internalized and recycled, it is likely that the maintenance, if not also the initial specification, of the PMD requires polarized endocytic recycling directed towards the posterior pole. The data presented indicate that Rab11 is responsible for such recycling: Rab11 is localized to the posterior pole of the oocyte and is required for the recycling of internalized transferrin (the ligand for transferrin receptor) to the plasma membrane of cultured oocytes. Independent evidence that Rab11 mediates polarized endocytic recycling comes from studies with vertebrates, where Rab11 recycles internalized molecules to the apical surface of polarized epithelial cells (Dollar, 2002).
What polarizes endocytic recycling to the posterior pole of the oocyte? The polarization of the endocytic pathways of other cells is triggered by Rho GTPase family members (i.e. Rho, Cdc42 and Rac), which are activated at specific regions of the cell cortex by a variety of intrinsic and extrinsic cues. Rho GTPases have also been strongly implicated in the polarization of exocytosis. Specifically, they have been shown to recruit the 'exocyst' to specific sites of the plasma membrane. The exocyst is a conserved complex of proteins to which vesicles of the secretory pathway fuse. Thus, through local activation of Rho GTPases, secretory vesicles are targeted to specific regions of the plasma membrane. By analogy, the Rho GTPases could localize Rab11 and polarize receptor recycling through local recruitment of an exocyst-like complex for Rab11-containing vesicles. Because Drosophila Rho GTPases are required for progression through early oogenesis, the analysis of their role in Rab11 localization and other aspects of oocyte polarization must await the identification of conditional mutants (Dollar, 2002).
Kinesin:ß-gal expression studies indicate that microtubule plus ends are not sharply focused onto the posterior pole of the oocyte in rab11P2148 mutant oocytes. The simplest interpretation of this finding is that microtubule plus ends are attached to the PMD, or to a neighboring membrane domain whose identity is established and/or maintained by Rab11. In wild-type oocytes, this domain is tightly defined such that Kin:ß-gal is concentrated at the posterior tip of the oocyte, while in rab11P2148 mutant oocytes, the domain is poorly defined and the Kin:ß-gal expression pattern is expanded. The slight enrichment of Kin:ß-gal at the posterior tip of rab11P2148 oocytes could reflect partial Rab11 activity and/or the polarizing activities of membrane trafficking pathways that may not rely on Rab11 (e.g. the secretory pathway), which targets newly synthesized molecules from the Golgi to the plasma membrane. Recent studies have identified two types of protein-protein interactions (CLIP-CLASP and APC-EB1) responsible for the stable association of microtubule plus ends with membranes. While the CLIP, CLASP, APC and EB1 protein families are all well-represented in the Drosophila genome, their role in the establishment of oocyte polarity has not yet been investigated (Dollar, 2002).
Apart from Rab11, the only protein known to play a specific role in microtubule plus end organization in Drosophila oocytes is Par-1, a kinase, whose suspected targets include the microtubule associated protein Tau. In strong par-1 mutants, microtubule plus ends, as revealed by Kin:ß-gal expression patterns, are not enriched at the posterior pole of the oocyte, but instead are concentrated tightly, forming a dot at the center of the cell. In weak par-1 mutants, a small amount of Kin:ß-gal is also found at the posterior pole. This small amount of Kin:ß-gal is always tightly localized to the cell tip, suggesting that Par-1 is not required for the specification of the PMD, but rather only for the efficient movement of already focused microtubule plus ends from the cell center to the PMD. Consistent with the idea that microtubule plus ends initially focus to a sharp point at the center of the cell and then move to the posterior pole, Kin:ß-gal and oskar mRNA show transient concentration at the center of the cell in wild-type oocytes. How Par-1 might promote the movement of microtubule plus ends from the cell center to the posterior pole is not clear. One possibility is that it promotes attachment of microtubule plus ends to a structure that is then moved to the posterior pole. Alternatively, Par-1 might stimulate a burst of microtubule growth, forcing growth toward the posterior end of the cell (Dollar, 2002).
The observation that osk mRNA transport to the posterior pole is delayed in rab11P2148 mutant oocytes suggests that Rab11 might also have a role in the movement of microtubule plus ends from the cell center to the posterior pole, and therefore, that such movement is membrane dependent. For example, microtubule plus ends could become attached to membrane compartments or vesicles at the cell center, and the vesicles may then be targeted to the posterior pole in a Rab11-dependent manner. Because osk mRNA arrives at the posterior pole as a fairly well-defined ball in rab11P2148 oocytes, Rab11 does not appear to be required for focusing microtubule plus ends at the cell center, but rather only for their timely movement and attachment to the posterior membrane domain (Dollar, 2002).
Although most osk transcripts are eventually transported to the posterior pole in rab11P2148 oocytes, they are not translated. Since Osk is required to anchor oskar mRNA at the posterior pole, the lack of oskar translation in rab11P2148 GLCs could explain the inability of the oskar mRNA ball to resolve into the thin posterior crescent. The nature of the osk translation block in rab11P2148 oocytes is not clear. One possibility is that key osk translation factors are localized to the posterior membrane domain established by Rab11. In rab11P2148 oocytes, this domain may be too poorly defined to support assembly of such factors into an active translation complex (Dollar, 2002).
All stem cells have the ability to balance their production of self-renewing and differentiating daughter cells. The germline stem cells (GSCs) of the Drosophila ovary maintain such balance through physical attachment to anterior niche cap cells and stereotypic cell division, whereby only one daughter remains attached to the niche. GSCs are attached to cap cells via adherens junctions, which also appear to orient GSC division through capture of the fusome, a germline-specific organizer of mitotic spindles. This study shows that the Rab11 GTPase is required in the ovary to maintain GSC-cap cell junctions and to anchor the fusome to the anterior cortex of the GSC. Thus, rab11-null GSCs detach from niche cap cells, contain displaced fusomes and undergo abnormal cell division, leading to an early arrest of GSC differentiation. Such defects are likely to reflect a role for Rab11 in E-cadherin trafficking as E-cadherin accumulates in Rab11-positive recycling endosomes (REs) and E-cadherin and Armadillo (ß-catenin) are both found in reduced amounts on the surface of rab11-null GSCs. The Rab11-positive REs, through which E-cadherin transits, are tightly associated with the fusome. It is proposed that this association polarizes the trafficking by Rab11 of E-cadherin and other cargoes toward the anterior cortex of the GSC, thus simultaneously fortifying GSC-niche junctions, fusome localization and asymmetric cell division. These studies bring into focus the important role of membrane trafficking in stem cell biology (Bogard, 2007).
The first clue that Rab11 plays important roles in early oogenesis in Drosophila came from immunostaining experiments that revealed strong expression of endogenous Rab11 and a fully functional Rab11::GFP in GSCs, cystoblasts and young (2-4- and 8-cell) germline cysts. Strikingly, the proteins were concentrated as discrete dots on the fusome, which electron microscopy and photobleaching studies have shown is highly vesicular and rapidly exchanged with other membrane stores. Triple-stain experiments showed that some of these dots also contained E-cadherin, which has been shown to transit though Rab11-positive recycling endosomes (REs) en route to the plasma membrane in some cells. High-magnification images showed that the Rab11 (and, more rarely, E-cadherin) dots were often nestled into cavities within the fusome. Such Rab11-harboring cavities are visible in the fusomes of all examined GSCs, cystoblasts and young germline cysts, not only in the ovary but also in the testes. In view of the well-described enrichment of Rab11 in REs, it is proposed that these Rab11- and E-cadherin-harboring cavities are REs and are therefore referred to as FREs (fusome-associated REs) (Bogard, 2007).
These studies indicate that Rab11 maintains GSC identity through polarized trafficking of E-cadherin and, possibly, other cargoes that reinforce essential GSC-niche contacts. These studies further indicate that Rab11 is required for fusome localization and asymmetric GSC division and suggest a feedback linkage between these events and E-cadherin trafficking. Although Rab11 has been implicated in the trafficking of E-cadherin in other cells, there are no other cases in which such trafficking has been correlated with a biological response. It will be of interest to determine whether Rab11 is required for the maintenance of stem cells in other systems and whether such maintenance involves E-cadherin trafficking or the trafficking of other adhesion molecules. It will also be of interest to determine the role of Rab11 in other E-cadherin-dependent cell behaviors, particularly as Rab11, at least in Drosophila, is expressed in only a small subset of E-cadherin-expressing cells (Bogard, 2007).
Rab11 is a small GTPase that regulates several aspects of vesicular trafficking. This study shows that Rab11 accumulates at the cleavage furrow of Drosophila spermatocytes and that it is essential for cytokinesis. Mutant spermatocytes form regular actomyosin rings, but these rings fail to constrict to completion, leading to cytokinesis failures. rab11 spermatocytes also exhibit an abnormal accumulation of Golgi-derived vesicles at the telophase equator, suggesting a defect in membrane-vesicle fusion. These cytokinesis phenotypes are identical to those elicited by mutations in giotto (gio) and four wheel drive (fwd) that encode a phosphatidylinositol transfer protein and a phosphatidylinositol 4-kinase, respectively. Double mutant analysis and immunostaining for Gio and Rab11 indicated that gio, fwd, and rab11 function in the same cytokinetic pathway, with Gio and Fwd acting upstream of Rab11. It is proposed that Gio and Fwd mediate Rab11 recruitment at the cleavage furrow and that Rab11 facilitates targeted membrane delivery to the advancing furrow (Giansanti, 2007; full text of article).
The Gio PITP is enriched at the furrow membrane and that it is required for Drosophila cytokinesis (Giansanti, 2006). This study shows that the furrow membrane is also enriched in Rab11 and that Rab11 localization at the equatorial membrane requires the wild-type activity of both giotto (gio) and four wheel drive (fwd). In addition, the wild-type functions of gio, fwd, and rab11 are all required for membrane-vesicle fusion during cytokinesis, because mutations in these genes result in an abnormal accumulation of Golgi-derived vesicles at the equator of telophase cells (Giansanti, 2006). Finally, the results strongly suggest that gio, fwd, and rab11 function in the same cytokinesis pathway. These observations suggest a model for the mechanisms underlying membrane addition to the cleavage furrow during spermatocyte cytokinesis. It is proposed that Gio mediates transfer of PtdIns monomers to the furrow membrane, causing a local enrichment in PtdIns molecules. The association of Gio with this membrane domain may facilitate recruitment of the PtdIns-4-kinase encoded by fwd, which would mediate phosphorylation of PtdIns to PtdIns(4)P, allowing their further phosphorylation to PtdIns(4,5)P2. Fwd may also mediate Rab11 recruitment at the cleavage furrow, allowing targeted Rab11-dependent vesicle fusion events necessary for completion of cytokinesis. It is realized that this is a rather speculative model. Its major drawback is that the subcellular localization and the molecular interactions of the Drosophila Fwd protein are currently unknown. However, studies in S. pombe have shown that one of the PtdIns-4-kinases present in this organism interacts with Cdc4p, a contractile ring protein essential for cytokinesis. This finding indicates that, at least in fission yeast, one of the PtdIns-4-kinases is associated with the cleavage furrow. In addition, a recent study has shown that one of the mammalian PtdIns-4-kinases interacts physically with Rab11 and is required for Rab11 localization in the Golgi complex. The same study has also shown that recruitment of this kinase to the Golgi does not require Rab11 (de Graaf, 2004). These results are consistent with the current findings, and they lead to the belief that Gio, Fwd, and Rab11 are all enriched at cleavage furrow, where they work in concert to ensure proper vesicle docking and fusion (Giansanti, 2007).
Mutations in rab11 cause frequent failures in meiotic cytokinesis of males without affecting cytokinesis of larval brain neuroblasts. The mutations analyzed are obviously hypomorphic since they cause lethality at the larval and pupal stages, whereas rab11 null alleles result in embryonic lethality. Thus, it is possible that the rab11 mutants analyzed retain a residual Rab11 activity that is sufficient for neuroblast cytokinesis but not meiotic cytokinesis. Alternatively, Rab11 may not be required for mitotic cytokinesis. A strong support for a specific involvement of Rab11 in meiotic cytokinesis comes from recent RNAi screens that have shown that Rab11 has little or no role in S2 cell cytokinesis (Giansanti, 2007).
Previous studies have shown that null mutations in fwd and fws disrupt spermatocyte cytokinesis but that they have no observable effects on larval neuroblast mitosis (Brill, 2000; Farkas, 2003; Giansanti, 2004). Thus, at least three proteins involved in membrane traffic, Rab11, Cog5 (encoded by four way stop or fws), and a PtdIns-4-kinase, seem to be specifically required for meiotic cytokinesis. This specificity is unlikely to depend on the peculiar features of the final steps of spermatocyte cytokinesis. In male meiotic cells, the cytoplasmic bridges generated by ring constriction are not severed by a canonical abscission process, as occurs in larval neuroblasts; they instead persist and are stabilized by the formation of a specialized structure called ring canal. Mutations in rab11, fws and fwd inhibit ring constriction and furrow ingression during early telophase and block cytokinesis well before the formation of a cytoplasmic bridge. These observations rule out the possibility that the spermatocyte-specific effects of these mutations reflect problems in the final step of cytokinesis when ring canals are assembled (Giansanti, 2007).
The specific role of Rab11, Cog5, and Fwd in spermatocyte cytokinesis may reflect a specifically high requirement for formation of new membrane at the advancing cleavage furrow. To fulfill this requirement, male meiotic cells may exploit all the extant pathways for membrane addition. These pathways would be redundant in mitotic cell where the requirements for membrane expansion at the advancing furrow are relatively low. Alternatively, the specific requirement of membrane trafficking functions for spermatocyte cytokinesis may reflect the organization of membrane stores within these cells. Spermatocytes contain a large ER that includes astral and parafusorial membranes, and they do not possess a detectable pericentriolar RE. Larval neuroblasts do exhibit a spindle envelope, but, in contrast to spermatocytes, they are devoid of astral membranes and possess pericentriolar REs (Giansanti, 2006). Thus, formation of new membrane during spermatocyte cytokinesis might utilize membrane trafficking activities that are at least in part distinct from those used by mitotic cells, depending on the organization of membrane stores within the two cell types (Giansanti, 2007).
Whatever the reason for their specific sensitivity to mutations that disrupt membrane-related functions, Drosophila spermatocytes are emerging as an extremely useful model system for studying membrane traffic during animal cell cytokinesis. There is indeed growing evidence that the analysis of mutations that disrupt spermatocyte cytokinesis can reveal membrane-trafficking genes that play redundant cytokinetic roles in other animal cell systems (Giansanti, 2007).
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date revised: 10 April 2008
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