Proteins that constitute the endosomal sorting complex required for transport (ESCRT) are necessary for the sorting of proteins into multivesicular bodies (MVBs) and the budding of several enveloped viruses, including HIV-1. The first of these complexes, ESCRT-I, consists of three proteins: Vps28p, Vps37p, and Vps23p or Tsg101 in mammals. A mutation was characterized in the Drosophila homolog of vps28. The dVps28 gene is essential: homozygous mutants die at the transition from the first to second instar. Removal of maternally contributed dVps28 causes early embryonic lethality. In such embryos lacking dVps28, several processes that require the actin cytoskeleton are perturbed, including axial migration of nuclei, formation of transient furrows during cortical divisions in syncytial embryos, and the subsequent cellularization. Defects in actin cytoskeleton organization also become apparent during sperm individualization in dVps28 mutant testis. Because dVps28 mutant cells contained MVBs, these defects are unlikely to be a secondary consequence of disrupted MVB formation and suggest an interaction between the actin cytoskeleton and endosomal membranes in Drosophila embryos earlier than previously appreciated (Sevrioukov, 2005).
In the Drosophila genome, a single gene, CG12770, exhibits significant homology to the yeast and mammalian Vps28 proteins. The cDNA GH04443 is derived from this locus and encodes a predicted protein of 210 amino acids that is 62% and 35% identical to its human (hVPS28) and yeast (ScVPS28) counterparts, respectively. There are no similarities to other protein sequence motifs in the database. An antibody raised against dVps28 recognizes a protein of the expected size that is widely expressed during Drosophila development and also in cultured Drosophila S2 cells. In S2 cells as well as in cells of the eye disc and in isolated spermatocysts, dVps28 protein was uniformly distributed throughout the cytosol with no obvious enrichment in any organelle (Sevrioukov, 2005).
To test whether the homology of Vps28 proteins extends to their biochemical activity, its binding to Vps23p/Tsg101 (Babst, 2000; Bishop, 2001) was examined. The Drosophila homolog dTsg101 is encoded by cDNA GH09529. Because antibodies are not yet available against endogenous dTsg101 protein, epitope-tagged versions of dTsg101 and dVps28 were coexpressed to test their interaction in S2 cells. Immunoprecipitation of dVps28 from S2 cells resulted in the coprecipitation of expressed HA-dTsg101, which was increased after coexpression of Myc-dVps28. These results indicated that, like its yeast and mammalian orthologs, dVps28 binds specifically to dTsg101 (Sevrioukov, 2005).
Consistent with ESCRT's conserved function from yeast to mammalian cells, loss of dVps28 function causes morphological changes in MVBs and developmental defects in the compound eye that indicate a subtle misregulation of cell signaling molecules. However, for the ligands and receptors, no significant changes were detected in their cell surface levels or their delivery to lysosomes in dVps28 cells, indicating that any changes were too subtle to be detected by the immunofluorescence methods used (Sevrioukov, 2005).
One possible explanation for this observation is that some Vps28 functions are partially fulfilled by another protein. It is not likely that this hypothetical protein is similar to Vps28p in sequence because no another Vps28-like molecule in the completed genome sequences of Drosophila melanogaster. Another possibility is that the dVps28l(2)k16503 allele does not completely inactivate dVps28 function. Although this possibility cannot be ruled out, it is unlikely for two reasons: (1) the dVps28l(2)k16503 allele is a strong mutation that causes lethality early in development at the transition from first to second instar, much earlier than a null mutation in the hrs gene, which regulates the sorting of receptors into MVBs and (2) the lethal phase and the loss of dVps28 protein were indistinguishable between larvae homozygous for dVps28l(2)k16503 and those that were hemizygous over a deficiency of the region. This indicates that the dVps28l(2)k16503 allele removes most if not all of dVps28 function (Sevrioukov, 2005).
Another possibility for the relatively mild phenotypes is the perdurance of dVps28 protein. This is suggested by the dramatic phenotypes after removal of the maternal contribution in mKO dVps28 embryos: many remained unfertilized and the remaining embryos were arrested in their development before reaching the cellular blastoderm. It is interesting to compare this phenotype to that of embryos lacking any maternal Hrs contribution. Hrs binds to mono-ubiquitinated membrane proteins, whose sorting into MVBs is thought to be mediated by Hrs binding to ESCRT-1. Consistent with this notion, mKO hrs embryos exhibit a reduced down-regulation of cell surface receptors and a resulting enhanced activation of the MAP kinase pathway. Importantly, these defects were observed after cellularization had been completed and during gastrulation. The finding that mKO dVps28 embryos exhibit major defects before completion of cellularization indicates functions of dVps28 in addition to the down-regulation of membrane proteins mediated by the interaction of ESCRT-I with Hrs (Sevrioukov, 2005).
Surprisingly, the earliest defect found in fertilized eggs was an uneven distribution of nuclei. In Drosophila, the first 13 nuclear divisions occur without accompanying cell divisions. The first five of these syncytial divisions occur close to the center of the embryo, with nuclei subsequently spreading out along the anterior-posterior axis. This process, referred to as axial expansion, depends on the function of the actin cytoskeleton. In mKO dVps28 embryos, this process often is disorganized resulting in an uneven distribution of nuclei (Sevrioukov, 2005).
Other dVps28 phenotypes emerged during cellularization. After axial expansion, the nuclei move to the embryo's cortex where the last four of the syncytial nuclear divisions occur. The resulting nuclei are surrounded by invaginating membranes in a process that requires the actin-myosin network. At this stage, two defects were evident in embryos lacking dVps28. Many of the cells that formed were droplet shaped instead of the usual cuboidal shape. The actin cytoskeleton is required for normal cellularization and interfering with is function, by cytochalasin, or altering the activity of the Rho or Cdc42 GTPases, interferes with normal cellularization. Furthermore pole cells, usually the first cells to form, were absent in most embryos. The lack of pole cells has previously been observed in embryos in which axial expansion is perturbed upon interference with the actin cytoskeleton (Sevrioukov, 2005).
All of these early phenotypes are consistent with a role of dVps28 in directly or indirectly organizing the actin cytoskeleton. A role for endosomes in actin remodeling during cellularization has previously been established in embryos mutant for nuf or rab11. The small GTPase Rab11 localizes to recycling endosomes, and Nuf is a homolog of Arfo2 that directly binds Rab11 and acts in recycling endosomes. Mutants eliminating either of these proteins cause gaps in which actin fails to be recruited to the furrows during cortical nuclear divisions, similar to the observations in mKO dVps28 embryos. In all three of these mutants, the actin defects may be a consequence of the failure to recruit Discontinuous actin hexagon (Dah: a membrane-associated protein that localizes to invaginating furrows in syncytial blastoderm embryos and during cellularization) to invaginating furrows. Dah has significant similarity to dystrobrevin and dystrophin. Dystrophin plays a critical role in anchoring the actin cytoskeleton to membranes, and consistent with a similar function for Dah, embryos lacking maternally contributed Dah fail to properly assemble the actin cytoskeleton at furrows (Sevrioukov, 2005 and references therein).
A subset of the defects in rab11 mutant embryos has been linked to a requirement for trafficking through the recycling endosome during cellularization. Consistent with this notion, defects in rab11 and nuf embryos only become apparent during cortical divisions. In mKO dVps28 embryos, by contrast, defects were detected in the distribution of nuclei, long before cellularization initiates. This indicates a function of dVps28 in actin remodeling independent of recycling to the cell surface and independent of Rab11 and Nuf. This is consistent with the finding that in mKO dVps28 embryos, recycling endosomes are not affected, as judged by Rab11 and Nuf localization, suggesting that dVps28 is required for furrow localization of Dah down-stream or in parallel to Rab11 and Nuf's function in recycling endosomes. Such a model is difficult to reconcile with the canonical function of Vps28 as an ESCRT-1 subunit involved in targeting proteins into the interior vesicles of late endosomes (Sevrioukov, 2005 and references therein).
It is unlikely that Dah mediates all effects of dVps28 on the actin cytoskeleton. No defects are observed in embryos lacking Dah before cycle 10, long after defects have become apparent in mKO dVps28 embryos. Furthermore, Dah is not required during spermatogenesis, another developmental context in which effects of dVps28 on the organization of the actin cytoskeleton were observed. Spermatogenesis in dVps28 mutant testis progresses until bundles of 64 syncytial spermatids are formed. These spermatids are separated by a process called individualization that requires complex membrane rearrangements. At the site of these rearrangements, syncytial membrane, cytoplasm, and vesicles accumulate in the cystic bulge. In wild-type testis, an actin-dependent process drives the cystic bulge away from the 64 spermatid nuclei toward the distal end. The loss of synchrony in the movement of the cystic bulge is the first detectable defect during spermatogenesis in dVps28 mutant testis. Because dah mutants have no phenotype in males, these results indicate an independent connection between dVps28 function and the actin cytoskeleton (Sevrioukov, 2005 and references therein).
Actin acts at many stages in the endocytic pathway. In yeast, the initial internalization step requires actin polymerization, and in mammalian cells, early endosomes move on the tip of actin tails. Additionally, late endocytic organelles require the actin cytoskeleton for fusion in yeast and mammalian cells. Furthermore, a screen of the yeast genome for mutations interfering with protein sorting to the vacuole identified several regulators of the actin cytoskeleton (Sevrioukov, 2005 and references therein).
Importantly, several of these mutants identified on the basis of a vacuolar sorting phenotype also exhibited defects in the organization of the actin cytoskeleton. For example, aberrant actin patches and a reduction of actin cables were observed in yeast lacking Vps36p, one of the subunits of the ESCRT-II complex. It will be interesting to see whether a similar functional connection between the actin cytoskeleton and the ESCRT-I complex may underlie the enigmatic phenotypes of Tsg101 mutations in mice that cause cell cycle arrest and early embryonic lethality (Sevrioukov, 2005 and references therein).
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