The reproducible pattern of organismal growth during metazoan development is the product of genetically controlled signaling pathways. Patterned activation of these pathways shapes developing organs and dictates overall organismal shape and size. Patches of tissue that are mutant for the Drosophila Tsg101 ortholog, erupted, cause dramatic overexpression of adjacent wild-type tissue. Tsg101 proteins function in endosomal sorting and are required to incorporate late endosomes into multivesicular bodies. Drosophila cells with impaired Tsg101 function show accumulation of the Notch receptor in intracellular compartments marked by the endosomal protein Hrs. This causes increased Notch-mediated signaling and ectopic expression of the Notch target gene unpaired (upd), which encodes the secreted ligand of the JAK-STAT pathway. Activation of JAK-STAT signaling in surrounding wild-type cells correlates with their overgrowth. These findings define a pathway by which changes in endocytic trafficking can regulate tissue growth in a non-cell-autonomous manner (Moberg, 2005). Tsg101 possesses the ability to bind monoubiquitinated substrates (Garrus, 2001; Sundquist, 2004). These substrates are predicted to be the ubiquitinated cytoplasmic tails of membrane bound proteins, and this interaction is predicted to deliver cargos to the lysosome via multivesicular bodies (reviewed in Katzmann, 2002).

Organismal patterning requires that the fates of individual cells within a multicellular organ be coordinated with the fates of surrounding cells. In most cases, this is achieved by secreted morphogens produced by a small group of “organizing” cells that then trigger a coordinated response among receiving cells. This type of signaling mediates the specialization of a subset of cells within a larger pool of precursors and shapes developing organs via local effects on cell proliferation (Moberg, 2005).

A number of secreted factors that control proliferation have been identified, including epidermal growth factor (EGF) and platelet-derived growth factor (PDGF). Other signaling pathways that can influence cell proliferation during development include those activated by secreted proteins such as Hedgehog, members of the Wnt and bone morphogenic protein (BMP) families, as well as the pathway downstream of the Notch receptor. For each of these pathways to regulate tissue growth and cell proliferation, they must eventually be able to influence mechanisms that regulate cell growth and division (Moberg, 2005).

Extracellular mitogens are also implicated in the aberrant growth observed in many cancers. In addition to the well-characterized role of hormones in promoting or sustaining the growth of certain carcinomas (e.g., breast and prostate), there is increasing recent awareness of the importance of growth-stimulating properties of stromal cells that are associated with most cancers of epithelial origin. Several studies have documented the increased propensity of tumor-associated fibroblasts to accelerate tumor progression in animal models and to promote tumorigenic changes in normal epithelial cells. While it is likely that many of these effects are the result of factors secreted by the stromal cells, the precise nature of these signals have, in most cases, not been defined (Moberg, 2005).

Studies of Drosophila imaginal discs have contributed significantly to understanding of the mechanisms that regulate tissue growth during organismal development. Studies in Drosophila have also begun to link signaling molecules that function in patterning the imaginal disc (e.g., Hh, Dpp, and Wg) with regulation of tissue growth and cell cycle progression. Genetic lesions in pathways that regulate imaginal disc growth can alter the size of adult structures such as the eye and the wing, and a number of genetic screens have used these phenotypes to identify mutations that result in tissue overgrowth. As is the case with the study of mammalian tumors, most of these mutations enhance growth cell-autonomously and provide little insight into mechanisms that might underlie interactions between tumors and stromal cells in mammals (Moberg, 2005).

Mutations in erupted (ept) are described that enable clones of mutant cells to stimulate the overgrowth of surrounding wild-type tissue. ept mutations disrupt the function of the Drosophila ortholog of the mammalian Tumor susceptibility gene 101 (Tsg101), whose role in tumorigenesis is controversial, but which has a defined role in the endocytic pathway. Mutations in Tsg101 activate Notch signaling and cause overproduction of the secreted mitogen Unpaired (Upd). These studies thus define a mechanism by which alterations in trafficking through the endocytic pathway can trigger the secretion of a growth factor and cause overproliferation of neighboring cells (Moberg, 2005).

Mammalian Tsg101 was initially discovered because antisense Tsg101 expression allowed fibroblasts to form colonies in soft agar and produce tumors in nude mice (Li, 1996). However, the role, if any, of Tsg101 in mammalian tumorigenesis has remained controversial. The similarity of Tsg101 to yeast Vps23p has led to a better understanding of Tsg101 as a component of an endosomal pathway that routes monoubiquitinated proteins into multivesicular bodies and the lysosome (reviewed in Katzmann, 2002). Mutations in the Drosophila ortholog of Tsg101, erupted, do indeed result in tissue overgrowth. Surprisingly, overgrowth occurs in the wild-type tissue that surrounds mutant cells. Notch trapped in ept mutant endosomes is activated, and it stimulates production of the secreted growth factor Upd, which causes stat92E-dependent proliferation and overgrowth of surrounding wild-type cells (Moberg, 2005).

Models of endosome-mediated receptor internalization emphasize the role of monoubiquitination as a signal for routing proteins through the MVB pathway (Katzmann, 2002). The data indicate that this pathway plays an important role in limiting Notch levels in cells of the developing eye, and that the excess Notch that accumulates upstream of the block in Tsg101 mutants is active and localizes to an Hrs-positive compartment. This might imply that ligand bound Notch is normally trafficked through endocytic compartments, but that not all ligand bound receptors succeed in transmitting a signal to the nucleus. Blocking this pathway at a particular step might then arrest Notch in a compartment from which it is able to signal. This is consistent with a proposed model in which endocytosed Notch is the preferred substrate of the Presenilin (Psn)-dependent γ-secretase (Gupta-Rossi, 2004), although no enrichment of Psn was observed in ept mutant cells. Delta accumulates ectopically in ept mutant eye disc cells, suggesting that the mechanism of Notch activation in endosomes may be ligand dependent. Further experiments are needed to test the role of Ub, Delta, and the γ-secretase in activating Notch in ept mutant cells (Moberg, 2005).

Certain asymmetric cell fates in the nervous system are controlled by selective endosomal routing of Delta (Emery, 2005). As several manipulations that block Notch entry into early endosomes also compromise signaling (Gupta-Rossi, 2004; Hori, 2004), Notch activation during normal development may also be controlled by trafficking to a particular compartment. These data suggest that this compartment lies between the cell membrane and the point in the MVB pathway at which Tsg101 acts, and includes the Hrs-positive endosome. eyFLP-mediated mitotic recombination of hrs alleles do not provoke overgrowth in the developing eye. If further study confirms that hrs mutant cells accumulate Notch but do not activate it, it would suggest that Notch activation occurs in an endosomal compartment downstream of the block in hrs mutants, but upstream of the block in ept mutants (Moberg, 2005).

hrs inactivation has been shown to affect localization of a number of receptors, including Notch, Egfr, Patched, Smoothened, and Thickveins. This study focused on the effects of ept on Notch, but it is likely that endosomal sorting of other receptors is also disrupted in ept cells. Indeed, the ability of ept alleles to enhance phenotypes of two EGFR pathway components, rolled/MAPK and Gap1, is consistent with findings that mammalian Tsg101 regulates Egfr endosomal sorting (Babst, 2000: Bishop, 2002; Lu, 2003). The extent to which other pathways contribute to ept mutant phenotypes, and the degree to which ept affects receptor trafficking in tissues other than the developing eye, clearly merits further investigation (Moberg, 2005).

Notch activation is an important determinant of the size of the adult eye. Manipulations that increase Notch activity or activate Notch at ectopic sites (e.g., by generating fringe mutant clones) result in increased growth of the eye imaginal disc and larger adult eyes. Activated Notch proteins also appear to promote cell proliferation in the context of certain cancers (reviewed in Maillard, 2003). While there are likely to be a number of cell-autonomous Notch targets that effect growth, data presented here identify Upd and the JAK-STAT module as likely mediators of non-cell-autonomous growth phenotypes associated with Notch activation, and they show that loss of Tsg101 is sufficient to activate this pathway in cells of the eye-antennal disc. In light of these findings, it will be of interest to test whether Notch is able to regulate the JAK-STAT pathway in epithelial tissues other than the fly eye (Moberg, 2005).

From the first description of Tsg101 as a gene required to restrict oncogenic transformation and anchorage-independent growth of cultured mammalian cells (Li, 1996), a satisfying mechanistic explanation of how Tsg101 regulates cell proliferation has proved elusive. Another ESCRT-1 subunit, HCRP-1/hVps37A, is also implicated as a growth inhibitory gene (Bache, 2004), but its role in proliferation control is similarly unclear. Mutation of the Drosophila Tsg101 homolog, erupted, affects tissue growth in two different ways: (1) clones of ept cells promote the overgrowth of surrounding wild-type tissue; (2) eye discs composed almost entirely of ept mutant tissue continue to grow during an extended larval stage, and they become tumorous masses. Despite an apparent slow-growth phenotype, these ept mutant cells continue to proliferate beyond the developmental stage at which they would otherwise exit the cell cycle. This contrasts with the behavior of normal cells that stop growing when the tissue reaches the appropriate size, and suggests that ept cells are unable to respond to signals that normally sense and restrict organ size. This phenotype differs from that induced by eye-specific overexpression of upd (Bach, 2003), suggesting that it is not due only to mitogenic effects of Upd. In addition to this growth defect, ept mutant cells also display phenotypes consistent with defects in apicobasal polarity. Considered together, these cell-autonomous defects are quite similar to those associated with mutations in the 'neoplastic tumor suppressor genes' (nTSGs) scribble, discs-large, and lethal(2) giant larvae (reviewed in Bilder, 2004). While this similarity need not reflect a common mechanism of growth control between Tsg101 and the nTSGs, it is perhaps significant that all of these genes can now be linked to the control of epithelial cell polarity via the Crb pathway (Moberg, 2005).

Mice lacking Tsg101 function die as early embryos, and inactivation of Tsg101 in cultured cells impairs proliferation (Wagner, 2003). However, the data suggest the possibility that mammalian cells with reduced Tsg101 function could promote the growth of neighboring cells in vivo. While there is no link yet established in mammalian cells between ESCRT-1 function and a putative Notch-upd-stat pathway, mammalian Notch signaling has been shown to induce transcription of a number of cytokine genes (reviewed in Maillard, 2005) whose encoded factors signal through downstream JAK-STAT pathways. Intriguingly, a recent report has also found evidence that the genomic interval containing human Tsg101 shows a high rate of allelic imbalance in nontumor-derived stroma associated with breast carcinomas (Ellsworth, 2004). Thus, an evaluation of the growth-regulating properties of Tsg101 mutant mammalian cells may require the use of more complex culture systems that incorporate both wild-type and mutant cells, possibly derived from different cell types (Moberg, 2005).

GENE STRUCTURE

cDNA clone length - 1753

Bases in 5' UTR - 333

Exons - 7

Bases in 3' UTR - 193

PROTEIN STRUCTURE

Amino Acids - 408

Structural Domains

Mammalian Tsg101 and the related S. cerevisiae Vps23p are components of a multiprotein complex termed the Endosomal Sorting Complex Required for Transport-1 (ESCRT-1) that also contains Vps28, and the hepatocellular carcinoma-related protein 1 (HCRP1)/Vps37. ESCRT-1 is required to sort vesicular cargos through the endosomal system. Human and Drosophila Tsg101 proteins are well conserved throughout their length (46% identical/61% similar) and have a shared domain structure, suggesting that they are functional homologs. Both contain an amino-terminal ubiquitin (Ub)-conjugating (UBC) domain similar to that found in the canonical E2 ubiquitin ligase S. cerevisiae Ubc4p. However, the active site cysteine in the UBC of Tsg101 family proteins has been replaced by a tyrosine (Y), and, as a consequence, these proteins lack Ub-conjugating activity, but retain the ability to bind monoubiquitinated substrates (Garrus, 2001; Sundquist, 2004). These substrates are predicted to be the ubiquitinated cytoplasmic tails of membrane bound proteins, and this interaction is predicted to deliver these cargos to the lysosome via multivesicular bodies (MVBs) (reviewed in Katzmann, 2002).

date revised: 15 January 2006

Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.