Krishna S et al. (1996) Worm Breeder's Gazette "A tre-1 in a Forest"

Padgett RW, Krishna S

[Worm Breeder's Gazette, 1996]

The TGF-b-like signalling response involves a crucial ligand-receptor interaction for transduction to occur. The TGF-b-like ligands share a highly conserved C-terminus and can bind to receptors in a dimeric fashion, linked by a cysteine bridge. Several types of receptors that are able to bind to TGF-b superfamily members in vitro have been reported. Of these, the type I and II receptors contain a serine/threonine kinase intracellular domain which is essential for proper signalling to occur. It has been demonstrated in Drosophila and in vertebrate systems that multiple type I receptors can interact with a single type II receptor. This presumably determines part of the specificity in the signalling of TGF-b superfamily members. For example, in the fly, the products of sax and tkv (type I receptors) interact with the punt (type II receptor) gene product and can send slightly different dpp signals. The type I and II receptors share significant aligment homology, but differ in several regions. They each have a poorly conserved extracellular domain, a variable hydrophobic stretch constituting a transmembrane domain, followed by the kinase domain. In addition, it is thought that the GS-box motif, which is the hallmark of a type I receptor, is required in the proper interaction of a type I and type II receptor upon ligand binding. This would then be followed by the subsequent phosphorylation of the type I receptor by the type II. It is then postulated that the activated receptor system further transduces the signal.

Krishna S et al. (1996) Worm Breeder's Gazette "TGF-B Superfamily Signaling in C. elegans"

Krishna S, Padgett RW

[Worm Breeder's Gazette, 1996]

Previous work examining the transduction of TGF-B superfamily signal at the level of the membrane has resulted in the formulation of a general model by which this may occur. Transmembrane receptors which interact specifically with TGF-B superfamily ligands have been isolated and have been demonstrated to be required for proper transduction in several organisms. A subset of these receptors contain a serine-threonine kinase domain, and can be categorized into two classes, type I and type II, which differ considerably with each other in their non-kinase regions. It is thought that the mature protease-cleaved dimeric ligand interacts with appropriate type II receptors followed by the formation of an oligomeric complex with specified type I receptors. This complex then conceivably transduces the appropriate signal to downstream molecules, eventually resulting in intracellular changes and/or altered expression patterns of specific gene targets. In the worm, the dauer and small pathways are examples of mechanisms controlled by TGF-B like ligands. In the dauering pathway, the products of daf-l, daf-4 are receptors and serve to transduce the signal. Additionally, the dwarfins comprise a group of genes which encode products that have been implicated in the TGF-B transduction pathway in the nematode (Savage et al., PNAS 1996; Savage et al., WBG 1995) and in Drosophila (Sekelsky et al., Genetics 139: 1347-1358, 1995) . In the worm, the sma-2, sma-3, and sma-4 genes are dwarfins that have been characterized by our group. The mutant phenotypes of these genes are a subset of those exhibited by daf-4. However, the sma phenotypes are not observed in other dauer mutants. Thus, daf-4 intersects the dauering and small pathways. Since daf-l does not exhibit the small phenotype, we hypothesized that there should be another type I receptor transducing a TGF-B-like signal to the sma gene products, that when mutated, results in small animals. Using molecular tools, we had identified tre-1 as a novel type I receptor in C. elegans. tre-1 demonstrates a large degree of homology with other type I receptors, particularly the thickveins gene in Drosophila. Searches of extant mutations in the region of tre-1 indicated that sma-6 was nearby. Given that four genes mutate to a small phenotype, other genes in the pathway may also yield the same phenotype. Based on these data, the possibility was raised that sma-6 may encode tre-1. Germline transformation experiments confirmed this hypothesis, whereby a 9kb genomic fragment containing the tre-1 gene and flanking regions extending into, but not encompassing, adjacent predicted transcripts was sufficient to rescue sma-6 mutants and revert F1 transformed progeny to wild-type length. We are currently continuing to characterize the expression of tre-1 and analyze interactions with other components of the small pathway. In addition, we have isolated superfamily ligands in the worm, including tig-1, tig-2, and tig-3 (Krishna, WBG, 1995). We are seeking mutations in the respective regions, and we are continuing to characterize these genes. These results help us to define the specificity of TGF-B signaling by demonstrating that different type I receptors are able to elicit varying biological responses. Furthermore, we have completed a large genetic screen in order to identify other mutations that yield small animals. Our results with sma-6 validate this screen, and we hope to isolate other novel components of the pathway. Based on these results, we are also examining sma-5 and sma-8 for potential participation in the signaling pathway.

Krishna S et al. (1997) International C. elegans Meeting "SPECIFICITY IN TGF-b SIGNALING"

Krishna S, Padgett RW

[International C. elegans Meeting, 1997]

TGF-b signaling involves the formation of a heteromeric complex between type I and II serine threonine kinase receptors. This complex is iniated by ligand-binding, and upon phosphorylation of the type I receptor by the type II kinase, signal is transduced from the receptors to SMADs, which are downstream components that are phosphorylated by the receptor complex. In this manner, the extracellular signal can produce intracellular changes. In C. elegans, the type II receptor daf-4 is involved in two parallel TGF-b-like pathways. Because the phenotype of the previously known type I receptor at the time, daf-1, could not account for those exhibited by mutations in the sma [2,3,4] genes (which were shown to function downstream of daf-4, Savage et. al.,PNAS 93, 790 794.), we hypothesized that another type I receptor participates in the Small pathway. Therefore, we have cloned and characterized sma-6, which encodes a type I receptor kinase which, presumably through association with DAF-4, transduces signal to the downstream SMAD components sma-[2, 3, 4]. We are currently examining the developmental role of sma-6 and the Small pathway, and we are using a null allele of sma-6 to facilitate this. The two characterized TGF b-like systems in C. elegans, the Small and Dauer pathways, provide an opportunity to examine how signaling specificity is generated and maintained in general TGF-b-like signaling mechanisms.

Krishna S et al. (1998) East Coast Worm Meeting "sma-6 and the Issue of Specificity in TGF-beta Signaling"

Krishna S, Padgett RW

[East Coast Worm Meeting, 1998]

In C. elegans, the TGF-beta-like type II receptor daf-4 intersects two distinct pathways. It is involved in the regulation of the dauering process in association with the type I receptor daf-1. In addition, it is also required in body size determination and male tail patterning, roles that do not require daf-1. We have cloned and characterized a novel type I receptor, and show that it is encoded by sma 6. Previously, we have demonstrated that the sma-2, -3, and 4 genes, members of the SMAD family, are downstream signaling components involved in only the Sma/Mab pathways. This study provides developmental genetic evidence that the interaction of the type II receptor with different type I receptors and their corresponding SMADs confers specificity in TGF-beta-like signaling. We show that there is minimal cross-talk between the Sma/Mab and dauer pathways and that sma-6 regulates male tail morphogenesis only through the small genes.

Krishna S et al. (1996) East Coast Worm Meeting "TGF-beta Superfamily Signalling in C. elegans"

Padgett RW, Krishna S

[East Coast Worm Meeting, 1996]

The TGF-b superfamily comprises a group of secreted proteins which are involved in fundamental biological processes including, but not limited to, axis specification, mesoderm induction, osteogenic differentiation, tissue regeneration, and neoplastic growth. Though there has been intense interest in recent years to determine the pathways by which TGF-b signalling occurs, the mechanism by which the signal is transduced from the ligand is still relatively poorly understood. Previous work examining the transduction of signal at the level of the membrane has resulted in the formulation of a general model by which this may occur. Transmembrane receptors which interact specifically with TGF-b superfamily ligands have been isolated and have been demonstrated to be required for proper transduction in several organisms. A subset of these receptors contain a serine-threonine kinase domain, and can be categorized into two classes, type I and type II, which differ considerably with each other in their non-kinase regions. It is thought that the mature protease-cleaved dimeric ligand interacts with appropriate type II receptors followed by the formation of an oligomeric complex with specified type I receptors. This complex then conceivably transduces the appropriate signal to downstream molecules, eventually resulting in intracellular changes and/or altered expression patterns of specific gene targets. In the worm, the dauer and small pathways are examples of mechanisms controlled by TGF-b-like ligands. In the dauering pathway, the products of daf-1, daf-4 are receptors and serve to transduce the signal. Additionally, the dwarfins comprise a group of genes which encode products that have been implicated in the TGF-b transduction pathway in the nematode (Savage et al., PNAS 1996; Savage et al., WBG 1995) and in Drosophila (Sekelsky et al., Genetics 139: 1347-1358, 1995) . In the worm, the sma-2, sma-3, and sma-4 genes are dwarfins that have been characterized by our group. The mutant phenotypes of these genes are a subset of those exhibited by daf-4. However, the sma phenotypes are not observed in other dauer mutants. Thus, daf-4 intersects the dauering and small pathways. Since daf-1 does not exhibit the small phenotype, we hypothesized that there should be another type I receptor transducing a TGF-b-like signal to the sma gene products, that when mutated, results in small animals. Using molecular tools, we had identified tre-1 as a novel type I receptor in C. elegans. tre-1 demonstrates a large degree of homology with other type I receptors, particularly the thickveins gene in Drosophila. Searches of extant mutations in the region of tre-1 indicated that sma-6 was nearby. Given that four genes mutate to a small phenotype, other genes in the pathway may also yield the same phenotype. Based on these data, the possibility was raised that sma-6 may encode tre-1. Germline transformation experiments confirmed this hypothesis, whereby a 9kb genomic fragment containing the tre-1 gene and flanking regions extending, but not encompassing, adjacent predicted transcripts was sufficient to rescue sma-6 mutants and revert F1 transformed progeny to wild-type length. We are currently continuing to characterize the expression of tre-1 and analyze interactions with other components of the small pathway. These results helps us to define the specificity of TGF-b signalling by demonstrating that different type I receptors are able to elicit varying biological responses. Furthermore, we have completed a large genetic screen (see abstract by Savage et al.) in order to identify other mutations that yield small animals. Our results with sma-6 validate this screen, and we hope to isolate other novel components of the pathway.

Krishna S et al. (1997) Worm Breeder's Gazette "sma-6 is Involved in Body Size Determination and Tail Morphogenesis"

Krishna S, Padgett RW, Collins D

[Worm Breeder's Gazette, 1997]

In C. elegans, two TGF-beta-like signaling systems have been identified which utilize a common type II receptor, daf-4. The phenotypes of daf-4 include constitutive dauer formation, small body size, and male tail abnormalities. daf-1 has previously been shown to encode a TGF-beta superfamily type I serine-threonine receptor kinase, and mutations in this gene effect constitutive dauering. Based on our work in characterizing the downstream SMAD components sma-[2,3,4], we had postulated the existence of another type I receptor which was involved in those phenotypes shared by these SMADs and daf-4, namely body size and tail development. We had therefore cloned a novel type I receptor using a molecular approach, and have shown that it is encoded by the gene sma-6. We are now interested in analyzing the developmental role of sma-6 in more detail. We have obtained novel alleles of sma-6, wk7, wk8, and wk9 in screens for small mutants, and we also have performed a non-complementation screen using the canonical e1482 allele. The e1482 allele contains a mutation in the extracellular region, 90A->V. This modification is most likely not severe as deduced from the amino acid replacement, and this reasoning is supported by the observation that e1482 animals are not as small as other mutants in the pathway. The wk7 allele, however, is the result of a mutation which changes residue 72 in the extracellular region into a premature termination. Because the expressed protein lacks most of the extracellular domain, and does not extend into the transmembrane and kinase domains, we conclude that wk7 represents a null allele of sma-6. Furthermore, the wk7 phenotype displays a body size that is consistent with those exhibited by mutations in sma-[2,3,4]. In addition, hemizygous wk7 animals do not show an observable difference in body size. wk7 does not exhibit antimorphic characteristics. We are currently determining the molecular change in the other sma-6 alleles, including a Tc-1 induced transformation (wk9). Because the phenotypes of the daf-4 and the sma-[2,3,4] genes, when mutant, also result in characteristic defects in tail development, we have analyzed the male tails of the sma-6 alleles. It can be hypothesized that another type I receptor is involved in this process, but our results argue against this. Animals homozygous for the e1482 allele do not exhibit the abnormalities, specifically crumpled spicules and ray fusions, present in other genes in the small pathway. However, the finding that these deformities are present in wk7,wk8, and wk9 males confirms the notion of daf 4, sma-2, sma-3, sma-4, and sma-6 functioning in a common pathway. Expression studies using a translation fusion of a sma-6 promoter and coding segment with lacZ are being examined. These results will be compared with antibodies we generated against the extracellular region of sma-6 to extend this result. These findings demonstrate the potential for disparate TGF-beta signaling pathways to intersect at a common type II receptor, and investigate the role of this novel receptor in C. elegans development. It will be meaningful to explore the applicability of this work to other TGF-beta-like systems.

Krishna S et al. (1996) Worm Breeder's Gazette "tig-1, tig-2: Novel TGF-b Homologs in C. elegans"

Krishna S, Padgett RW

[Worm Breeder's Gazette, 1996]

The TGF-b superfamily comprises a group of secreted proteins which are involved in fundamental biological processes including, but not limited to, axis specification, mesoderm induction, osteogenic differentiation, tissue regeneration, and neoplastic growth. There is significant primary structure homology shared among all the members, and the superfamily can be further partitioned into groups based upon homology and function. These families include the dpp/BMP group, the activin/inhibin family, the TGF-b subset, the MIS family, and others. Though there has been intense interest in recent years to determine the pathways by which TGF-b signalling occurs, the mechanism by which the signal is transduced from a TGF-b superfamily members is still relatively poorly understood. Membrane receptors which specifically bind with TGF-b-like ligands have been identified and are being characterized in several organisms. A subset of these receptors contain a serine-threonine kinase domain, and can be categorized into two classes, type I and type II, which differ considerably with each other in their non-kinase regions. A general model for the transduction of the TGF-b-like signal involves an initial proteolytic modification of the ligand followed by binding of mature cysteine-bridged dimeric form with the type II receptor. Upon binding, interaction between the two types of receptors can occur, and mutual phosphorylation is implicated in receptor activation. In this activated state, the receptor kinases presumably are then capable of transducing the signal through the phosphorylation of other targets. This cascade would eventally result in biochemical changes and altered expression patterns of specific genes. In our laboratory, we have been trying to elucidate of the TGF-b signal transduction system in C. elegans. We have identified candidate genes at each step of the transduction process in the nematode using a variety of methods. Putative homologs at the metalloprotease (Finelli et al., this Gazette), ligand, and receptor (Krishna et al., this Gazette) level have been identified and are being characterized. Additionally, attempts to isolate downstream components have yielded the dwarfin family of potential downstream mediators (Das et al., this Gazette), including sma-2, sma-3, and sma-4, which have been implicated in the pathway through both molecular and genetic observations (Savage et al., this Gazette).

Krishna S et al. (2016) Dev Cell "PIKfyve Regulates Vacuole Maturation and Nutrient Recovery following Engulfment."

Florey O, Overholtzer M, Yang W, Thompson CB, Palm W, Lee Y, Krishna S, Xu H, Bandyopadhyay U

[Dev Cell, 2016]

The scavenging of extracellular macromolecules byengulfment can sustain cell growth in a nutrient-depleted environment. Engulfed macromolecules are contained within vacuoles that are targeted for lysosome fusion to initiate degradation and nutrient export. We have shown that vacuoles containing engulfed material undergo mTORC1-dependent fission that redistributes degraded cargo back into the endosomal network. Here we identify the lipid kinase PIKfyve as a regulator of an alternative pathway that distributes engulfed contents in support of intracellular macromolecular synthesis during macropinocytosis, entosis, and phagocytosis. We find thatPIKfyve regulates vacuole size in part through its downstream effector, the cationic transporter TRPML1. Furthermore, PIKfyve promotes recovery of nutrients from vacuoles, suggesting a potential link between PIKfyve activity and lysosomal nutrient export. During nutrient depletion, PIKfyve activity protects Ras-mutant cells from starvation-induced cell death and supports their proliferation. These data identify PIKfyve as a critical regulator of vacuole maturation and nutrient recovery during engulfment.

Krishna S et al. (1999) Development "Specificity of TGFbeta signaling is conferred by distinct type I receptors and their associated SMAD proteins in Caenorhabditis elegans."

Maduzia LL, Padgett RW, Krishna S

[Development, 1999]

In C. elegans, the TGFbeta-like type II receptor daf-4 is required for two distinct signaling pathways. In association with the type I receptor daf-1, it functions in the dauer pathway. In addition, it is also required for body size determination and male tail patterning, roles which do not require daf-1. In an effort to determine how two different signals are transmitted through daf-4, we looked for other potential signaling partners for DAF-4. We have cloned and characterized a novel type I receptor and show that it is encoded by sma-6. Mutations in sma-6 generate the reduced body size (Sma) and abnormal mail tail (Mab) phenotypes identical to those observed in daf-4 and sma-2, sma-3, sma-4 mutants (C. elegans Smads), indicating that they function in a common signaling pathway. However, mutations in sma-6, sma-2, sma-3, or sma-4 do not produce constitutive dauers, which demonstrates that the unique biological functions of daf-4 are mediated by distinct type I receptors functioning in parallel pathways. We propose that the C. elegans model for TGFbeta-like signaling, in which distinct type I receptors determine specificity, may be a general mechanism of achieving specificity in other organisms. These findings distinguish between the manner in which signaling specificity is achieved in TGFbeta-like pathways and receptor tyrosine-kinase (RTK) pathways.

Suzuki Y et al. (1999) Worm Breeder's Gazette "Mediators and Modulators of dbl-1 Functions in Body Size Control"

Wood WB, Suzuki Y, Morris GA

[Worm Breeder's Gazette, 1999]

Size regulation of body components is a poorly understood aspect of animal growth and development. We have previously shown that the activity of the dbl-1 gene, a C. elegans gene encoding a TGF-b ligand, is critical for cell-size and body-size control during post-embryonic development of the worm (1). Identification of additional downstream targets that mediate dbl-1 functions and of upstream regulators that modulate the activity of dbl-1 could provide new insights into the mechanisms of size regulation. Loss-of-function (lf) mutations in dbl-1 result in a Sma phenotype, whereas the overexpression of dbl-1 results in a Lon phenotype (1). These results suggest that mutations affecting the activity of dbl-1 could be identified by screening for these convenient phenotypes. To identify potential targets of the body size control pathway, we are carrying out screens for suppressors of the Sma and Lon phenotypes caused by dbl-1(lf) mutations and dbl-1 overexpression, respectively. We plan genetic and molecular characterization of genes defined by these suppressors. Previous studies on the epistatic relationships between sma genes and lon genes placed lon-2 and lon-3 upstream and lon-1 downstream of the genes encoding the receptors and the Smad proteins of the body size control pathway (2). In similar experiments, we have shown that dbl-1(lf) mutations are epistatic to lon-2 and lon-3 mutations, suggesting that these two lon genes act upstream of dbl-1 as well, and are thus possible modulators of dbl-1 activity. lon-1 and lon-2 are being studied in other laboratories (3, 4, 5). We are attempting to clone lon-3 and have obtained cosmid rescue. Rescuing arrays can cause a Sma phenotype, suggesting the possibility that lon-3, like dbl-1, is a dose-dependent regulator of body size. We hope to elucidate how dbl-1 and lon-3 interact. 1. Suzuki, Y., Yandell, M. D., Roy, P. J., Krishna, S., Savage-Dunn, C., Ross, R. M., Padgett, R. W. and Wood, W. B. (1999). Development 126: 241-250. Similar results have been obtained by Morita, K. and Ueno, N. (1999). Development, in press. 2. Savage, S., Padgett, R., and Baird, S. (1994) WBG 13(2): 38 3. Shetgiri, P., Krishna, S., Savage, C., and Padgett, R. W. (1997) International Worm Meeting Abstract 539 4. Vellai, T. and Fodor, A. (1997) WBG 14(5): 59. 5. de Bono, M. and Bargmann, C. (1997) International Worm Meeting Abstract 121