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Jeb signals through the Alk receptor tyrosine kinase to drive visceral muscle fusion
Umeå universitet, Medicinsk fakultet, Umeå centrum för molekylär patogenes (UCMP) (Medicinska fakulteten).
Umeå universitet, Medicinsk fakultet, Umeå centrum för molekylär patogenes (UCMP) (Medicinska fakulteten).
Umeå universitet, Medicinsk fakultet, Umeå centrum för molekylär patogenes (UCMP) (Medicinska fakulteten).
Visa övriga samt affilieringar
2003 (Engelska)Ingår i: Nature, ISSN 0028-0836, E-ISSN 1476-4687, Vol. 425, nr 6957, s. 512-516Artikel i tidskrift (Refereegranskat) Published
Ort, förlag, år, upplaga, sidor
2003. Vol. 425, nr 6957, s. 512-516
Identifikatorer
URN: urn:nbn:se:umu:diva-3564PubMedID: 14523447Scopus ID: 2-s2.0-0141929353OAI: oai:DiVA.org:umu-3564DiVA, id: diva2:142329
Tillgänglig från: 2008-10-29 Skapad: 2008-10-29 Senast uppdaterad: 2023-03-24Bibliografiskt granskad
Ingår i avhandling
1. Identification of downstream targets of Alk signaling in Drosophila melanogaster
Öppna denna publikation i ny flik eller fönster >>Identification of downstream targets of Alk signaling in Drosophila melanogaster
2008 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
Abstract [en]

The Drosophila gene Anaplastic lymphoma kinase (Alk) is homologous to mammalian ALK, a member of the Alk/Ltk family of receptor tyrosine kinases. In Drosophila Alk is crucial for development of the embryonic visceral mesoderm, where it is the receptor for Jelly Belly (Jeb) ligand. Jeb binding stimulates an Alk-driven, extracellular signal-regulated, kinase-mediated signaling pathway, which results in the expression of the downstream gene duf/kirre. The visceral mesoderm is made up from two different cell types, founder cells and fusion competent myoblasts. The Jeb-Alk signal transduction pathway drives specification of the founder cells of the Drosophila visceral muscle. In this work we aimed to identify genes specifically expressed in the founder cells and/or fusion competent myoblasts. Four genes from a number of candidiates were investigated further. These genes are Hand, expressed in founder cells, goliath, parcas and delilah, which are expressed in fusion competent myoblasts. Hand is a basic helix-loop-helix (bHLH) transcription factor. Alk-mediated signal transduction drives the expression of the bHLH transcription factor hand in vivo, and loss of Alk function results in a complete lack of hand expression in the visceral mesoderm, while Alk gain of function results in an expansion of hand expression. There are no obvious defects in the visceral muscle fusion process hand mutant animals, suggesting that Hand is not critical for visceral muscle fusion per se.

I have studied another molecule Goliath, a putative RING finger E3 ligase. goliath is specifically expressed in the FCM of visceral and somatic muscles. goliath mutant animals do not display any obvious muscle phenotypes, perhaps reflecting a redundant role with CG10277, which encodes a second Goliath family protein in Drosophila. Deletion mutation of the CG10277 locus does not result in muscle defects either, and generation of double mutants of goliath and CG10277 will be required to determine their function in vivo.

In addition, I have studied another bHLH transcription factor Delilah and its role in muscle development. We show that delilah is expressed in visceral muscle, somatic muscles and in tendon cells. Delilah mutant animals display a held out wing phenotype and are unable to fly. Inducible RNAi against delilah results in a similar phenotype. Delilah is transcriptionally regulated by mef2 and biniou, early regulators of muscle development. While delilah appears to function in tendon cells, we were unable to find any obvious phenotype in either visceral or somatic muscles. In order to further investigate the underlying mechanism of Delilah function we have used Tandem affinity purification (TAP) methodology followed by mass spectrometry to identify Delilah binding partners. This analysis suggests a number of candidate functional partners for the Delilah protein.

Ort, förlag, år, upplaga, sidor
Umeå: Umeå centrum för molekylär patogenes (UCMP) (Medicinska fakulteten), 2008. s. 65
Serie
Umeå University medical dissertations, ISSN 0346-6612 ; 1223
Nyckelord
Drosophila, Alk, Jeb, visceral muscles, somatic muscles, Delilah, Goliath, Hand
Nationell ämneskategori
Biokemi och molekylärbiologi
Identifikatorer
urn:nbn:se:umu:diva-1894 (URN)978-91-7264-668-1 (ISBN)
Disputation
2008-11-17, Betula, Bldg 6M, Umea University, Umea, 09:00 (Engelska)
Opponent
Handledare
Tillgänglig från: 2008-10-29 Skapad: 2008-10-29 Senast uppdaterad: 2010-01-18Bibliografiskt granskad
2. Investigating the function of the Receptor Tyrosine Kinase ALK during Drosophila melanogaster development
Öppna denna publikation i ny flik eller fönster >>Investigating the function of the Receptor Tyrosine Kinase ALK during Drosophila melanogaster development
2004 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
Abstract [en]

The Drosophila melanogaster gene Anaplastic Lymphoma Kinase (DAlk) is homologous to mammalian Alk, which is a member of the Alk/Ltk family of receptor tyrosine kinases (RTKs). In humans the t(2;5) translocation involving the Alk locus encodes an active form of Alk that is the causative agent in Non-Hodgkin’s Lymphoma (Morris et al., 1994). Alk has also been associated with other cancers such as inflammatory myofibroblastic tumours (IMTs). The physiological function of the Alk RTK has not been described in any system until very recently, and is still not defined in vertebrates. The molecular similarity between Drosophila Alk and mammalian Alk suggested that mutation of Alk in flies may affect similar functional and developmental processes, and thus lead to some understanding of Alk function in vivo.

By employing an EMS mutagenesis screen we were able to obtain loss-of-function mutants in the Drosophila DAlk gene. Eleven independent DAlk mutants were identified and characterized. DAlk is normally expressed in the developing gut and in the CNS. DAlk mutant animals have a lethal phenotype and die at late embryonic stages or as 1st instar larva. In DAlk mutant embryos there is a complete failure in the development of the midgut whereas the CNS appears normal. The midgut consists of visceral musculature that is syncytial and is formed by fusion of multiple myoblasts. This is a dynamic process where two types of myoblasts, i.e. fusion-competent-myoblasts and founder-cells that function as seeds for muscle formation, fuse. In DAlk homozygous embryos there is no founder cell specification, which explains the failure of midgut formation in these embryos.

Recently a novel secreted molecule Jelly Belly (Jeb) was identified. Jeb is expressed in the tissue neighbouring the DAlk expressing cells of the developing visceral mesoderm. Jeb mutant embryos show a phenotype that is similar to that of DAlk mutant embryos. We have been able to show that Jeb is the ligand for DAlk in the developing visceral mesoderm and that Jeb binding stimulates a DAlk driven ERK signaling pathway. This leads to the expression of Dumbfounded (duf)/kin of Irregular chiasm-C (kirre), a founder-cell specific immunoglobulin that has an important role in myoblast aggregation and fusion.

The functional Drosophila midgut is made up of the visceral muscle that encircles the endodermal tube. This tube formation includes migration of cells originating in the anterior and posterior parts of the embryo, first along the anterior-posterior axis using the visceral mesoderm as a template, then dorsally and ventrally. In DAlk mutant embryos there is no visceral muscle fusion and both the visceral mesoderm and the endoderm fail to undergo dorsal-ventral migration.

Ort, förlag, år, upplaga, sidor
Umeå: Umeå centrum för molekylär patogenes (UCMP) (Medicinska fakulteten), 2004. s. 66
Serie
Umeå University medical dissertations, ISSN 0346-6612 ; 934
Nyckelord
DAlk, receptor tyrosine kinase, Jeb, visceral muscle fusion, ERK, Drosophila, endoderm
Forskningsämne
molekylär cellbiologi
Identifikatorer
urn:nbn:se:umu:diva-411 (URN)91-7305-775-4 (ISBN)
Disputation
2004-12-10, Hörsal E04, 6L, Norrlands Universitetssjukhus, Umeå, 09:00 (Engelska)
Opponent
Tillgänglig från: 2004-11-01 Skapad: 2004-11-01 Senast uppdaterad: 2009-05-08Bibliografiskt granskad
3. Protein tyrosine kinases and the regulation of signalling and adhesion in drosophila melanogaster
Öppna denna publikation i ny flik eller fönster >>Protein tyrosine kinases and the regulation of signalling and adhesion in drosophila melanogaster
2007 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
Abstract [en]

In order to build a multi-cellular organism and to regulate cellular functions, cells need to communicate with each other, as well as tightly regulate their behaviour in response to environmental changes. For these purposes all eukaryotic cells express a large number of membrane spanning receptors that either themselves contain catalytic activity or via cytoplasmic effector enzymes, function to transmit “signals” from the cell exterior to induce appropriate responses within the cell. Protein tyrosine kinases (PTKs) are important signalling molecules, represented by the transmembrane receptor tyrosine kinases (RTKs) in addition to the cytoplasmic non-receptor PTKs, which alter cell behaviour by phosphorylating target proteins. An additional requirement for proper signalling and multicellular organisation is the adhesion between cells as well as adhesion of cells to the extracellular matrix (ECM).

Adhesion between cells and the ECM is mainly mediated by the integrin family of cell surface receptors, which functions as a structural link between the ECM and the actin cytoskeleton as well as important centres for signalling. Mammalian studies have implicated the cytoplasmic Focal Adhesion Kinase (FAK), as a major transmitter of signalling emanating from integrins, regulating cell migration, survival, proliferation and differentiation. In our studies of the sole FAK family member in Drosophila, Fak56, we have concluded that the deletion of Fak56 from the fruit fly genome causes no obvious defects in integrin-mediated adhesion, migration or signalling in vivo. Consequently, in contrast to the embryonic lethality observed in mouse knockouts, Fak56 mutant flies are both viable and fertile. However, we do find a clear genetic interaction between Fak56 and Drosophila integrins. Additionally, overexpression studies indeed indicate Fak56 as a negative regulator of integrin adhesion, given that excess Fak56 protein phenocopies loss of integrin function, causing phenotypes such as muscle detachment and wing blistering.

In Drosophila, as well as in mammals, FAK family proteins are highly abundant in the CNS and in our studies we have identified a requirement of Fak56 in synaptic transmission at neuromuscular junctions. Lack of Fak56 causes a weakening of action potential conduction, resulting in sensitivity to high-frequency mechanical and electrical stimulation, manifested by epileptic-like seizures and paralysis in Fak56 mutants, a phenotype known as Bang Sensitivity (BS) in flies. We also show that Fak56 phosphorylation is directly modulated in response to alterations in intracellular calcium levels, supporting a role for Fak56 in neurotransmission.

Fak56 is directly activated by the Drosophila Anaplastic Lymphoma Kinase, DAlk, receptor which was identified in our lab. We characterised DAlk as a novel RTK that is expressed in the embryonic CNS and mesoderm where it drives activation of the ERK/MAPK pathway. Indeed, we found DAlk to ectopically induce protein tyrosine phosphorylation and specifically phosphorylation of ERK, resulting in autonomous cell transformation and uncontrolled tissue growth. Subsequently, we identified a requirement for DAlk function during Drosophila embryogenesis, where it displays an essential role in gut development. Specifically, we identified the secreted molecule Jelly belly (Jeb) as a ligand for DAlk and showed that Jeb-DAlk interaction activates an ERK-mediated signalling pathway essential for visceral muscle specification and fusion, and consequently formation of the gut.

The potent ability of PTKs to regulate cell behaviour, together with the strong linkage between RTK dysregulation and tumour formation, renders the negative regulation of kinase activity an important area of research. We have identified the Drosophila homologue of Cbl-interacting protein of 85kDa, dCIN85, an adaptor molecule which in mammalian cells has shown involvement in RTK endocytosis and downregulation, as well as in the regulation of actin cytoskeleton dynamics. In the fruit fly, dCIN85 displays essential functions, given that dCIN85 loss of function mutants display a grand-child less phenotype. Generation of a dCIN85 antibody, together with isoform-specific transgenic flies, have allowed us to observe a punctuate localization pattern of the SH3-domain containing dCIN85 variants, representing Rab5-positive endosomal structures. This, in addition to the confirmation of a direct dCIN85-dCbl interaction, indicates an evolutionary conservation of dCIN85 function. Interestingly, dCIN85 co-localises with dRICH1, a Cdc42 specific RhoGAP, in differentiated photoreceptor cells in eye imaginal discs. This may imply a role for dCIN85 in the regulation of the specialised endocytic recycling processes required for the assembly/maintenance of tight junctions and establishment of cell polarity in epithelial tissues.

Ort, förlag, år, upplaga, sidor
Umeå: Umeå centrum för molekylär patogenes (UCMP) (Medicinska fakulteten), 2007. s. 104
Serie
Umeå University medical dissertations, ISSN 0346-6612 ; 1080
Nyckelord
Drosophila, Signalling, Adhesion, Protein Tyrosine Kinase, Development, Endocytosis
Nationell ämneskategori
Biokemi och molekylärbiologi
Identifikatorer
urn:nbn:se:umu:diva-971 (URN)978-91-7264-246-1 (ISBN)
Disputation
2007-02-09, Hörsal E04, 6L, Norrlands Universitetssjukhus, Umeå, 09:00 (Engelska)
Opponent
Handledare
Tillgänglig från: 2007-01-19 Skapad: 2007-01-19 Senast uppdaterad: 2009-05-19Bibliografiskt granskad

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