Professor Jeff Williams FRSE
Image 1. An electron micrograph of a Dictyostelium fruiting body.
The quantum evolutionary leap, from unicellularity to multiucellularity, occurred many times and in many different ways in the eukaryotes. Dictyostelium discoideum is a free-living soil amoeba that undergoes a remarkable, facultative transition to multicellularity when subjected to starvation. The initially separate cells aggregate together in response to pulses of cyclic AMP (cAMP) that they synthesise and secrete as a chemoattractant. They first form themselves into a migratory slug that displays most of the defining features of a multi-cellular organism. During their subsequent development, to form a fruiting body, the cells differentiate as either spore cells or stalk cells (Fig 1).
Dictyostelium is much valued as a model eukaryotic cell, to study processes such as chemotaxis and phagocytosis, but we wish to understand how its remarkable developmental cycle is effected. The answers are in part known but there remain very large gaps in our understanding. cAMP and another small organic molecules, the polyketide DIF, play key roles. cAMP induces the differentiation of prespore cells and they secrete DIF. DIF then induces a proportion of the uncommitted cells to differentiate as prestalk cells. We are tracing this signaling pathway upwards, from a DIF-responsive promoter that directs expression of EcmA, an extracellular matrix protein. We have identified a Myb transcription factor, MybE, and a bZip transcription factor, DimB, that co-operatively mediate prestalk gene induction by DIF (Fig 2). When cells are exposed to DIF DimB is rapidly serine-threonine phosphorylated at a site near its C terminus and it moves to the nucleus and binds to the ecmA prestalk promoter.
Image 2. Gene expression directed by a multimerised, minimal promoter fragment.
Two other components in the signalling pathways for stalk cell differentiation are STAT (Signal Transducer and Activator of Transcription) proteins. They are conserved in structure and function with the STAT proteins that mediate cytokine signalling in animal cells. Cytokines control many vital animal cell functions and the study of STATs in a diverged but genetically amenable organism such as Dictyostelium is providing insights into mammalian STAT signalling pathways (Fig 3). One of the two STAT proteins, STATa, is activated, i.e. it is tyrosine phosphorylated dimirises and moves to the nucleus, in response to cAMP signalling. STATa directs the differentiation of the tip-organiser cells; a sub-set of the prestalk cells that control slug integrity and behaviour. The second, STATc, is activated by a non-paradigmatic pathway wherein DIF acts to cause repression of a STATc specific protein tyrosine phosphatase: PTP3. This repression is bought about by DIF induced serine phosphorylation of PTP3 at two sites.
Image 3. The crystal structure of a Dictyostelium STAT protein.
STATs dimerise via their SH2 domains. By interacting with specific phosphotyrosine residues in this way, SH2 domains provide regulatable protein-protein interaction domains. Dictyostelium is the only nonmetazoan with functionally characterized SH2 domains, but the cognate tyrosine kinases are unknown. There are no orthologs of the animal tyrosine kinases, but there are very many tyrosine kinase-like kinases (TKLs), a group of kinases which, despite their family name, are mainly classified as serine-threonine kinases. STATc is activated by phosphorylation on Tyr922 when cells are exposed to the prestalk inducer differentiation inducing factor (DIF-1), a chlorinated hexaphenone. We have recently shown that after of exposure cells to DIF-1, Pyk2, a tyrosine-specific TKL, directly activates STATc (Proc Natl Acad Sci U S A. 2012,109(28), 1931-7). This result has significant implications for understanding the evolutionary origins of SH2 domain-phosphotyrosine signaling. It also has mechanistic implications. Our previous work suggested that a predictedly constitutive STATc tyrosine kinase activity is counterbalanced in vivo by the DIF-1-regulated activity of PTP3, a Tyr922 phosphatase. Now we have shown that the STATc-Pyk2 complex is formed constitutively, by an interaction between the STATc SH2 domain and phosphotyrosine residues on Pyk2 that are generated by autophosphorylation. Also, as predicted, Pyk2 is constitutively active as a STATc kinase. This observation provides further evidence for this highly atypical, possibly ancestral, STAT regulation mechanism.
Thus the STATc, and DimB DIF signaling pathways have both been traced to the regulated phosphorylation of specific sites within pathway intermediates and the cognate kinases are being purified. One overriding goal now is to understand how all of the different signal transduction pathways that regulate these transcription factors are structured and integrated to direct cellular differentiation.
MrfA is the transcription factor that directs differentiation of a sub-set of prestalk cells located in the extreme anterior of the slug. In very recent findings, that bridge the prokaryote-eukaryote divide, a bacteriophage tail spike auto-proteolytic cleavage mechanism has been shown to be strictly homologous to a cleavage mechanism that liberates the active portion of MrfA and related eukaryotic membrane-tethered transcription factors. This remarkable finding has recently been obtained for MrfA: the Dictyostelium orthologue of the vertebrate Myelin-gene Regulatory Factor, of mouse, human and, in a collaboration with Hiroshi Senoo and Masashi Fukuzawa, for the Dictyostelium orthologue:MrfA (Senoo et al, (2013, PMID: 24046445 Journal of Cell Science, epub date 17th Sept). There are other potential explanations for this observation but the most obvious possibility is that of lateral transfer of the 60 amino acid cleavage domain.
Fig 1 An electron micrograph of a late stage in the development of a Dictyostelium culminant (fruiting body). They are typically about 1mm high and contain 100,000 cells: 20,000 dead, highly vacuolated stalk cells supporting 80,000 spore cells. Work from this laboratory showed that the prestalk and stalk cells are heterogeneous, with different cell types located in different parts of the developing structures. This is indicated by false colouring, in red, of a sub-set of the prestalk cells that are just entering the entrance to the stalk (image with the kind courtesy of Larry Blanton and Mark Grimson).
Fig 2 Cell type specific expression of a lacZ reporter construct containing a Myb and a bZIP transcription factor binding site, both contained within just 22 nucleotides of promoter DNA. The 22-mer, from within the promoter of the DIF-inducible ecmA gene, was multimerised four times and inserted upstream of a heterologous cap site and TATA box. This was in turn linked to a lacZ reporter. Stable transformants were generated using the construct and the cells were developed to various stages then stained for ß-galactosidase activity.
Fig 3 The crystal structure of Dictyostelium STATa, determined in collaboration with Dr. Christoph Mueller and his group at the EMBL and compared with the structure of metazoan STAT3. STATs dimerise via mutual interactions of their single SH2 domains (SH2 in the figure) and single tyrosine phosphorylation sites (pTyr in the figure). The target DNA for STAT3 is shown in grey and the interacting amino acids are asterisked. The STATa structure proved highly informative because dimeric transcription factors usually only dimerise in the presence of their target DNA. STATa crystallised without target DNA and this showed for the first time that unliganded STATs exist as a highly extended, semi-linear structure.
Fig. 4 Crystal structure of an intramolecular chaperone mediating triple-beta-helix folding. (Schulz EC, et al., Nat.Struct.Mol.Biol. (2010) 17 , 210). The 60 amino acid phage tail spike domain functions to direct trimerisation of the protein and a serine-lysine dyad within the domain then catalyses endo-proteolytic cleavage. This is precisely the mechanism used to process the MRF proteins and this homology extends to the amino acid level.
Tsuyoshi Araki, Linh Hai Vu, Norimitsu Sasaki, Takefumi Kawata, Ludwig Eichinger, Jeffrey G Williams: Two Dictyostelium Tyrosine Kinase-Like kinases function in parallel, stress-induced STAT activation pathways:Mol Biol Cell 2015 Feb 15;26(4):805-20. doi: 10.1091/mbc.E14-08-1319. Epub 2014 Dec 17
Linh Hai Vu, Tsuyoshi Araki, Jianbo Na, Christoph S Clemen, Jeffrey G Williams, Ludwig Eichinger: Identification of the Protein Kinases Pyk3 and Phg2 as Regulators of the STATc-Mediated Response to Hyperosmolarity: PLoS ONE 01/2014; 9(2):e90025.
Clémence Habourdin, Gérard Klein, Tsuyoshi Araki, Jeffrey G Williams, Laurence Aubry: The arrestin-domain containing protein AdcA is a response element to stress: Cell Communication and Signaling 11/2013; 11(1):91.
Hiroshi Senoo, Tsuyoshi Araki, Masashi Fukuzawa, Jeffrey G Williams: A new kind of membrane-tethered eukaryotic transcription factor that shares an auto-proteolytic processing mechanism with bacteriophage tail-spike proteins: Journal of Cell Science J Cell Sci. 2013 Nov 15;126(Pt 22):5247-58. doi: 10.1242/jcs.133231. Epub 2013 Sep 17
Yoko Yamada, Yuzuru Kubohara, Haruhisa Kikuchi, Yoshiteru Oshima, Hong-Yu Wang, Susan Ross, Jeffrey G Williams: The Dictyostelium prestalk inducer DIF-1 directs phosphorylation of a bZIP transcription factor: The International journal of developmental biology 09/2013; 57(5):375-81.
Tsuyoshi Araki, Takefumi Kawata, Jeffrey G Williams: Identification of the kinase that activates a nonmetazoan STAT gives insights into the evolution of phosphotyrosine-SH2 domain signaling: Proceedings of the National Academy of Sciences 06/2012; 109(28):E1931-7.
Tsuyoshi Araki, Jeffrey G Williams: Perturbations of the actin cytoskeleton activate a Dictyostelium STAT signalling pathway: European journal of cell biology 05/2012; 91(5):420-5.
Hiroshi Senoo, Hong-Yu Wang, Tsuyoshi Araki, Jeffrey G Williams, Masashi Fukuzawa: An orthologue of the Myelin-gene Regulatory Transcription Factor regulates Dictyostelium prestalk differentiation: The International journal of developmental biology 01/2012; 56(5):325-32.
Beatriz Nuñez-Corcuera, Joanna L Birch, Yoko Yamada, Jeffrey G Williams: Transcriptional repression by a bZIP protein regulates Dictyostelium prespore differentiation: PLoS ONE 01/2012; 7(1):e29895.
Yoko Yamada, Beatriz Nuñez-Corcuera, Jeffrey G Williams: DIF-1 regulates Dictyostelium basal disc differentiation by inducing the nuclear accumulation of a bZIP transcription factor: Developmental Biology 03/2011; 354(1):77-86.
Yamada Yoko, Kay Robert R, Bloomfield Gareth, Ross Susan, Ivens A, Williams JG. Dev Biol. 2010 Mar 15;339(2):390-7. doi: 10.1016/j.ydbio.2009.12.045. Epub 2010 Jan 18 A new Dictyostelium prestalk cell sub-type: Developmental Biology 03/2010; 339(2):390-7.
Jeffrey Williams: Dictyostelium finds new roles to model Genetics 07/2010; 185(3):717-26.
Tsuyoshi Araki, Wouter N van Egmond, Peter J M van Haastert, Jeffrey G Williams: Dual regulation of a Dictyostelium STAT by cGMP and Ca2+ signaling: Journal of Cell Science
2010 Mar 15;123(Pt 6):837-41. doi: 10.1242/jcs.064436. Epub 2010 Feb 16.
Judith Langenick, Tsuyoshi Araki, Yoko Yamada, Jeffrey G Williams: A Dictyostelium homologue of the metazoan Cbl proteins regulates STAT signalling: Journal of Cell Science 11/2008; 121(Pt 21):3524-30
Tsuyoshi Araki, Judith Langenick, Marianne Gamper, Richard A Firtel, Jeffrey G Williams: Evidence that DIF-1 and hyper-osmotic stress activate a Dictyostelium STAT by inhibiting a specific protein tyrosine phosphatase: Development 05/2008; 135(7):1347-53.
Pascale Gaudet, Jeffery G Williams, Petra Fey, Rex L Chisholm: An anatomy ontology to represent biological knowledge in Dictyostelium discoideum: BMC Genomics 02/200
Araki, T., Langenick, J., Gamper, M., Firtel, R.A. and Williams, J. G. (2008) Evidence that DIF-1 and hyper-osmotic stress activate a Dictyostelium STAT by inhibiting a specific protein tyrosine phosphatase. Development. 135:1347-53. PMID: 18305004
Langenick, J., Araki, T., Yamada, Y. and Williams, J. G. (2008) A Dictyostelium homologue of the metazoan Cbl proteins regulates STAT signalling. J Cell Sci. 200: 3524-30. PMID: 18840649
Yamada, Y.*, Wang, H. Y.*, Fukuzawa, M. , Barton, G. J. and Williams, J. G. (2008) A new family of transcription factors. Development. 135:3093-101. *equal co-authors PMID: 18701541
Gaudet, P., Williams, J.G., Fey, P., Chisholm, R.L. (2008) An anatomy ontology to represent biological knowledge in Dictyostelium discoideum. BMC Genomics. 9:130.
Wang, H. Y. and Williams, J. G. (2009). Identification of a target for CudA, the transcription factor which directs formation of the Dictyostelium tip organiser. Int J Dev Biol 54, 161-5.
Araki, T., van Egmond, W. N., van Haastert, P. J. and Williams, J. G. (2010). Dual regulation of a Dictyostelium STAT by cGMP and Ca2+ signalling. J Cell Sci 123, 837-41. PMID: 20159963
Yamada, Y., Kay, R. R., Bloomfield, G., Ross, S., Ivens, A. and Williams, J. G. (2010). A new Dictyostelium prestalk cell sub-type. Dev Biol 339, 390-7. PMID: 20080085
Sugden, C., Ross, S., Bloomfield, G., Ivens, A., Skelton, J., Mueller-Taubenberger, A. and Williams, J. G. Two novel SH2 domain proteins interact to regulate Dictyostelium gene expression during growth and early development. (2010) J Biol Chem, 285, 2297-22935 PMID: 20457612
Williams, J.G. (2010) Dictyostelium finds new roles to model. Genetics 185, 717-26.
Wang, H.Y. and William, J.G. (2010) Synergy between two transcription factors directs gene expression in Dictyostelium tip-organiser cells. Int J Dev Biol 54, 1301-7.
Nunez-Corcuera, B., Birch, J. and William, J. G. (2011) A SET/MYND chromatin re-modelling protein regulates Dictyostelium prespore patterning. Int J Dev Biol 55, 205-8.
Sugden, C., Ross, S., Annesley, S.J., Cole, C., Bloomfield, G., Ivens, A., Skelton, J., Fisher, P.R., Barton, G. and Williams, J. G. (2011) A Dictyostelium SH2adaptor protein required for correct DIF-1 signaling and pattern formation. Dev Biol 353, 290-301.
Yamada, Y., Nunez-Corcuera, B. and William,s J.G. (2011) DIF-1 regulates Dictyostelium basal disc differentiation by inducing the nuclear accumulation of a bZIP transcription factor. Dev Biol 354, 77-86.
Araki, T. Kawata, T. and Williams, J.G. Identification of the kinase that activates a nonmetazoan STAT gives insights into the evolution of phosphotyrosine-SH2 domain signaling Proc Natl Acad Sci U S A PLUS 2012,109, 1931-7.
Sugden Christopher, Urbaniak Michael D, Araki Tsuyoshi, Williams Jeffrey The Dictyostelium prestalk inducer Differentiation Inducing Factor-1 (DIF-1) triggers unexpectedly complex global phosphorylation changes. Mol Biol Cell 2015 Feb 15;26(4):805-20. doi: 10.1091/mbc.E14-08-1319. Epub 2014 Dec 17