Our laboratory is investigating molecular processes critical for developing a terminally differentiated organism from a homogeneous population of totipotent cells. We are using molecular, genetic, and biochemical techniques and the model eukaryotic system Dictyostelium to define cell autonomous and non-autonomous signal transduction pathways that specify cell fate and pattern formation. In Dictyostelium, stimulation of a family of 7-transmembrane (7-TM) domain receptors by its ligand, the secreted chemoattractant-morphogen cAMP, establishes the fundamental developmental organization, an anterior (prestalk)/posterior (prespore) axis. As a chemoattractant, cAMP activates cAMP receptor/G protein signaling to mobilize individual cells to form multicellular aggregates; later in development, cAMP receptor signaling regulates cell fate specification. We focus on signaling pathways, both common and unique to the family of cAMP receptors (CARs), to elucidate mechanisms and circuits that may be conserved in more complex developmental programs. Wnt-signaling regulates two primary, but discrete pathways in the metazoa. As a morphogen, Wnt functions as an effective inhibitor of GSK3, a developmental switch that regulates cell fate determination. Wnts also organize planar cell polarity and coordinate cell migration, but a dependent role for GSK3 is not clear. In many respects, extracellular cAMP in Dictyostelium is a functional Wnt analog. Dictyostelium cells will polarize in response to cAMP. In addition, cAMP regulates the activation/de-activation of GSK3 which, in turn, establishes developmental patterning. Previously, we had shown that ZAK1 is an activating tyrosine kinase of GSK3 and now identify ZAK2, the other tyrosine kinase in the cAMP-activation pathway for GSK3; no additional family members exist. We now show that tyrosine phosphorylation/activation of GSK3 by ZAK2 and ZAK1 are differentially required to regulate GSK3 within distinct differentiated cell populations. ZAK2/GSK3 also acts in a non-autonomous manner to regulate alternative cell fate decisions that further parallels mechanisms of Wnt signaling. Finally we show that efficient polarization of Dictyostelium toward cAMP depends on ZAK-mediated tyrosine phosphorylation of GSK3. We suggest that combinatorial regulation of GSK3 can differentially guide cell polarity, directional cell migration, and cell fate specification in Dictyostelium and potentially other systems. In the absence of Wnt, the transcriptional co-factor ?O-catenin is de-stabilized via phosphorylation by protein kinase GSK3?O in complex with Axin. In the !?canonical!? Wnt signaling pathway, Disheveled (Dvl) is required to functionally inhibit the activity of the GSK3?O/Axin complex to stabilize ?O-catenin. Here, we now examine time-dependent responses to Wnt stimulation in mammalian cells. We show that GSK3?O/Axin complexes are rapidly (t1/2 ~3 minutes) disrupted upon Wnt stimulation and that these changes in GSK3?O/Axin association precede ?O-catenin stabilization and Axin degradation. We further demonstrate an association of G??o with Fz that is also very rapidly (t1/2 <1 min.) altered upon Wnt-3a stimulation. The Wnt-dependent affects on both G??o/Fz and GSK3?O/Axin2 are pertussis toxin sensitive, implicating a role for G proteins in signaling to ?O-catenin. Finally, we show that direct activation of G proteins in vivo with GTP??S will disrupt GSK3?O/Axin2 complexes and suggest that G??o and G??q signaling contributes to Wnt-mediated stabilization of ?O-catenin. Members of the SWI2/SNF2 family of !?helicase-like!?, DNA-dependent ATPases modulate transcription of chromatin templates. Proteins in the CHD branch of this family contain a chromodomain and a DNA-binding motif, in addition to the central helicase-like domain, and studies implicate CHD members in control of gene expression. We report the characterization of a presumptive CHD1 ortholog in DD. discoideum. Expression of CHD1 is induced as cells become competent to enter the multicellular stage of the D. discoideum life-cycle, coincident with de-repression of the earliest developmentally regulated genes. Disruption of CHD1 leads to severe developmental defects including aberrant aggregation, reduction in prespore-specific gene expression, inappropriate patterning of both prespore and prestalk cells, and developmental arrest prior to the formation of terminally differentiated fruiting bodies. During aggregation, cells expressing prestalk genes normally sort to the top of the aggregate and form the extending tip, an organizer for further differentiation and for prestalk and prespore patterning. In the absence of CHD1, prestalk cells remain dispersed through the aggregate, even as extensions project from the mound. We propose that a primary defect in cells lacking CHD1 is the inability of prestalk cells to establish a functional tip. Consequently, prespore-specific gene expression is not maximally activated and cellular differentiation cannot proceed. These studies suggest that CHD1 is essential for differentiation and patterning of the major cell types in D. discoideum, and perhaps suggests a similar regulatory role for metazoan development. The LIM domain is a unique motif of two conserved, zinc finger-like modules, CX2CX16-23HX2CX2CX2CX16-21CX2C. Structural stability of LIM domains depends on the coordinated tetrahedral binding of two zinc atoms via cysteine and histidine residues. LIM proteins act as adapters, mediating protein-protein interactions in processes such as transcription regulation, cell fate determination and actin cytoskeleton organization. LIM-regulated cytoskeletal reorganization is required for cell motility, cell-cell interaction, cell-substratum attachment, development, and other dynamic processes. We have identified two novel LIM domain proteins, LimF and CH-LIM, from Dictyostelium that act as binding partners and that coordinate with Rab21 to regulate phagocytosis. LimF, a 23 kDa protein consisting of three tandem LIM domains, was used as a two-hybrid bait to screen for interacting proteins. CH-LIM and Rab21 were isolated from this screen and interaction was confirmed by GST pull-down binding assays. CH-LIM is a 76 kDa protein consisting of three LIM domains and an N-terminal Calponin Homology (CH) domain. The CH domain is a 100 amino acid motif which also serves as a protein binding interface for many structural and signaling molecules. Rab21 is a member of the small G protein family of signaling molecules often associated with membrane trafficking. Tagged versions of each protein, GFP-LimF, GFP-CH-LIM, and GFP-Rab21, were expressed in cells to identify their intracellular localizations. LimF, CH-LIM, and Rab21 all exhibited association with intracellular vacuoles. To investigate in vivo function, we generated limF and ch-lim knock-out and overexpression cell lines and strains that express activating or inhibiting mutations of Rab21. Overexpression of LimF, loss of CH-LIM, or expression of the constitutively active Rab21 increases the rate of phagocytosis above that of wild-type. Conversely, overexpression of CH-LIM, loss of LimF, or expression of constitutively inactive Rab21 decreases the rate of phagocytosis. Studies using cells that carry mutations in multiple genes suggest that the activating function of Rab21-GTP requires LimF, and that, in turn, activated LimF represses the inhibiting function of CH-LIM on phagocytosis. We suggest that these components participate in a single complex to regulate the activity of the phagocytosis.
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