Dictyostelium   Printer friendly

Functional Analysis

Profilin isoforms in Dictyostelium discoideum [1]
Eukaryotic cells contain a large number of actin binding proteins of different functions, locations and concentrations. They bind either to monomeric actin (G-actin) or to actin filaments (F-actin) and thus regulate the dynamic rearrangement of the actin cytoskeleton. The Dictyostelium discoideum genome harbours representatives of all G-actin binding proteins including actobindin, twinfilin, and profilin. A phylogenetic analysis of all profilins suggests that two distinguishable groups emerged very early in evolution and comprise either vertebrate and viral profilins or profilins from all other organisms. The newly discovered profilin III isoform in D. discoideum shows all functions that are typical for a profilin. However, the concentration of the third isoform in wild type cells reaches only about 0.5% of total profilin. In a yeast-2-hybrid assay profilin III was found to bind specifically to the proline-rich region of the cytoskeleton-associated vasodilator-stimulated phosphoprotein (VASP). Immunolocalization studies showed similar to VASP the profilin III isoform in filopodia and an enrichment at their tips. Cells lacking the profilin III isoform show defects in cell motility during chemotaxis. The low abundance and the specific interaction with VASP argue against a significant actin sequestering function of the profilin III isoform.

Figure 1: Evolutionary relationship of profilins. Two data sets of 52 non-plant profilins (left) and 69 plant profilins (not shown in detail) were analyzed and depicted as two separate radial trees. Plant profilins form a clearly separated and more closely related group of their own, mammalian and viral profilins cluster together as well as all non mammalian profilins which form a separate branch. Mammalian profilins IV are distinct and also belong to this branch. In contrast, the three D. discoideum and the two P. polycephalum profilin isoforms are closer related to the mammalian and viral profilins than to the majority of other profilins from lower organisms.

Horizontal gene transfer
In an article in Currtent Biology Arthur Guljamow et al. reported on a horizontal gene transfer of two cytoskeletal elements from an eukaryote to a cyanobacterium [2]. Which consequences such a horizontal transfer on the relationship of the mamalian profilin IV to their relatives in the lower organism remain to be highlighted.

Filamin-regulated F-actin Assembly Is Essential for Morphogenesis and Controls Phototaxis in Dictyostelium [3]
To follow its development cycle morphogenesis, chemotaxis and phototaxis are important for Dictyostelium. Dictyostelium strains lacking the F-actin cross-linking protein filamin (ddFLN) have a severe phototaxis defect at the multicellular slug stage. Filamins are rod-shaped homodimers that cross-link the actin cytoskeleton into highly viscous, orthogonal networks. Each monomer chain of filamin is comprised of an F-actin-binding domain and a rod domain. In rescue experiments only intact filamin re-established correct phototaxis in filamin minus mutants, whereas C-terminally truncated filamin proteins that had lost the dimerization domain and molecules lacking internal repeats but retaining the dimerization domain did not rescue the phototaxis defect. For correct phototaxis ddFLN is only required at the tip of the slug because expression under control of the cell type-specific extra cellular matrix protein A (ecmA) promoter and mixing experiments with wild type cells supported phototactic orientation. Likewise, in chimeric slugs wild type cells were primarily found at the tip of the slug, which acts as an organizer in Dictyostelium morphogenesis.

Figure 2: Distribution of GFP-tagged FLN proteins in living HG1264 cells. FLN-GFP is primarily present in the cell cortex and less so throughout the cytosol.

The annexins of Dictyostelium discoideum [4]
Annexins are a highly conserved ubiquitous family of calcium ion and phospholipid-binding proteins present in nearly all eukaryotic cells. Analysis of the Dictyostelium genome revealed the presence of two annexin genes, the annexin C1 gene (nxnA) giving rise to two isoforms of 47 and 51 kDa, and the annexin C2 gene (nxnB) coding for a 56-kDa protein with 33% sequence identity to annexin C1. Annexin C2 is expressed at very low and constant levels throughout development; it is present in about 35-fold lower amounts compared to annexin C1. The results of the study of their cellular distribution and dynamics were studied thoroughly.

Figure 3: Sequence comparison of annexin C1 isoforms (DDB0191502, DDB0232009) and Annexin C2 (DDB0232028). The sequence identity between the longer isoform of annexin C1 and annexin C2 is 33%. The sequences could be obtained at the dictyBase, The Dictyostelium WWW Server

The Dictyostelium repertoire of seven transmembrane domain receptors [5]
The availability of fully sequenced genomes allows the in silico analysis of whole gene families in a given genome. A particularly large and interesting gene family is the G-protein-coupled receptor family. These receptors detect a variety of extra cellular signals and transduce them, generally via heterotrimeric G-proteins, to effector proteins inside the cell and thus elicit a physiological response. G-protein-coupled receptors are found in all eukaryotes and constitute in vertebrates 3–5% of all genes. They are also very important drug targets and many drugs are directed against these receptors. The Dictyostelium discoideum genome contains a surprisingly high number of 55 such receptors, approximately 0.5% of the encoded genes. Besides the four well-studied cAMP receptors the genome encodes eight additional cAMP receptor-like proteins and one of these is distinguished by a novel domain structure, one secretin-like receptor, and 17 GABAB-like and 25 Frizzled-like receptors. The existence of the latter three types of receptors in D. discoideum was surprising because they had not been observed outside the animal kingdom before. Their presence suggests unprecedented complex and so far unknown signalling activities in this lower eukaryote.

Figure 4: The Dictyostelium family of GABAB-like receptors dendogram.

GrlJ, a Dictyostelium GABAB-like receptor with roles in post-aggregation development [6]
The G-protein-coupled receptor (GPCR) family represents the largest and most important group of targets for chemotherapeutics. They are extremely versatile receptors that transduce signals as diverse as biogenic amines, purins, odorants, ions and pheromones from the extra cellular compartment to the interior via biochemical processes involving GTPbinding proteins. Until recently, the cyclic AMP receptors (cARs) were the only known G protein coupled receptors in Dictyostelium discoideum. The completed genome sequence revealed the presence of several families of GPCRs in Dictyostelium, among them members of the family 3 of GPCRs, the GABAB/ glutamate like receptor family, which in higher eukaryotes is involved in neuronal signalling.

Figure 5: Hydrophobicity plot of GrlJ, program ProtScale using the scale of Kyte&Doolittle, windows width = 19, at the Expasy server [7]. The arrows indicate the seven transmembrane domains of the protein.

A G protein-coupled receptor, RpkA, is involved in cell-density sensing [8]
One mechanism multicellular structures use for controlling cell number involves the secretion and sensing of a factor, such as leptin or myostatin, in mammals. Dictyostelium cells secrete autocrine factors for sensing cell density prior to aggregation and multicellular development such as CMF (conditioned-medium factor), which enables starving cells to respond to cAMP pulses. Its actions are mediated by two receptors. CMFR1 activates a G protein independent signalling pathway regulating gene expression. An unknown Ga1-dependent receptor activates phospholipase C (PLC), which regulates the lifetime of Ga2-GTP. Here, we describe RpkA, an unusual seven-transmembrane receptor that is fused to a C-terminal PIP5 kinase domain and that localizes in membranes of a late endosomal compartment. Loss of RpkA resulted in formation of persistent loose aggregates and altered expression of cAMP regulated genes. The developmental defect can be rescued by full-length RpkA and the transmembrane domain only. The PIP5 kinase domain is dispensable for the developmental role of RpkA. rpkA2 cells secrete and bind CMF but are unable to induce downstream responses. Inactivation of Ga1, a negative regulator of CMF signalling, rescued the developmental defect of the rpkA2 cells, suggesting that RpkA actions are mediated by Ga1.

Figure 7: Development of AX2 and RpkA cells: top row AX2 cells 6 hr and 24 hr after starvation, and second row rpkA- cells, respectively.

GxcDD, a putative RacGEF, is involved in Dictyostelium development [9]
Rho subfamily GTPases are implicated in a large number of actin-related processes. They shuttle from an inactive GDP-bound form to an active GTP-bound form. This reaction is catalysed by Guanine nucleotide exchange factor (GEFs). GTPase activating proteins (GAPs) help the GTPase to return to the inactive GDP-bound form. The social amoeba Dictyostelium discoideum lacks a Rho or Cdc42 ortholog but has several Rac related GTPases. Compared to our understanding of the downstream effects of Racs our understanding of upstream mechanisms that activate Rac GTPases is relatively poor.

GxcDD (Guanine exchange factor for Rac GTPases) is a 180-kDa multidomain protein containing a type 3 CH domain, two IQ motifs, three PH domains, a RhoGEF domain and an ArfGAP domain. Inactivation of the gene results in defective streaming during development under different conditions and a delay in developmental timing. The characterization of single domains revealed that the CH domain of GxcDD functions as a membrane association domain, the RhoGEF domain can physically interact with a subset of Rac GTPases, and the ArfGAP-PH tandem accumulates in cortical regions of the cell and on phagosomes. A conformational change may be required for activation of GxcDD, which would be important for its downstream signalling.

The data indicate that GxcDD is involved in proper streaming and development. We propose that GxcDD is not only a component of the Rac signalling pathway in Dictyostelium, but is also involved in integrating different signals. We provide evidence for a Calponin Homology domain acting as a membrane association domain. GxcDD can bind to several Rac GTPases, but its function as a nucleotide exchange factor needs to be studied further.

  1. Rajesh Arasada, Annika Gloss, Budi Tunggal, Jayabalan M. Joseph, Daniela Rieger, Subhanjan Mondal, Jan Faix, Michael Schleicher, Angelika A. Noegel, Profilin isoforms in Dictyostelium discoideum, Biochimica et Biophysica Acta 1773, 631641 (2007)
  2. Arthur Guljamow, Holger Jenke-Kodama, Harald Saumweber, Philippe Quillardet, Lionel Frangeul, Anne Marie Castets, Christiane Bouchier, Nicole Tandeau de Marsac and Elke Dittmann, Horizontal gene transfer of two cytoskeletal elements from a eukaryote to a cyanobacterium, Current Biology 17, R757-59 (2007).
  3. Nandkumar Khaire, Rolf Müller, Rosemarie Blau-Wasser, Ludwig Eichinger, Michael Schleicher,Matthias Rief, Tad A. Holak., and Angelika A. Noegel, Filamin-regulated F-actin Assembly Is Essential for Morphogenesis and Controls Phototaxis in Dictyostelium, J. Biol. Chem. 282, 1948-55 (2007)
  4. Marija Marko, Yogikala Prabhu, Rolf Müller, Rosemarie Blau-Wasser, Michael Schleicher, Angelika A. Noegel, The annexins of Dictyostelium, European Journal of Cell Biology 85, 1011–1022 (2006).
  5. Yogikala Prabhu, Ludwig Eichinger, The Dictyostelium repertoire of seven transmembrane domain receptors, European Journal of Cell Biology 85, 937–946 (2006).
  6. Yogikala Prabhu, Rolf Muller, Christophe Anjard, Angelika A Noegel, GrlJ, a Dictyostelium GABAB-like receptor with roles in post-aggregation development, BMC Developmental Biology 7, 44 (2007).
  7. Gasteiger E., Hoogland C., Gattiker A., Duvaud S., Wilkins M.R., Appel R.D., Bairoch A., Protein Identification and Analysis Tools on the ExPASy Server, (In) John M. Walker (ed): The Proteomics Protocols Handbook, pp. 571-607, Humana Press (2005).
  8. Deenadayalan Bakthavatsalam, Derrick Brazill, Richard H. Gomer, Ludwig Eichinger, Francisco Rivero, and Angelika A. Noegel, A G Protein-Coupled Receptor with a Lipid Kinase Domain Is Involved in Cell-Density Sensing, Current Biology 17 892897 (2007).
  9. Subhanjan Mondal, Dhamodharan Neelamegan, Francisco Rivero1 and Angelika A Noegel, GxcDD, a putative RacGEF, is involved in Dictyostelium development, BMC Cell Biology 8, 23 (2007).

January 29, 2009
Institute of Biochemistry I, Cologne
Suggestions and wishes: Gudrun Konertz
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