Biomedical subjects

Leonard Bosgraaf

Publications and source records attributed to Leonard Bosgraaf.

13 recordsLinked to original sources

PubMed2006

The regulation of myosin II in Dictyostelium.

Dictyostelium conventional myosin (myosin II) is an abundant protein that plays a role in various cellular processes such as cytokinesis, cell protrusion and development. This review will focus on the signal transduction pathways that regulate myosin II during cell movement. Myosin II appears to have two modes of action in Dictyostelium: local stabilization of the cytoskeleton by myosin filament association to the actin meshwork (structural mode) and force generation by contraction of actin filaments (motor mode). Some processes, such as cell movement under restrictive environment, require only the structural mode of myosin. However, cytokinesis in suspension and uropod retraction depend on motor activity as well. Myosin II can self-assemble into bipolar filaments. The formation of these filaments is negatively regulated by heavy chain phosphorylation through the action of a set of novel alpha kinases and is relatively well understood. However, only recently it has become clear that the formation of bipolar filaments and their translocation to the cortex are separate events. Translocation depends on filamentous actin, and is regulated by a cGMP pathway and possibly also by the cAMP phosphodiesterase RegA and the p21-activated kinase PAKa. Myosin motor activity is regulated by phosphorylation of the regulatory light chain through myosin light chain kinase A. Unlike conventional light chain kinases, this enzyme is not regulated by calcium but is activated by cGMP-induced phosphorylation via an upstream kinase and subsequent autophosphorylation.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed2006

Characterization of the GbpD-activated Rap1 pathway regulating adhesion and cell polarity in Dictyostelium discoideum.

The regulation of cell polarity plays an important role in chemotaxis. GbpD, a putative nucleotide exchange factor for small G-proteins of the Ras family, has been implicated in adhesion, cell polarity, and chemotaxis in Dictyostelium. Cells overexpressing GbpD are flat, exhibit strongly increased cell-substrate attachment, and extend many bifurcated and lateral pseudopodia. These cells overexpressing GbpD are severely impaired in chemotaxis, most likely due to the induction of many protrusions rather than an enhanced adhesion. The GbpD-overexpression phenotype is similar to that of cells overexpressing Rap1. Here we demonstrate that GbpD activates Rap1 both in vivo and in vitro but not any of the five other characterized Ras proteins. In a screen for Rap1 effectors, we overexpressed GbpD in several mutants defective in adhesion or cell polarity and identified Phg2 as Rap1 effector necessary for adhesion, but not cell polarity. Phg2, a serine/threonine-specific kinase, directly interacts with Rap1 via its Ras association domain.

Animals↗

PubMed2006

Myosin light chain kinase A is activated by cGMP-dependent and cGMP-independent pathways.

Stimulation of Dictyostelium cells with the chemoattractant cAMP results in transient phosphorylation of the myosin regulatory light chain (RLC). We show that myosin light chain kinase A (MLCK-A) is responsible for RLC phosphorylation during chemotaxis, and that MLCK-A itself is transiently phosphorylated on threonine-166, dramatically increasing its catalytic activity. MLCK-A activation during chemotaxis is highly responsive to cellular cGMP levels and the cGMP-binding protein GbpC. MLCK-A- cells have a partial cytokinesis defect, and do not phosphorylate RLC in response to concanavalin A (conA), but cells lacking cGMP or GbpC divide normally and phosphorylate in response to conA. Thus MLCK-A is activated by a cGMP/GbpC-independent mechanism activated during cytokinesis or by conA, and a cGMP/GbpC-dependent pathway during chemotaxis.

Animals↗

PubMed2005

Paxillin is required for cell-substrate adhesion, cell sorting and slug migration during Dictyostelium development.

Paxillin is a key regulatory component of focal adhesion sites, implicated in controlling cell-substrate interactions and cell movement. We analyse the function of a Dictyostelium discoideum paxillin homologue, PaxB, which contains four highly conserved LD and four LIM domains, but lacks two characteristic tyrosine residues, that form the core of vertebrate SH2-binding domains. PaxB is expressed during growth and all stages of development, but expression peaks during slug formation. Using a paxB-gfp knockin strain we show the existence of focal adhesions and characterise their dynamics. During multicellular development PaxB is not only found in focal adhesions at the cell-substrate interface, but also in the tips of filopodial structures predominantly located at the trailing ends of cells. paxB- strains are less adhesive to the substrate, they can aggregate but multicellular development from the mound stage onwards is severely impeded. paxB- strains are defective in proper cell type proportioning, cell sorting, slug migration and form-defective fruiting bodies. Mutation of a conserved JNK phosphorylation site, implicated in the control of cell migration, does not have any major effects on cell sorting, slug migration or morphogenesis in Dictyostelium. PaxB does not appear to function redundantly with its closest relative Lim2 (paxA), which when deleted also results in a mound arrest phenotype. However, analysis of paxA- and paxB- single and double null mutants suggest that PaxB may act upstream of Lim2.

Amino Acid Sequence↗

PubMed2005

RasGEF-containing proteins GbpC and GbpD have differential effects on cell polarity and chemotaxis in Dictyostelium.

The regulation of cell polarity plays an important role in chemotaxis. Previously, two proteins termed GbpC and GbpD were identified in Dictyostelium, which contain RasGEF and cyclic nucleotide binding domains. Here we show that gbpC-null cells display strongly reduced chemotaxis, because they are unable to polarise effectively in a chemotactic gradient. However, gbpD-null mutants exhibit the opposite phenotype: cells display improved chemotaxis and appear hyperpolar, because cells make very few lateral pseudopodia, whereas the leading edge is continuously remodelled. Overexpression of GbpD protein results in severely reduced chemotaxis. Cells extend many bifurcated and lateral pseudopodia, resulting in the absence of a leading edge. Furthermore, cells are flat and adhesive owing to an increased number of substrate-attached pseudopodia. This GbpD phenotype is not dependent on intracellular cGMP or cAMP, like its mammalian homolog PDZ-GEF. Previously we showed that GbpC is a high-affinity cGMP-binding protein that acts via myosin II. We conclude that cGMP activates GbpC, mediating the chemoattractant-induced establishment of cell polarity through myosin. GbpD induces the formation of substrate-attached pseudopodia, resulting in increased attachment and suppression of polarity.

Actins↗

PubMed2004

Unusual Guanylyl Cyclases and cGMP Signaling in Dictyostelium discoideum.

cGMP is used as a second messenger in many eukaryotes. cGMP signaling requires at least three components: Guanylyl cyclases synthesize cGMP from GTP. Specific cGMP-binding proteins propagate the signal, usually by phosphorylation of their target proteins. Finally, phosphodiesterases terminate the cGMP signal by hydrolyzing cGMP to 5'cGMP. Recently, all guanylyl cyclases and most of the cGMP target proteins and phosphodiesterases of the cellular slime mold Dictyostelium discoideum have been identified. Characterization of these enzymes show them to be structurally and evolutionarily distinct from their bacterial and metazoan counterparts. In this chapter we review the properties of the Dictyostelium guanylyl cyclases and discuss their role in the unusual cGMP pathway of Dictyostelium.

Animals↗

PubMed2004

Chemotaxis: signalling modules join hands at front and tail.

Chemotaxis is the result of a refined interplay among various intracellular molecules that process spatial and temporal information. Here we present a modular scheme of the complex interactions between the front and the back of cells that allows them to navigate. First, at the front of the cell, activated Rho-type GTPases induce actin polymerization and pseudopod formation. Second, phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) is produced in a patch at the leading edge, where it binds pleckstrin-homology-domain-containing proteins, which enhance actin polymerization and translocation of the pseudopod. Third, in Dictyostelium amoebae, a cyclic-GMP-signalling cascade has been identified that regulates myosin filament formation in the posterior of the cell, thereby inhibiting the formation of lateral pseudopodia that could misdirect the cell.

Animals↗

PubMed2003

Pleckstrin homology domain diffusion in Dictyostelium cytoplasm studied using fluorescence correlation spectroscopy.

The translocation of pleckstrin homology (PH) domain-containing proteins from the cytoplasm to the plasma membrane plays an important role in the chemotaxis mechanism of Dictyostelium cells. The diffusion of three PH domain-green fluorescent protein (GFP) fusions (PH2-GFP, PH10-GFP, and PH-CRAC (cytosolic regulator of adenylyl cyclase)-GFP) in the cytoplasm of vegetative and chemotaxing Dictyostelium cells has been studied using fluorescence correlation spectroscopy to gain a better understanding of the functioning of the domains and to assess the effect of initiation of chemotaxis on these domains in the cell. PH2-GFP was homogeneously distributed in vegetative as well as chemotaxing cells, whereas PH10-GFP and PH-CRAC-GFP showed translocation to the leading edge of the chemotaxing cell. The diffusion characteristics of PH2-GFP and PH-CRAC-GFP were very similar; however, PH10-GFP exhibited slower diffusion. Photon counting histogram statistics show that this slow diffusion was not due to aggregation. Diffusion of the three PH domains was affected to similar extents by intracellular heterogeneities in vegetative as well as chemotaxing cells. From the diffusion of free cytoplasmic GFP, it was calculated that the viscosity in chemotaxing cells was 1.7 times lower than in vegetative cells. In chemotaxing cells, PH2-GFP showed increased mobility, whereas the mobilities of PH10-GFP and PH-CRAC-GFP remained unchanged.

Animals↗

PubMed2003

Roc, a Ras/GTPase domain in complex proteins.

We identified a novel group of the Ras/GTPase superfamily, termed Roc, that is present as domain in complex proteins together with other domains, including leucine-rich repeats (LRRs), ankyrin repeats, WD40 repeats, kinase domains, RasGEF and RhoGAP domains. Roc is always succeeded by a novel 300-400-amino-acid-long domain, termed COR. Proteins with Roc/COR are present in prokaryotes, Dictyostelium, plants and metazoa.

Amino Acid Sequence↗

PubMed2002

A novel cGMP signalling pathway mediating myosin phosphorylation and chemotaxis in Dictyostelium.

Chemotactic stimulation of Dictyostelium cells results in a transient increase in cGMP levels, and transient phosphorylation of myosin II heavy and regulatory light chains. In Dictyostelium, two guanylyl cyclases and four candidate cGMP-binding proteins (GbpA- GbpD) are implicated in cGMP signalling. GbpA and GbpB are homologous proteins with a Zn2+-hydrolase domain. A double gbpA/gbpB gene disruption leads to a reduction of cGMP-phosphodiesterase activity and a 10-fold increase of basal and stimulated cGMP levels. Chemotaxis in gbpA(-)B(-) cells is associated with increased myosin II phosphorylation compared with wild-type cells; formation of lateral pseudopodia is suppressed resulting in enhanced chemotaxis. GbpC is homologous to GbpD, and contains Ras, MAPKKK and Ras-GEF domains. Inactivation of the gbp genes indicates that only GbpC harbours high affinity cGMP-binding activity. Myosin phosphorylation, assembly of myosin in the cytoskeleton as well as chemotaxis are severely impaired in mutants lacking GbpC and GbpD, or mutants lacking both guanylyl cyclases. Thus, a novel cGMP signalling cascade is critical for chemotaxis in Dictyostelium, and plays a major role in myosin II regulation during this process.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed2002

Identification of four candidate cGMP targets in Dictyostelium.

In Dictyostelium, a transient increase in intracellular cGMP is important for cytoskeletal rearrangements during chemotaxis. There must be cGMP-binding proteins in Dictyostelium that regulate key cytoskeletal components after treatment with chemoattractants, but to date, no such proteins have been identified. Using a bioinformatics approach, we have found four candidate cGMP-binding proteins (GbpA-D). GbpA and -B have two tandem cGMP-binding sites downstream of a metallo beta-lactamase domain, a superfamily that includes cAMP phosphodiesterases. GbpC contains the following nine domains (in order): leucine-rich repeats, Ras, MEK kinase, Ras guanine nucleotide exchange factor N-terminal (RasGEF-N), DEP, RasGEF, cGMP-binding, GRAM, and a second cGMP-binding domain. GbpD is related to GbpC, but is much shorter; it begins with the RasGEF-N domain, and lacks the DEP domain. Disruption of the gbpC gene results in loss of all high-affinity cGMP-binding activity present in the soluble cellular fraction. GbpC mRNA levels increase dramatically 8 h after starvation is initiated. GbpA, -B, and -D mRNA levels show less dramatic changes, with gbpA mRNA levels highest 4 h into starvation, gbpB mRNA levels highest in vegetative cells, and gbpD levels highest at 8 h. The identification of these genes is the first step in a molecular approach to studying downstream effects of cGMP signaling in Dictyostelium.

Amino Acid Sequence↗

PubMed2002

A model for cGMP signal transduction in Dictyostelium in perspective of 25 years of cGMP research.

The chemoattactant mediated cGMP response of Dictyostelium cells was discovered about twenty-five years ago. Shortly thereafter, guanylyl cyclases, cGMP-phosphodiesterases and cGMP-binding proteins were detected already in lysates, but the encoding genes were discovered only very recently. The deduced proteins appear to be very different from proteins with the same function in metazoa. In this review we discuss these new findings in perspective of the previously obtained biochemical and functional data on cGMP in Dictyostelium.

Animals↗

PubMed2002

Identification and characterization of two unusual cGMP-stimulated phoshodiesterases in dictyostelium.

Recently, we recognized two genes, gbpA and gbpB, encoding putative cGMP-binding proteins with a Zn(2+)-hydrolase domain and two cyclic nucleotide binding domains. The Zn(2+)-hydrolase domains belong to the superfamily of beta-lactamases, also harboring a small family of class II phosphodiesterases from bacteria and lower eukaryotes. Gene inactivation and overexpression studies demonstrate that gbpA encodes the cGMP-stimulated cGMP-phosphodiesterase that was characterized biochemically previously and was shown to be involved in chemotaxis. cAMP neither activates nor is a substrate of GbpA. The gbpB gene is expressed mainly in the multicellular stage and seems to encode a dual specificity phosphodiesterase with preference for cAMP. The enzyme hydrolyses cAMP approximately 9-fold faster than cGMP and is activated by cAMP and cGMP with a K(A) value of approximately 0.7 and 2.3 microM, respectively. Cells with a deletion of the gbpB gene have increased basal and receptor stimulated cAMP levels and are sporogeneous. We propose that GbpA and GbpB hydrolyze the substrate in the Zn(2+)-hydrolase domain, whereas the cyclic nucleotide binding domains mediate activation. The human cGMP-stimulated cAMP/cGMP phosphodiesterase has similar biochemical properties, but a completely different topology: hydrolysis takes place by a class I catalytic domain and GAF domains mediate cGMP activation.

3',5'-Cyclic-GMP Phosphodiesterases↗