Regulation of CNS and motor axon guidance in Drosophila by the receptor tyrosine phosphatase DPTP52F.

Receptor-linked protein tyrosine phosphatases (RPTPs) regulate axon guidance and synaptogenesis in Drosophila embryos and larvae. We describe DPTP52F, the sixth RPTP to be discovered in Drosophila. Our genomic analysis indicates that there are likely to be no additional RPTPs encoded in the fly genome. Five of the six Drosophila RPTPs have C. elegans counterparts, and three of the six are also orthologous to human RPTP subfamilies. DPTP52F, however, has no clear orthologs in other organisms. The DPTP52F extracellular domain contains five fibronectin type III repeats and it has a single phosphatase domain. DPTP52F is selectively expressed in the CNS of late embryos, as are DPTP10D, DLAR, DPTP69D and DPTP99A. To define developmental roles of DPTP52F, we used RNA interference (RNAi)-induced phenotypes as a guide to identify Ptp52F alleles among a collection of EMS-induced lethal mutations. Ptp52F single mutant embryos have axon guidance phenotypes that affect CNS longitudinal tracts. This phenotype is suppressed in Dlar Ptp52F double mutants, indicating that DPTP52F and DLAR interact competitively in regulating CNS axon guidance decisions. Ptp52F single mutations also cause motor axon phenotypes that selectively affect the SNa nerve. DPTP52F, DPTP10D and DPTP69D have partially redundant roles in regulation of guidance decisions made by axons within the ISN and ISNb motor nerves.

Complex genetic interactions among four receptor tyrosine phosphatases regulate axon guidance in Drosophila.

Four receptor-linked protein tyrosine phosphatases are selectively expressed on central nervous system axons in the Drosophila embryo. Published data show that three of these (DLAR, DPTP69D, DPTP99A) regulate motor axon guidance decisions during embryonic development. Here we examine the role of the fourth neural phosphatase, DPTP10D, by analyzing double-, triple-, and quadruple-mutant embryos lacking all possible combinations of the phosphatases. This analysis shows that all four phosphatases participate in guidance of interneuronal axons within the longitudinal tracts of the central nervous system. In the neuromuscular system, DPTP10D works together with the other three phosphatases to facilitate outgrowth and bifurcation of the SNa nerve, but acts in opposition to the others in regulating extension of ISN motor axons past intermediate targets. Our results provide evidence for three kinds of genetic interactions among the neural tyrosine phosphatases: partial redundancy, competition, and collaboration.

L-type Ca2+ channels and purinergic P2X2 cation channels participate in calcium-tyrosine kinase-mediated PC12 growth cone arrest.

During development and regeneration of the nervous system, growth cones of the various nerve cells navigate and direct neurite elongation by detecting and responding to cues in the environment. To investigate changes in growth cone behaviour due to calcium influx we used nerve growth factor (NGF)-induced growth cones of PC12 (rat pheochromocytoma cells) cells as a model. High external concentrations of potassium and ATP depress growth cone motility, induce club-shaped growth cones and reduce filopodia length and the number and relative F-actin contents of single growth cones (r.a.c.), respectively. The cellular responses are mediated by a sustained increase in the intracellular free Ca2+ concentrations ([Ca2+]i) as monitored by calcium-sensitive fluorescent dyes and confocal microfluorimetry. The responses are not detectable in the presence of the protein tyrosine kinase inhibitor genistein. Immunocytochemistry revealed an increased level of tyrosine-phosphorylated proteins in cell bodies and growth cones but not in cell nuclei. Paxillin, a cytoskeleton-associated protein located in neurites and growth cones, was detected among the phosphotyrosine proteins. The sustained (> 30 s) Ca2+ influx through voltage-gated L-type but not N- or P-type Ca2+ channels induced the F-actin loss and tyrosine phosphorylation. Ca2+ entry through P2X2 ligand-gated channels caused the same effects. Our data suggest the following mechanism: increased [Ca2+]i levels activate tyrosine kinases located close to the ion channels which then leads to changes in morphology due to tyrosine phosphorylation of proteins, e. g. paxillin.

Differential cytoskeletal changes during growth cone collapse in response to hSema III and thrombin.

Growth cones are known as the site of action of many factors that influence neurite growth behavior. To assess how different collapsing agents influence the growth cone cytoskeleton, we used recombinant human Semaphorin III (hSema III) and the serine protease thrombin. Embryonic chick dorsal root ganglion neurons showed a dramatic depolymerization of actin filaments within 5 min upon hSema III exposure and virtually no influence on microtubules (MT). Only at later time points (20-30 min) was the polymerization/depolymerization rate of MT significantly affected. Thrombin induced a morphologically and kinetically similar growth cone collapse. Moreover, thrombin induced an early and selective depolymerization of dynamic MT, accompanied by the formation of loops of stable MT bundles. Selective changes in the phosphorylation pattern of tau were associated with microtubule assembly in thrombin-induced responses. Our data provide evidence that different signal transduction pathways lead to distinct changes of the growth cone cytoskeleton.

Quantitative estimation of F-actin in single growth cones.

Growth cones at distal ends of elongating neurites are characterized by a bunch of motile filopodia. Filamentous actin (F-actin) is the supporting cytoskeletal structure of growth cone filopodia. Normal growth cone motility requires balanced polymerization and depolymerization rates of F-actin. If this balance is disturbed, growth cone shape is altered and extension may fail. Image acquisition by confocal scanning microscopy was used as a very efficient tool to optically isolate single growth cones from the rest of the cell to study morphological and physiological behavior. The relative F-actin content (r.a.c.) of a single growth cone area was defined as a parameter describing different growth cone states. To estimate r.a.c., a double-labeling technique was applied. F-actin was selectively labeled by fluorescent rhodamine-conjugated phalloidin and total protein was unspecifically labeled by 5-(4, 6-dichlorotriazin-2-yl)aminofluorescein (DTAF). The r.a.c. was calculated by rationing and averaging digitized rhodamine and DTAF fluorescence of single growth cone areas. Subsequently, r.a.c. was used as a numeric descriptor of the variable F-actin underlying morphological structures of growth cones. The method allowed an analysis of local changes in growth cone morphology measured as a change in F-actin due to signaling events. It can be used to quantify ligand-receptor effects at subcellular areas of intact cells.

Bradykinin-induced collapse of rat pheochromocytoma (PC12) cell growth cones: a role for tyrosine kinase activity.

Pathfinding of growing nerve processes is guided by extracellular guidance cues. Here we report growth cone collapse of NGF-differentiated PC12 cells in culture evoked by the neuropeptide bradykinin. The growth cone response is mediated by B2 bradykinin receptors. Two different effects were distinguished. (1) Disappearance of filopodia occurred together with a loss of fibrillar actin (F-actin) in the growth cones at picomolar concentrations of bradykinin. The relative F-actin content was measured by means of rhodamine-phalloidin fluorescence using confocal microscopy. (2) Bradykinin-induced Ca2+ release and retraction of the neurite occurred at nanomolar concentrations. Ca2+ responses at single growth cones were measured using a 1:1 mixture of fura-red and fluo-3 Ca2+-sensitive dyes. The [Ca2+]i rise is not a prerequisite for the observed effects, because F-actin loss and retraction occurred during inhibition of Ca2+ responses. In contrast, inhibition by genistein pointed to a tyrosine kinase activity in the bradykinin-evoked cellular events. Subsequent analysis of phosphotyrosine proteins revealed that bradykinin stimulated tyrosine phosphorylation of the cytoskeleton-associated protein paxillin and the nonreceptor protein tyrosine kinase pp60(c-src). Paxillin and pp60(c-src) co-precipitated after bradykinin treatment. Immunostaining experiments showed punctate distribution of paxillin along PC12 neurites and in growth cones. Taken together, our data suggest that pp60(c-src) and paxillin are putative components of the intracellular signaling pathway of bradykinin-mediated neurite retraction and provide evidence for a crosstalk between G-protein- and tyrosine kinase-dependent pathways in these cellular events.

Detection of a trigger zone of bradykinin-induced fast calcium waves in PC12 neurites.

Bradykinin and caffeine were used as two different agonists to study inositol 1,4,5-trisphosphate (IP3)-sensitive and caffeine/ryanodine-sensitive intracellular Ca2+ release in the outgrowing neurites of nerve-growth-factor (NGF)-treated rat phaeochromocytoma cells (PC12). Changes in neuritic intracellular free Ca2+ ([Ca2+]i) in single cells were measured after loading with a 1:1 mixture of the acetoxymethyl (AM) ester of the Ca2+-sensitive dyes Fura-red and Fluo-3, in combination with confocal microscopy. Bradykinin-induced Ca2+ release was blocked by U73211, a specific phospholipase C inhibitor. Caffeine-induced Ca2+ release was very low in neurites at rest. It increased after the cells were preloaded with Ca2+. The Ca2+ signal evoked at high concentrations of bradykinin (>500 nM) arose from a trigger zone in the proximal part of the neurite, as a bi-directional wave towards the growth cone and cell body. The speed of neuritic Ca2+ waves was reduced in cells loaded with the Ca2+ chelator 1, 2-bis(2-aminophenoxy)ethane-tetraacetic acid/AM. Preloading of Ca2+ stores led to increased bradykinin-induced Ca2+ release, as seen for caffeine, and faster Ca2+ wave speeds. Caffeine evoked a simultaneous [Ca2+]i rise along the neurites of Ca2+ preloaded cells. Higher Ca2+ signal amplitudes and faster Ca2+ wave speeds, but no longer-lasting IP3-induced [Ca2+]i signals, correlated with increased caffeine-induced Ca2+ release in the neurites. At low concentrations of bradykinin (<1.0 nM), the Ca2+ signals ceased to propagate as complete Ca2+ waves. Instead, repetitive stochastic Ca2+ release events (neuritic Ca2+ puffs) were observed. Neuritic Ca2+ puffs spread across only a few microns, at a slower speed than neuritic Ca2+ waves. These Ca2+ puffs represent elementary Ca2+ release units, whereby the released Ca2+ ions form these elementary events into the shape of a Ca2+ wave.