20 research outputs found
Growth cone responses to growth and chemotropic factors
Abstract During nervous system development axons reach their target areas under the influence of numerous guidance cues that affect rate and direction of growth. This report addresses the unsettled question of whether and to what extent growth velocity and turning responses (attraction, repulsion) are interdependent. We exposed individual growth cones of fetal rat dorsal root ganglion neurons in culture asymmetrically to gradients of seven different factors and recorded their growth rates and turning angles. Growth cones exhibited divergent patterns of turning and growth responses. For example, hepatocyte growth factor (HGF), insulin-like growth factor-1 (IGF-1) and thrombin all promoted growth, but HGF was a powerful attractant, thrombin a potent repellent and IGF-1 did not elicit turning. Galanin and neuropeptide Y also affected growth and â or turning differentially. Finally, nerve growth factor in the culture medium not only inhibited the turning responses to HGF, but also converted growth promotion of HGF and IGF-1 into inhibition. Overall, our studies indicate that: (i) turning and advance are regulated independently, except that strong attractive or repulsive responses generally are accompanied by growth promotion; (ii) asymmetric growth factor application per se does not elicit attraction; (iii) regulation of the two parameters may occur through a single receptor; and (iv) the effects of combined growth factors may not be additive and can be inhibitory
Functional Complexity of the Axonal Growth Cone: A Proteomic Analysis
The growth cone, the tip of the emerging neurite, plays a crucial role in establishing the wiring of the developing nervous system. We performed an extensive proteomic analysis of axonal growth cones isolated from the brains of fetal Sprague-Dawley rats. Approximately 2000 proteins were identified at â„99% confidence level. Using informatics, including functional annotation cluster and KEGG pathway analysis, we found great diversity of proteins involved in axonal pathfinding, cytoskeletal remodeling, vesicular traffic and carbohydrate metabolism, as expected. We also found a large and complex array of proteins involved in translation, protein folding, posttranslational processing, and proteasome/ubiquitination-dependent degradation. Immunofluorescence studies performed on hippocampal neurons in culture confirmed the presence in the axonal growth cone of proteins representative of these processes. These analyses also provide evidence for rough endoplasmic reticulum and reveal a reticular structure equipped with Golgi-like functions in the axonal growth cone. Furthermore, Western blot revealed the growth cone enrichment, relative to fetal brain homogenate, of some of the proteins involved in protein synthesis, folding and catabolism. Our study provides a resource for further research and amplifies the relatively recently developed concept that the axonal growth cone is equipped with proteins capable of performing a highly diverse range of functions
Hippocampal pyramidal neurons and/or their axonal growth cones labeled with antibodies to Golgi proteins (top row).
<p>The antibody specificities are indicated above. cis/medial/trans, known Golgi location of the antigens. pk, neuronal perikaryon; large arrow, axonal growth cone; small arrows, reticular structures.</p
Proteins with the most abundant spectral counts under the following GO Terms: (A) âGrowth Coneâ (cytoplasmic component, CC) and âAxon Guidanceâ (biological process, BP); (B) âActin Cytoskeletal Organizationâ (BP) and âRegulation of Microtubule Polymerization/Depolymerizationâ (BP); (C) âVesicle Targeting/Dockingâ (BP) and âVesicle Fusionâ (BP); and (D) âHexose Catabolic Processâ (BP) and âATP Biosynthetic Processâ (BP).
<p>Note that scales are different for each panel.</p
KEGG Pathways in Growth Cones.
<p>(20 pathways identified with the highest statistical significance).</p>aâd<p>: Pathways not included for further consideration because they share elements with several other pathways, and, especially, because their key identifying proteins [FcÎł receptor (a); claudins, occludins (b); insulin receptor (c); ErbB (d)] were not detected.</p
Abundance of non-typifying proteins in fetal brain fractions including GCPs, as determined by Western blot.
<p><b>A</b> shows the relative abundance in fetal brain homogenate (H), low-speed supernatant (LSS) and GCPs (40 ”g protein/lane) of six non-typifying proteins, a cytosolic marker (Ldh) and a growth cone marker (Gap43). Hsp90 is Hsp90ab1. <b>B</b> illustrates the presence of immunoreactivity of six additional proteins in fetal brain LSS and GCPs (equal amounts of protein loaded). <b>C</b> shows the quantitative analysis of the data in A. Net fluorescence intensities of the bands shown in A were normalized to H for each experiment and then averaged (experiments done in triplicate). The resulting relative density units shown equal the fold increase in immunoreactivity relative to H (means ± s.e.m.). p values are shown where GCP enrichment over H was significant.</p
Frequencies of the most abundant spectra identifying proteins under the following GO Terms: (A) âTranslationâ and âTranslational Initiationâ (biological process, BP); (B) âER to Golgi Vesicle-Mediated Transportâ (BP)/âGolgi Vesicle Buddingâ (BP); (C) âProtein Foldingâ (BP); and (D) âUbiquitin Protein Ligase Activityâ (BP); âProteasomal Protein Catabolic Process (BP)/âProteasome Complexâ (cellular component, CC).
<p>Frequencies of the most abundant spectra identifying proteins under the following GO Terms: (A) âTranslationâ and âTranslational Initiationâ (biological process, BP); (B) âER to Golgi Vesicle-Mediated Transportâ (BP)/âGolgi Vesicle Buddingâ (BP); (C) âProtein Foldingâ (BP); and (D) âUbiquitin Protein Ligase Activityâ (BP); âProteasomal Protein Catabolic Process (BP)/âProteasome Complexâ (cellular component, CC).</p
Functional annotation clusters (âBiological Processâ, BP) in the growth cone proteome (DAVID).
<p>The 13 most enriched clusters and characteristic examples of their GO Terms are shown, together with their enrichment scores, in the colored boxes. Bars show GO Term enrichment (burgundy) and % count (number of included protein species relative to total number of protein species in GCPs; blue). Benjamini scores are shown on the far right. The DAVID annotation cluster analysis left out a number of highly enriched GO Terms that were grouped functionally (AâE) and are listed also.</p
Functional annotation (âCellular Componentâ) analysis of the growth cone proteome.
<p>GO Terms are listed in functional groups. Enrichment values (relative to random expression) are shown in red; % count (grey) indicates the number of protein species associated with each GO Term relative to the total number of protein species in net GCPs. For the enrichment values, p<0.01; Benjamini scores <0.05.</p