Figure 1.
Relative distribution of assigned spectra between growth cones (blue) and background (green).
Identified proteins (listed by gene name) are grouped as “markers” of axonal (A) growth cones, axonal or dendritic (D) cytoskeleton, neurons (N), glia (G), the nucleus or serum. Total numbers of identifying spectra detected for each protein are indicated on the right.
Figure 2.
The 43 highest-ranked proteins based on identifying spectra (≥400) detected in the net GCP preparation.
The proteins are grouped functionally. CRMP, collapsing response mediator proteins. Percent values indicate the cumulative number of spectra identifying the proteins shown in each group relative to the total number of spectra detected in growth cones.
Figure 3.
Functional annotation (“Cellular Component”) analysis of the growth cone proteome.
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.
Figure 4.
Functional annotation clusters (“Biological Process”, BP) in the growth cone proteome (DAVID).
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 (A–E) and are listed also.
Table 1.
Excluded Annotation Clusters* and Sample GO Terms (BP FAT).
Table 2.
KEGG Pathways in Growth Cones.
Figure 5.
KEGG pathways in axonal growth cones.
The five relevant KEGG pathway categories and their subcategories (enriched subcategories are shaded) are listed on the left. The numbers following each subcategory title indicate the number of pathways detected in GCPs/total number of identified pathways in mammals. Bars indicate the average enrichment factor for the subcategory (green), the enrichment factor for each pathway (blue), and the number of protein species of the pathway detected in GCPs (ochre). P values (<0.05) are shown on the right.
Figure 6.
Proteins with the most abundant spectral counts under the following GO Terms: (A) “Growth Cone” (cytoplasmic component, CC) and “Axon Guidance” (biological process, BP); (B) “Actin Cytoskeletal Organization” (BP) and “Regulation of Microtubule Polymerization/Depolymerization” (BP); (C) “Vesicle Targeting/Docking” (BP) and “Vesicle Fusion” (BP); and (D) “Hexose Catabolic Process” (BP) and “ATP Biosynthetic Process” (BP).
Note that scales are different for each panel.
Figure 7.
Frequencies of the most abundant spectra identifying proteins under the following GO Terms: (A) “Translation” and “Translational Initiation” (biological process, BP); (B) “ER to Golgi Vesicle-Mediated Transport” (BP)/“Golgi Vesicle Budding” (BP); (C) “Protein Folding” (BP); and (D) “Ubiquitin Protein Ligase Activity” (BP); “Proteasomal Protein Catabolic Process (BP)/“Proteasome Complex” (cellular component, CC).
Figure 8.
Frequencies of the most abundant spectra identifying proteins under the following GO Terms: “Cellular Response to Oxidative Stress” (BP) and “Terpenoid/Sterol Biosynthetic Processes” (BP).
Figure 9.
Abundance of non-typifying proteins in fetal brain fractions including GCPs, as determined by Western blot.
A shows the relative abundance in fetal brain homogenate (H), low-speed supernatant (LSS) and GCPs (40 µg protein/lane) of six non-typifying proteins, a cytosolic marker (Ldh) and a growth cone marker (Gap43). Hsp90 is Hsp90ab1. B illustrates the presence of immunoreactivity of six additional proteins in fetal brain LSS and GCPs (equal amounts of protein loaded). C 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.
Figure 10.
Hippocampal pyramidal neurons and/or their axonal growth cones labeled with antibodies to proteins involved in protein synthesis (top row).
The antibody specificities are indicated above. Dual-fluorescence images including labeling for filamentous actin are shown in the bottom row. pk, neuronal perikaryon; large arrows, axonal growth cones; small arrows, reticular structures.
Figure 11.
Hippocampal pyramidal neurons and their axonal growth cones labeled with antibodies to proteins involved in protein folding (top row).
The antibody specificities are indicated above. Additional filamentous-actin label is shown in the bottom row. pk, neuronal perikaryon; large arrows, axonal growth cones. Small arrows indicate: left, reticular structures (Pdia6); right, edge labeling (Hsp90ab1).
Figure 12.
Hippocampal pyramidal neurons and/or their axonal growth cones labeled with antibodies to Golgi proteins (top row).
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.
Figure 13.
Hippocampal pyramidal neurons and/or their axonal growth cones labeled with antibodies to proteins involved in proteasomal degradation (top row).
The antibody specificities are indicated above. Additional filamentous-actin label is shown in the bottom row. pk, neuronal perikaryon; large arrow, axonal growth cone; small arrow, Psma-positive, large puncta.
Figure 14.
Hippocampal pyramidal neurons and their axonal growth cones labeled with antibodies to proteins involved in functions of the nucleus (top row).
The antibody specificities are indicated above. Additional filamentous-actin label is shown in the bottom row. Large arrows, axonal growth cones.
Table 3.
Proteome of the Growth Cone versus Transcriptome of the Growing Axon.