Axon development is regulated at genetic and proteomic interfaces between the integrin adhesome and the RPM-1 ubiquitin ligase signaling hub

Abstract

Integrin signaling plays important roles in development and disease. An adhesion signaling network called the integrin adhesome has been principally defined using bioinformatics and proteomics. To date, the adhesome has not been studied using integrated proteomic and genetic approaches. Here, proteomic studies in C. elegans identified physical associations between the RPM-1 ubiquitin ligase signaling hub and numerous adhesome components including Talin, Kindlin and beta-integrin. C. elegans RPM-1 is orthologous to human MYCBP2, a prominent player in nervous system development associated with a neurodevelopmental disorder. Using neuron-specific, CRISPR loss-of-function strategies, we show that core adhesome components affect axon development and interact genetically with RPM-1. Mechanistically, Talin opposes RPM-1 in a functional 'tug-of-war' on growth cones that is required for accurate axon termination. Thus, our findings orthogonally validate the adhesome via multi-component genetic and physical interfaces with a key neuronal signaling hub and identify new links between the adhesome and brain disorders.

Keywords: adhesome; axon development; axon outgrowth; axon termination; genetics; growth cone; integrin; kindlin; neurobehavioral deficit; neurodevelopmental disorder; proteomics; talin.

Figure 1.. RPM-1 AP-proteomics identifies several integrin adhesome components.

A) Schematic of RPM-1 ubiquitin ligase constructs used for AP-proteomics from C. elegans. Both GS::RPM-1 and GS::RPM-1 LD capture binding proteins (e.g. FSN-1), while GS::RPM-1 LD enriches ubiquitination substrates. B) Example of single AP-proteomics experiment showing individual proteins identified by LC-MS/MS. Shown are results from GS::RPM-1 sample compared with GS::GFP (negative control). Highlighted (shades of green) are integrin receptors, PAT-3/β-Integrin and PAT-2/α-Integrin, and two canonical integrin signaling complexes, UNC-112/Kindlin complex and TLN-1/Talin complex. Additional adhesome components (blue) were identified. Also highlighted are RPM-1 (magenta) and known RPM-1 binding proteins (gray). Dashed line delineates 1.5x fold enrichment. C) Results from single AP-proteomics experiment showing GS::RPM-1 compared to GS::RPM-1 LD ubiquitination substrate ‘trap’. Highlighted are previously validated substrates (CDK-5 and UNC-51, purple). D-F) Examples of LC-MS/MS peptide spectrum for D) PAT-3/β-Integrin, E) TLN-1/Talin, and F) UNC-112/Kindlin.

Figure 2.. Two core adhesome signaling complexes and numerous adhesome components with links to human neurobehavioral deficits are present in RPM-1 proteomics.

A) Illustration of integrin signaling with the two transmembrane receptors PAT-3 β-Integrin and PAT-2 α-Integrin (green), the TLN-1 Talin complex (dark green) and the UNC-112 Kindlin complex (light green). B) Summary of RPM-1 AP-proteomics data from C. elegans integrated with a computationally predicted protein-protein interaction network (lines) and genetic links to human neurobehavioral abnormalities (orange halo). Adhesome components enriched in RPM-1 AP-proteomics are highlighted in color with increasing circle size denoting significance (GS::RPM-1 test samples versus GS::GFP negative controls). Highlighted (shades of green) are Integrins, UNC-112 Kindlin complex and TLN-1 Talin complex. Also shown are additional adhesome components detected (blue) or absent (gray) in RPM-1 AP-proteomics. Orthologous human protein annotated in brackets. Data is presented from 7 independent RPM-1 AP-proteomics experiments. Significance determined using Student’s t-test. ns = not significant

Figure 3.. Adhesome components are expressed in C. elegans mechanosensory neurons and localized to axons.

A-C) Alpha-fold predictions showing adhesome signaling components that were GFP-tagged using CRISPR engineering in C. elegans. Structural predictions indicate GFP is not likely to interfere with protein folding. A) C. elegans PAT-3::GFP, B) UNC-112::GFP, and C) GFP::TLN-1. D) Schematic of C. elegans mechanosensory neurons highlighting region imaged to visualize axon termination sites for PLM neurons (light gray box). E) Representative images showing that PAT-3::GFP, UNC-112::GFP and GFP::TLN-1 are expressed in PLM neurons and localized to axons. Pmec-7::RFP (jsIs973) labels mechanosensory neurons. F) Schematic highlights cell body and initial axon segment (light gray box) of PLM neurons. G) Confocal images show PAT-3::GFP, UNC-112::GFP and GFP::TLN-1 primarily localized to PLM axon. For E and G, note that integrin components also show prominent expression in muscles. Scale bars 10μm.

Figure 4.. Impairing adhesome components with a CRISPR-based, cell-specific protein degradation system results in premature axon termination.

A) Illustration of CRISPR-based cell-specific protein degradation system used to impair integrin adhesome components in C. elegans mechanosensory neurons (mecDEG). Adapted from Wang et al. 2017. B) Schematic of mechanosensory neurons with imaged regions highlighted (light gray boxes). C) Representative images of PLM axons for indicated genotypes visualized using Pmec-7::RFP (jsIs973). Premature axon termination defects (arrows) occur when mecDEG targets GFP that is CRISPR engineered onto adhesome components (PAT-3::GFP, UNC-112::GFP and GFP::TLN-1). Vulva (asterisks) used as anatomical reference point for premature termination. D) Quantitation of premature axon termination defects in PLM neurons for indicated genotypes. Note premature axon termination occurs with mecDEG targeting of adhesome components and in tln-1 (ok1648) hypomorphic mutants. Means (bars) are shown for 5 or more counts (black dots) with 20 or more animals/count for each genotype. Error bars indicate SEM. Significance assessed using Student’s t-test with Bonferroni correction for multiple comparisons. ***p<0.001. Scale bar 10μm.

Figure 5.. Opposing genetic interactions between adhesome components and rpm-1 are required for accurate axon termination.

A) Schematic of C. elegans mechanosensory neurons with imaged regions highlighted (light gray boxes). B) Representative images of PLM axons for indicated genotypes visualized using Pmec-7::RFP (jsIs973). rpm-1 mutants display failed axon termination defects (arrowhead) and mecDEG targeting of adhesome components (PAT-3::GFP, UNC-112::GFP and GFP::TLN-1 CRISPR) results in premature termination defects (arrows). rpm-1 double mutants with adhesome component degradation (e.g. rpm-1; PAT-3::GFP + mecDEG) predominantly display failed termination defects. Vulva (asterisks) used as anatomical reference point for premature termination. C) Quantitation of failed axon termination defects (dark gray) and premature termination defects (light gray) in PLM neurons for indicated genotypes. Note premature termination defects are strongly suppressed in double mutants lacking rpm-1 and adhesome components. Mean (bars) are shown for 5 or more counts (20 or more animals/count) for each genotype. Error bars indicate SEM. Significance assessed using Student’s t-test with Bonferroni correction. ***p<0.001. Scale bars 10μm.

Figure 6.. Developmental time-course studies demonstrate that tln-1 and rpm-1 affect axon termination by engaging in a genetic ‘tug-of-war’ over growth cones.

A) Timeline of C. elegans larval development and key time points evaluated. B) Representative images of PLM axons for indicated genotypes visualized using Pmec-7::RFP (jsIs973). Shown are axonal growth cones (brackets), premature termination sites (arrows) and failed termination defects (arrowheads). Note double mutants (rpm-1; GFP::TLN-1 + mecDEG) display failed growth cone collapse with persistent, enlarged growth cones. In double mutants, growth cones are initially in locations corresponding to premature axon termination (1 to 16h PH) but failed axon termination becomes the primary phenotype as development progresses (44h PH). Note AVM cell body is only visible at 44h PH on one side of the animal. C) Summary of quantitative results for growth cone frequency during development for indicated genotypes. D-E) Quantitative analysis of growth cone frequency at D) 7h PH and E) 16h PH. F) Quantitation of growth cone size at 3h PH for indicated genotypes. G) Quantitation of failed axon termination defects (dark gray) and premature termination defects (light gray) in PLM neurons for indicated genotypes at specified time points in development. For C-G, minimum of 21 PLM neurons scored for each time point and genotype. Mean (square, bar or line) are shown for each genotype. For F, dots represent a single animal. Error bars indicate SEM. For C-E and G, significance assessed using Fisher's exact test. For F, significance assessed using Student’s t-test with Bonferroni correction for growth cone size. ns=not significant, *p<0.05, **p<0.01, ***p<0.001. Scale bars are 5μm (1-7h PH, white bars) or 10μm (16-44h PH, teal bars).