Figure 1.
Isolation of ventral plasma membrane complexes.
A) Workflow for the MS-based proteomics analysis of isolated ventral plasma membrane complexes. B) Schematic of the method to lyse cells after chemical cross-linking.
Figure 2.
Changes to actin stress fibres and vinculin-containing ACs upon microtubule disruption.
Serum starved HFFs were treated with DMSO and nocodazole (Noc) for 4 hours and fixed either with A) paraformaldehyde supplemented with glutaraldehyde to preserve microtubules, and immunostained for actin (green), vinculin (red) and tubulin (blue) or with B) paraformaldehyde and immunostained for vinculin. Vinculin staining was quantified as a percentage of the total cell area and displayed as a Beeswarm-Boxplot (line, median; box, interquartile range; whiskers, maximum and minimum; n = 51 to 55 cells).
Figure 3.
Changes in isolated ventral plasma membrane complexes upon microtubule disruption.
A) Serum-starved HFFs plated on either fibronectin (FN) or poly-D-lysine (PDL) were treated with DMSO or nocodazole (Noc) for 4 hours, and ventral plasma membrane complexes were isolated for Western blotting for AC components (α5-integrin, talin, vinculin, paxillin, FAK, pY397-FAK and ILK) and non-AC components (BAK and transferrin receptor). B) Protein bands were quantified and normalised to FN, DMSO (mean ± SD; n = 3).
Figure 4.
Unsupervised hierarchical clustering analysis of ventral plasma membrane complexes upon microtubule disruption.
Complete output of unsupervised hierarchical clustering analysis of proteins identified by MS. Correlations at selected dendrogram nodes are indicated. Three clusters were identified: i) proteins present in equal abundance in both fibronectin (FN) and poly-D-lysine (PDL) samples; ii) proteins enriched in PDL; and iii) proteins enriched in FN. Clusters were subjected to pathway enrichment analysis and significantly enriched KEGG pathway terms (Bonferroni-corrected p-value<0.05) are displayed as pie charts.
Figure 5.
Identification of statistically enriched adhesion complex proteins.
Volcano plot of QSpec output, log10(Bayes factor) and ln(fold change, FN/PDL). Horizontal dashed line corresponds to Bayes factor of 10 and vertical dashed line corresponds to a fold change (FN/PDL) of 3. Proteins statistically enriched to FN lie in the top right quadrant (log10(Bayes factor) ≥1, and ln(fold change, FN/PDL) ≥1.1), which represents a conservative false discovery rate estimate of less than 5% [41]. Proteins statistically enriched to FN were subjected to pathway enrichment analysis and significantly enriched KEGG pathway terms (Bonferroni-corrected p-value<0.05) are displayed as a pie chart. FN, fibronectin; PDL, poly-D-lysine.
Figure 6.
Unsupervised hierarchical clustering analysis of proteins statistically enriched to FN.
Heat map dendogram displaying the hierarchical clustering of the fold change of FN, Noc relative to FN, DMSO. Three clusters were identified and analysed by gene ontology enrichment analysis to identify significantly enriched ‘cellular component’ terms (Bonferroni-corrected p-value<0.05). In general, the three clusters were: i) proteins that decreased in abundance upon nocodazole treatment and were enriched in ECM-related terms; ii) proteins that did not change and were enriched in ECM-related terms; and iii) proteins that increased and were enriched in AC-related terms. FN, fibronectin; PDL, poly-D-lysine; Noc, nocodazole.
Figure 7.
Protein-protein interaction network analysis of FN-enriched ventral plasma membrane complexes.
Node colour was determined by a blue-red colour gradient corresponding to the log2(Noc/DMSO) values, with red nodes indicating an increase and blue nodes a decrease in protein abundance upon nocodazole treatment. Nodes were sorted according to their cell localisation and node size was proportional to the normalised spectral counts. The network was ordered according to the interaction network distance relative to the plasma membrane integrins, 1-hop, 2-hop, etc. Corresponding gene names are displayed underneath each node for clarity. FN, fibronectin; Noc, nocodazole.
Figure 8.
Validation of changes in AC components observed by MS.
A) Isolated ACs were probed by Western blotting for αv-integrin, tensin-1, filamin A, ELKS and PDLIM5. B) To investigate the pattern of changes to integrins upon loss of microtubules further, HFFs treated with DMSO or nocodazole (Noc) were fixed and immunostained for α5-integrin, αvβ3-integrin and vinculin and quantified as a percentage of the total cell area (line, median; box, interquartile range; whiskers, maximum and minimum; ****p<0.0001, Student's t-test; n = 54). C) Immunostained cells showing the cellular distribution of α5-integrin (red), αvβ3-integrin (green) and vinculin (blue). Blue and yellow insets are 4× enlargements of corresponding boxes.
Figure 9.
Changes to the maturation state of ACs upon microtubule disruption.
Serum-starved HFFs were treated with DMSO and nocodazole (Noc) for 4 hours, fixed and immunostained for A) α5-integrin (red), tensin-1 (green) and vinculin (blue) and B) α5-integrin (red), vinculin (green) and pTyr (blue). Blue and yellow insets are 4× enlargements of corresponding boxes.
Figure 10.
Force-dependence of the microtubule-induced AC modulation.
Serum-starved HFFs were treated with DMSO, Y-27632, nocodazole (Noc) or a combination of Noc and Y-27632, fixed and immunostained for α5-integrin (red), vinculin (green) and actin (blue). Blue and yellow insets are 4× enlargements of corresponding boxes.