Tropomyosin concentration but not formin nucleators mDia1 and mDia3 determines the level of tropomyosin incorporation into actin filaments

The majority of actin filaments in human cells exist as a co-polymer with tropomyosin, which determines the functionality of actin filaments in an isoform dependent manner. Tropomyosin isoforms are sorted to different actin filament populations and in yeast this process is determined by formins, however it remains unclear what process determines tropomyosin isoform sorting in mammalian cells. We have tested the roles of two major formin nucleators, mDia1 and mDia3, in the recruitment of specific tropomyosin isoforms in mammals. Despite observing poorer cell-cell attachments in mDia1 and mDia3 KD cells and an actin bundle organisation defect with mDia1 knock down;depletion of mDia1 and mDia3 individually and concurrently did not result in any significant impact on tropomyosin recruitment to actin filaments, as observed via immunofluorescence and measured via biochemical assays. Conversely, in the presence of excess Tpm3.1, the absolute amount of Tpm3.1-containing actin filaments is not fixed by actin filament nucleators but rather depends on the cell concentration of Tpm3.1. We conclude that mDia1 and mDia3 are not essential for tropomyosin recruitment and that tropomyosin incorporation into actin filaments is concentration dependent

Supplementary Figure 4 from Targeted single cell isolation of spontaneously escaping live melanoma cells for comparative transcriptomics

Supplementary Figure 4 shows an Epithelial cohort gallery</p

Supplementary Video 1A from Targeted single cell isolation of spontaneously escaping live melanoma cells for comparative transcriptomics

Supplementary Video 1A shows representative daily images of a vehicle-treated spheroid. Scale bar: 200 µm, number at top left: days post-implantation.</p

Supplementary Figure 8 from Targeted single cell isolation of spontaneously escaping live melanoma cells for comparative transcriptomics

Supplementary Figure 8 shows a summary of GSEA pathway analyses using Hallmarks gene set showing top 25 enriched pathways for each comparison</p

Supplementary Video 2B from Targeted single cell isolation of spontaneously escaping live melanoma cells for comparative transcriptomics

Supplementary Video 2B shows representative images of a Sulfasalazine-treated spheroid. Scale bar: 200 µm, number at top left: days post-implantation.</p

Supplementary Figure 5 from Targeted single cell isolation of spontaneously escaping live melanoma cells for comparative transcriptomics

Supplementary Figure 5 shows an Escaping cell cohort gallery</p

Supplementary Figure 9 from Targeted single cell isolation of spontaneously escaping live melanoma cells for comparative transcriptomics

Supplementary Figure 9 shows Escaped cell analyses for naltrindole and sulfasalazine spheroid assays</p

Supplementary Table 4 from Targeted single cell isolation of spontaneously escaping live melanoma cells for comparative transcriptomics

Supplementary Table 4 shows Differentially expressed genes for amoeboid vs. escaping analysis</p

Supplementary Table 6 from Targeted single cell isolation of spontaneously escaping live melanoma cells for comparative transcriptomics

Supplementary Table 6 shows Sorting events for each cell included in the final data set</p