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

Abstract

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.

Figure 1

mDia1 and mDia3 are not required for tropomyosin recruitment to actin filaments in cells at steady state. (a) Expression of actin nucleators in BJeH cells assessed via qPCR. (b,c) mDia1 (b) and mDia3 (c) were depleted separately in BJeH cells via lentiviral-mediated delivery of shRNAmir, knock down was confirmed in 3 different passages via western blotting. (d,e) non-targeting shRNA, mDia1 KD and mDia3 KD cells were fixed and co-stained for (d) F-actin and Tpm3.1/3.2 or (e) Tpm1.6/1.7/2.1 and Tpm4.2. mDia1 KD and mDia3 KD cells made poorer cell-cell junctions (stemless arrow heads) than non-targeting shRNA control cells (stemmed arrow heads). Per group a zoomed in image of one junction is displayed on the right. (f) Partitioning of tropomyosins and actin with F-actin pellet was measured via a biochemical assay in mDia1 and mDia3 KD cells as well as non-targeting shRNA expressing cells. Graph shows data from 3 independent experiments presented as mean ± SD. (g) Protein levels of tropomyosins were measured via western blotting, normalised to α-tubulin and compared between mDia1 KD, mDia3 KD and non-targeting shRNA expressing BJeH cells. Graph shows quantification across 3 independent experiments presented as mean ± SD. **P < 0.01, *P < 0.05. Scale bar shows 10 μm unless stated otherwise.

Figure 2

mDia1 and mDia3 are not required to recover Tpm3.1/3.2 containing stress fibre structures after Lat A washout. (b) mDia1 KD, (c) mDia3 KD and (a) non-targeting shRNA transfected BJeH cells were treated with DMSO or LatA and fixed and stained for F-actin and Tpm3.1/3.2 (CG3) at different time points after wash out (w/o). Directly after LatA treatment Tpm3.1/3.2 co-localises with F-actin foci (stemmed arrows). Non-targeting shRNA and mDia3 KD cells show dorsal stress fibres at 30 min and 1 hour after LatA w/o (stemless arrow heads) while mDia1 KD cells do not. Zoomed in images highlights the reduction in dorsal stress fibres in mDia1 KD cells 30 minutes after LatA wash out.

Figure 3

mDia1 and mDia3 are not required to recover Tpm1.6/1.7/2.1 and Tpm4.2 containing stress fibre structures after Lat A washout. (b) mDia1 KD, (c) mDia3 KD and (a) non-targeting shRNA transfected BJeH cells were treated with DMSO or LatA and fixed and stained for Tpm1.6/1.7/2.1 (tm311) and Tpm4.2 (δ9d) at different time points after wash out (w/o). Directly after LatA treatment Tpm1.6/1.7/2.1 and Tpm4.2 co-localises with F-actin foci (arrows).

Figure 4

Dual knock down of mDia1 and mDia3 has no significant impact on tropomyosin recruitment or elicit significant compensation from other nucleators. (a) mDia1 and mDia3 were depleted separately or simultaneously in BJeH cells via shRNA, knock down was confirmed in via western blotting, using α-tubulin as a loading control. (b,c) non-targeting shRNA, mDia1 KD, mDia3 KD and mDia1 + mDia3 KD cells were fixed and co-stained for (b) F-actin and Tpm3.1/3.2 or (c) Tpm1.6/1.7/2.1 and Tpm4.2. (d) Partitioning of tropomyosins and actin with F-actin pellet was measured via a biochemical assay in mDia1 KD, mDia3 KD and mDia1 + mDia3 KD cells as well as non-targeting shRNA expressing cells. Graph shows data from 3 independent experiments presented as mean ± SD. (e) Protein levels of tropomyosins were measured via western blotting, normalised to α-tubulin and compared between mDia1 KD, mDia3 KD and non-targeting shRNA expressing BJeH cells. Graph shows quantification across 3 independent experiments presented as mean ± SD. (f) A qPCR screen was performed to test for compensation from other nucleators in mDia1 KD, mDia3 KD and mDia1 + mDia3 KD cells, with non-targeting shRNA expressing cells as a control. Graph shows results from 3 independent experiments expressed as mean ± SD. ***P < 0.001, **P < 0.01, *P < 0.05.

Figure 5

Tpm3.1 recruitment to actin filaments is concentration dependent. (a) Genotypes of WT and γ9d +/− MEFs. (b,c) Tropomyosin protein levels were compared between WT and γ9d +/− MEFs via western blotting. (b) Representative tropomyosin blots with α-tubulin as a loading control and (c) quantification across 4 independent experiments presented as mean ± SD. (d and e) Partitioning of tropomyosins and actin with F-actin pellet was measured via a biochemical assay in WT and γ9d +/− MEFs. G indicates globular actin (soluble cytosol fraction), F indicates filamentous actin fraction (pellet). (d) Representative blots and (e) Quantification across 3 independent experiments presented as mean ± SD. ***P < 0.001.