Specialization of actin isoforms derived from the loss of key interactions with regulatory factors (2022)
Figure 2

Figure 2 - Effects of silent mutations on actin expression levels, cell viability and cytoskeletal organization.

In this figure, the shape of the dots allows to identify the strains on the different graphs (circles for Sc, squares for ScNI, triangles for Sc[Ca], inversed triangles for Sc[Sp] and diamonds for Sc[At]). The color of the dots indicates the percentage of identity of the nucleotide sequences to the actin gene of S. cerevisiae, ranging from 100% (blue) to 76% (orange).

A Actin expression levels shown by western blotting for strains expressing S. cerevisiae's actin protein from various coding sequences, with tubulin (Tub1p) as a loading control.

B Quantification of actin expression levels, showing a decrease when more silent mutations are present. Data are presented as mean +/- SD (n = 4 for Sc, n = 8 for ScNI; n = 12 for Sc[Ca], Sc[Sp] and Sc[At]; 2 biological replicates with n/2 technical replicates each). *P<0.05 (Brown-Forsythe and Welch ANOVA tests, with Dunnett's T3 multiple comparisons tests).

C Doubling times of yeast strains cultures, grown at 25 ºC in YPD medium. Data are presented as mean +/- SD (n = 6 for Sc, n = 3 for ScNI, Sc[Ca], Sc[Sp] and Sc[At]; technical replicates). *P<0.05 (Brown-Forsythe and Welch ANOVA tests, with Dunnett's T3 multiple comparisons tests).

D Level of actin expression as a function of growth constant does not show any clear correlation. Rather, there is an apparent level of actin expression (0.25 < expression < 0.35) below which growth rates drastically reduce. Data are presented as mean +/- SD (for actin expression values, n = 4 for Sc, n = 8 for ScNI; n = 12 for Sc[Ca], Sc[Sp] and Sc[At]; 2 biological replicates with n/2 technical replicates each; for growth constants, n = 6 for Sc, n = 3 for ScNI, Sc[Ca], Sc[Sp] and Sc[At]; technical replicates). r is a Pearson correlation coefficient considered non-significant if its two-tailed p-value is > 0.05.

E Phalloidin staining depicting F-actin organization. Images are maximum intensity projections of 3D stacks. Scale bar: 3 µm.

F In vivo actin network deviation indexes, defined to evaluate the patch-cable balance compared to S. cerevisiae haploid cells (value is 0 in S. cerevisiae's cells, 1 when cells contain only actin patches and -1 when cells contain only cables). Data are presented as mean +/- SD (n = 30 for all conditions). ***P<0.001 (Brown-Forsythe and Welch ANOVA tests, with Dunnett's T3 multiple comparisons tests).

G In vivo actin network deviation indexes as a function of actin expression levels does not show any clear correlation. Rather, we observe a threshold of actin expression levels (0.25 < expression < 0.35) below which actin cytoskeleton organization is affected. Data are presented as mean +/- SD (for actin expression values, n = 4 for Sc, n = 8 for ScNI; n = 12 for Sc[Ca], Sc[Sp] and Sc[At]; 2 biological replicates with n/2 technical replicates each; for indexes, n = 30 for all conditions). Pearson correlation coefficient r is considered non-significant if p > 0.05.

H Polarity indexes, defined to assess whether cell polarity is normal or affected (value is 1 when all patches of medium to large budded cells are present in the bud, and -1 refers when all patches are in the mother cell. Data are presented as mean +/- SD (n = 30 for all conditions). **P<0.01, ***P<0.001 (Brown-Forsythe and Welch ANOVA tests, with Dunnett's T3 multiple comparisons tests).

Data information: Abbreviations: ns - non-significant, Sc - wild-type S. cerevisiae cells, ScNI - S. cerevisiae cells where the actin gene has been replaced with the wild-type gene but without the intron, Sc[X] - S. cerevisiae cells where the actin gene has been replaced with a gene carrying silent mutations based on the sequences from species X (for the list of species and coding see Table EV1 or Fig 1).