Temperature sensitive point mutations in fission yeast tropomyosin have long range effects on the stability and function of the actin-tropomyosin copolymer

Fig. 1. CAR formation and constriction in cells containing temperature sensitive Tpmcdc8alleles. Mid log phase cells were mounted onto the microscope as described previously and held at the designated temperature for 20 min before undertaking observations. (A) Maximum projection of 21-z slice images from timelapse experiments (9 min between frames) where CAR formation and constriction was monitored in rlc1.gfp cells containing either a cdc8+, cdc8.110, or cdc8.27 allele. 0 time denotes the start of the experiment and does not relate to other mitotic events. Cells formed either normal contractile rings (red border); aberrant rings that after a delay went on to constrict (yellow border - asterisk); aberrant rings that detached from the cortex and subsequently constricted to form a misplaced septum (green border); or aberrant rings that collapsed upon constriction (blue border). 9 min between frames. Scale bars–5 μm. (B) Histograms from analysis of CAR constriction of >45 cells from each cell type showing frequency of each Actin Ring (AR) phenotype described in (A) in cdc8.110, or cdc8.27 cells when incubated at different temperatures. Colours are consistent with (A). n denotes number of…

Fig. 2. CD spectra and melting curves. (A) Coomassie-Blue stained SDS-PAGE gel showing purified acetylated wild-type and mutant Tpmcdc8 proteins. (B) CD spectra for unacetylated TpmCdc8 at 10 and 50 °C. Normalised CD signal at 222 nm for AcTpm and unacetylated Tpm as a function of temperature (C) WT-, A18T- and E31K-Tpm and (D) WT-, E129K- and A18TE31K-Tpm. (E) First derivative plots for the four AcTpms melting curves.

Fig. 3. Actin binding assays of TpmCdc8. (A) Representive SDS/PAGE gels of pellet and supernatant fractions from the co-sedimentation assay of AceTpm-E129K. Ratio of density was measured by densitometry of co-sedimentation SDS/PAGE gels. The free Tpm concentration plotted against the ratio of density of actin to Tpm from the SDS-PAGE gel for Nt-acetylated (empty shapes) and non-acetylated (filled shapes) (B) WT-Tpmcdc8 and (C) E129K-Tpmcdc8. Binding curves were generated by fitting the Hill equation to the data and the K50% are the best fit values and standard error of the fit. Fits of wild type proteins (B) are shown in (C) as dotted lines for comparison.

Fig. 4. Positions of the subsitituted residues. (A) Hydrophobicity Cluster Analysis showing residues of an α-helix of TpmCdc8. The positions of mutations investigated in this study are shown circled in red. Red residues represent negatively charged amino acids, blue positively charged, and green hydrophobic amino acids. The black lines outlining hydrophobic patches indicate the hydrophobic core of the coiled coil. (B) Diagram illustrating a heptad repeat of 7 residues (A-G) between 2 α-helices. Residues in positions A and D are normally small compact hydrophobic residues involved in stabilising the coiled-coil interactions, and residues E and G are often charged and can form of a salt bridges between the two chains. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)