Analysis of biophysical and functional consequences of tropomyosin-fluorescent protein fusions

Abstract

The dynamic nature of actin polymers is modulated to facilitate a diverse range of cellular processes. These dynamic properties are determined by different isoforms of tropomyosin which are recruited to distinct subpopulations of actin polymers to differentially regulate their functional properties. This makes tropomyosin an attractive target for labelling discrete actin populations. We have assessed the effect of different fluorescent labelling strategies for this protein. Although tropomyosin-fluorescent fusions decorate actin in vivo, they are either nonfunctional or perturb regulation of actin nucleation and cell cycle timings. Thus, conclusions and physiological relevance should be carefully evaluated when using tropomyosin fusions.

Keywords: Cdc8; Schizosaccharomyces pombe; acetylation; actin cytoskeleton; fission yeast.

Figure 1

Tropomyosin proteins used in this study. (A) Predictive models of Tpm^Cdc8 (upper panel) and Cerulean3‐Tpm^Cdc8 (lower panel) dimers. (B) Coomassie blue‐stained SDS/PAGE analysis of purified wild‐type and Cerulean3‐tagged Tpm^Cdc8 proteins. (C) CD Spectra of purified acetylated wt (magenta), amino‐terminally Cerulean3‐tagged (black), and both acetylated (blue) and unacetylated (red) carboxyl‐terminally Cerulean3‐tagged Tpm^Cdc8 proteins. A CD spectrum of a fluorescent protein (cyan) is included for comparison. *Denotes amino‐terminally acetylated protein.

Figure 2

FP Fusions affect Tpm filament formation not themostability. (A) Normalised differential CD absorbance data for unacetylated Tpm^Cdc8 (green), acetylated Tpm^Cdc8 (magenta), Cerulean3–Tpm^Cdc8 (black), aceteylated Tpm^Cdc8–Cerulean3 (blue) and unaceylated Tpm^Cdc8–Cerulean3 (red) at 222 nm. (B) First derivative plots at 222 nm of data described in (A). (C) Viscosity of 20 μm Tpm^Cdc8 proteins and buffer (grey dashed) at increasing NaCl concentrations (0–200 mm) at 23 °C.

Figure 3

Actin‐binding assays of FP Tpm fusions. SDS/PAGE gels of pellet and supernatant fractions from cosedimentation assays of (A) Cerulean3‐Tpm^Cdc8, (B) Tpm^Cdc8‐Cerulean3 and (C) Nt‐acetylated Tpm^Cdc8‐Cerulean3. (D) Binding curve of the free Tpm^Cdc8 concentration against the ratio of density of actin, for Cerulean3‐Tpm^Cdc8, measured by densitometry of cosedimentation SDS/PAGE gels. Curves represent Hill equation lines of best fit.

Figure 4

Ability of FP Tpm fusions to associate with and support CAR function. Localisation of Cerulean‐Tpm^Cdc8 (A and C) and Tpm^Cdc8‐Cerulean3 (B and D) in wild‐type (A and B) and cdc8.110 (C and D) cells at at 36 °C. (E) Growth curves of cdc8.110 cells containing plasmids encoding for wild‐type Tpm ^cdc8+ (magenta), Cerulean3‐Tpm ^cdc8 (black), Tpm ^cdc8 ‐Cerulean3 (blue) or an empty vector (grey) cultured at 36 °C. Scale bars: 5 μm.

Figure 5

Myosin regulatory function of FP Tpm fusions. Montages of timelapse frames of (A) cdc8.110 myo2.mCherry pREP41Tpm ^cdc8+, (B) cdc8.110 myo2.mCherry pREP41Cerulean3‐Tpm ^cdc8, and (C) cdc8.110 myo2.mCherry pREP41Tpm ^cdc8‐Cerulean3 cells grown at 36 °C. Images show mCherry (A–C) and Cerulean (B‐lower panels) signals Timings are shown in minutes. (D) Micrographs of mNeongreen signal from cdc8.110 myo52.mNeongreen cells expressing different Tpm ^cdc8 constructs. Scale bars: 5 μm.