Fluorescent protein tags for human tropomyosin isoform comparison

Figures

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  Fig. 1.. Tropomyosin fusion mammalian expression constructs used in this work. Tropomyosin (TPM) N-terminally fused via a 40-amino acid flexible linker (40AA) to: (A) mNeonGreen (mNG); (B) mCherry; (C) mStayGold(E138D) (mSG); and (D) mScarlet3-H (mS3-H). For each, variants for nine different human tropomyosin isoforms were generated, namely TPM1.6, TPM1.7, TPM1.8, TPM1.12, TPM2.1, TPM2.2, TPM3.1, TPM3.2 and TPM4.2. All constructs are under the control of a CMV promoter (pCMV) for expression in mammalian cells.

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  Fig. 2.. mNeonGreen-40AA-tagged tropomyosin isoforms colocalise with actin. (A) Representative spinning-disk microscopy images of human tropomyosin isoforms fused via a 40-amino acid linker to mNeonGreen in hTERT-RPE1 cells. Note that the images shown are after fixation and staining with rhodamine-phalloidin. Scale bar: 30 µm (n=8). (B) Annotated images of cells expressing mNeonGreen-TPM1.6 (left) and mNeonGreen-TPM3.1 (right). Arrows point to stress fibres, triangles are adjacent to cortical actin, and asterisks indicate likely focal adhesions. Scale bar: 30 µm. (C) Strong colocalisation between mNG-TPM probes and phalloidin was detected in all isoforms using Pearson's correlation coefficient analysis. Each datapoint represents an image. (D) Fluorescence intensity plots of lines (shown in images) along stress fibres from cells expressing mNeonGreen-TPM1.6 (left) and mNeonGreen-TPM1.7 (right). Scale bars: 10 µm.

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  Fig. 3.. mCherry-40AA-tagged tropomyosin isoforms show typical tropomyosin fluorescence. Representative spinning-disk microscopy images of human tropomyosin isoforms fused to mCherry via a 40-amino acid linker, expressed in live hTERT-RPE1 cells. Scale bar: 30 µm (n=8).

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  Fig. 4.. Tropomyosin isoforms tagged with mNeonGreen and mCherry via a 40-amino acid linker allow direct in vivo comparison of isoform localisation. Representative spinning-disk microscopy images of live hTERT-RPE1 cells co-expressing two different human tropomyosin isoforms fused to mCherry and mNeonGreen via a 40-amino acid linker. Scale bar: 30 µm (n=11). Colocalisation for each image shown was quantified by Pearson's correlation coefficient (r) method.

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  Fig. 5.. Individual frames from time-lapse imaging of cells expressing mNG-TPM1.6 and mCherry-TPM1.7 show normal cytoskeletal activity. Images were taken every 5 min; scale bars: 30 µm (n=10).

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  Fig. 6.. Tagging with flexible 40-amino acid linkers facilitates direct super-resolution comparison of human tropomyosin isoforms. Representative super-resolution microscopy of fixed hTERT-RPE1 cells co-expressing tropomyosin isoforms fused via a 40-amino acid linker to mCherry and mNeonGreen. (A) AiryScan microscopy images. (B) Structured illumination microscopy images. Scale bars: 30 µm (n=5).

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  Fig. 7.. Tropomyosins tagged with a photostable green fluorescent protein via a 40-amino acid linker are a new tool for applications requiring prolonged laser exposure. (A) Representative spinning-disk microscopy images of live hTERT-RPE1 cells expressing tropomyosin isoforms fused via a 40-amino acid linker to mStayGold(E138D) (n=9). (B) Individual frames from time-lapse imaging of cells expressing mStayGold(E138D)-TPM1.7. Images were taken every 5 min (n=10). Scale bars: 30 µm.

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  Fig. 8.. Tagging of an improved photostability red fluorescent protein with tropomyosins via a 40-amino acid linker. Representative spinning-disk microscopy images of live hTERT-RPE1 cells expressing different tropomyosin isoforms fused to mScarletH-3 via a 40-amino acid linker (n=5).