mNG-tagged fusion proteins and nanobodies to visualize tropomyosins in yeast and mammalian cells

Abstract

Tropomyosins are structurally conserved α-helical coiled-coil proteins that bind along the length of filamentous actin (F-actin) in fungi and animals. Tropomyosins play essential roles in the stability of actin filaments and in regulating myosin II contractility. Despite the crucial role of tropomyosin in actin cytoskeletal regulation, in vivo investigations of tropomyosin are limited, mainly due to the suboptimal live-cell imaging tools currently available. Here, we report on an mNeonGreen (mNG)-tagged tropomyosin, with native promoter and linker length configuration, that clearly reports tropomyosin dynamics in Schizosaccharomyces pombe (Cdc8), Schizosaccharomyces japonicus (Cdc8) and Saccharomyces cerevisiae (Tpm1 and Tpm2). We also describe a fluorescent probe to visualize mammalian tropomyosin (TPM2 isoform). Finally, we generated a camelid nanobody against S. pombe Cdc8, which mimics the localization of mNG–Cdc8 in vivo. Using these tools, we report the presence of tropomyosin in previously unappreciated patch-like structures in fission and budding yeasts, show flow of tropomyosin (F-actin) cables to the cytokinetic actomyosin ring and identify rearrangements of the actin cytoskeleton during mating. These powerful tools and strategies will aid better analyses of tropomyosin and F-actin cables in vivo.

A monomeric StayGold fluorescent protein

“…2d and Supplementary Figs. [9][10][11][12][13][14] are available for download. Raw images (>650 GB in total) from the microscopy-based studies (Fig.…”

“…9). In a second imaging experiment, we observed the cytoskeleton of fixed human retinal pigment epithelial-1 (RPE-1) cells expressing the actin-binding protein tropomyosin 9 (a naturally dimeric coiled-coil protein 10 ) fused to either mNeonGreen 11 , StayGold 1 or mStayGold (Supplementary Fig. 10).…”

Unraveling the mechanisms and evolution of a two-domain module in IQGAP proteins for controlling eukaryotic cytokinesis

“…Surprisingly, the rng2-CHDΔ cells formed WT-like actin rings, as revealed by phalloidin staining ( Figure 6A ), despite the ability of CHD to bind F-actin. 31 , 44 Time-lapse analysis using the mNeonGreen (mNG)-tagged tropomyosin Cdc8 as a biosensor for the actin ring 49 confirmed this result ( Figures 6B – 6D ). The rng2-CykFΔ cells also assembled actin rings ( Figures 6A and 6B ), but the ring emergence and removal were significantly delayed ( Figures 6B – 6D ).…”

Functional redundancy and formin-independent localization of tropomyosin isoforms in Saccharomyces cerevisiae

“…Visualization of tropomyosin in live cells has been a major challenge because fluorescently-tagged tropomyosin fusions are not completely functional and thus, need careful interpretation for accurate insights about Tpm localization and dynamics in vivo 42 . We have recently shown that mNeonGreen-Tpm (mNG-Tpm) fusion proteins clearly report localization of Tpm isoforms to actin structures in vivo in fungal and mammalian cells 40 (Fig. S1A, S1B) but their functionality compared to native Tpm remained a question of interest.…”

“…While genetic studies have indicated a possible preference between Tpm isoforms and formin isoforms 32 , whether Tpm1 and Tpm2 localize to distinct actin filaments/cables in S. cerevisiae has remained unaddressed mainly due to absence of functional Tpm fluorescent fusions and live-cell imaging data for Tpm1 and Tpm2. Recent work from our lab made it possible to visualize the live dynamics of Tpm across species using mNG-Tpm fusion proteins 40 and revealed their localization to actin cables and the actomyosin ring during the cell cycle 40 . However, their combined action and roles in actin cable stability in vivo have not been fully explored.…”