Salactin, a dynamically unstable actin homolog in Haloarchaea

ABSTRACT Across the domains of life, actin homologs are integral components of many essential processes, such as DNA segregation, cell division, and cell shape determination. Archaeal genomes, like those of bacteria and eukaryotes, also encode actin homologs, but much less is known about these proteins’ in vivo dynamics and cellular functions. We identified and characterized the function and dynamics of Salactin, an actin homolog in the hypersaline archaeon Halobacterium salinarum. Live-cell time-lapse imaging revealed that Salactin forms dynamically unstable filaments that grow and shrink out of the cell poles. Like other dynamically unstable polymers, Salactin monomers are added at the growing filament end, and its ATP-bound critical concentration is substantially lower than the ADP-bound form. When H. salinarum’s chromosomal copy number becomes limiting under low-phosphate growth conditions, cells lacking Salactin show perturbed DNA distributions. Taken together, we propose that Salactin is part of a previously unknown chromosomal segregation apparatus required during low-ploidy conditions. IMPORTANCE Protein filaments play important roles in many biological processes. We discovered an actin homolog in halophilic archaea, which we call Salactin. Just like the filaments that segregate DNA in eukaryotes, Salactin grows out of the cell poles towards the middle, and then quickly depolymerizes, a behavior known as dynamic instability. Furthermore, we see that Salactin affects the distribution of DNA in daughter cells when cells are grown in low-phosphate media, suggesting Salactin filaments might be involved in segregating DNA when the cell has only a few copies of the chromosome.


SUPPLEMENTAL TABLES
Supplemental Table S1.Percent identity of Salactin to other polymerizing actin fold proteins.

Supplemental Figure S4 .
Dynamics of Salactin fused to different tags.(A) Kymograph of Salactin-HaloTag (strain hsJZ86) also expressing the native salactin copy.(B) Kymograph of Salactin-HaloTag expressed in ∆salactin cells (strain hsJZ106).(C) Fluorescent images of Salactin-msfGFP expressed in ∆salactin cells (strain hsJZ95).The pink arrow points toward the appearance of filaments.(D) Histogram of the relative frequencies for polymerization rates comparing wild type + Salactin-msfGFP (strain hsJZ52), wild type + Salactin-Halotag (strain hsJZ86), ∆salactin + Salactin-HaloTag (strain hsJZ106).The wild type + Salactin-HaloTag has a significantly (p-value <0.0001) slower polymerization rate, while the other two have comparable polymerization rates.(E) RNA-seq from total mRNA extracted from strains ura3 and hsJZ52.Salactin transcriptional measurements showing the overexpression levels were calculated by dividing the number of sequenced salactin reads by the total reads from each dataset.N = 184 for WT (salactin-msfGFP), 110 for WT (Salactin-Halotag), and 126 for ∆salactin (Salactin-Halotag).Supplemental Figure S5.SDS-PAGE gels of the purification protocol at the beginning (induction) and end (purified protein).Pre-and post-induction of Salactin expressed in Escherichia coli (left).Final purified protein used for in vitro assays (right).All gels are stained with SYPRO Orange.Supplemental Figure S6.Malachite Green Assay using 4 µM Salactin in different salt conditions (450 mM, 975 mM, 1.5 M, 2.29 M KCl).As noted in the main text, the higher ATPase activity suggests polymerization is favored at higher salt concentrations.Representative pelleting SDS-PAGE gels of Salactin in ATP (left) and AMPPNP (right) across different protein concentrations.All gels are stained with SYPRO Orange.Supplemental Figure S8.Pelleting of Salactin in ATP compared to ADP.SDS-Page gel of 10 µM Salactin in 2.5 mM ATP, 50 µM ATP, 2.5 mM ADP, and HP buffer (left).Measured SYPRO Orange intensity of the four different conditions (right).All gels are stained with SYPRO Orange.S = supernatant, P = pellet.Supplemental Figure S9.(A) Growth curves of ∆ura3 and ∆salactin cells in rich media.Data is from 6 biological replicates.(B) Representative image of ∆ura3 and ∆salactin on an agar motility plate (10% CM, 0.3% agar) 5 days after inoculation (left).∆ura3 is in the bottom right corner, ∆salactin is in the upper right corner of the plate, and NRC-1 (wild-type cells) are on the left.Quantitation of 9 plates (N = 9) across 5 days indicated no motility defect in the ∆salactin strain relative to the ∆ura3 strain (p-value >0.05 for each of the five days) (right).Supplemental Figure S10.(A) Area of ∆ura3 and ∆salactin cells in standard phosphate media at stationary phase.N = 1337 for ∆ura3 and 1506 for ∆salactin.(B) Zoomed-in image of ∆salactin cells in low phosphate demonstrating that cells now show clear foci of DNA as indicated by the yellow arrowheads.Supplemental Figure S11 -Montage of Salactin-msfGFP (strain hsJZ52) filament dynamics in low phosphate media.Images are on the same scale and the scale bar on the first panel (2 µm) applies to all panels.Video of Salactin-msfGFP expressed on top of the native copy, visualized by Near-TIRF fluorescent microscopy.Images were taken with a 488nm laser every 30 seconds for 1 hour.Video is 600x actual speed.Scale bar = 4 µm.Movie corresponds to the montage in Figure 2A.SM2.Video of Salactin-HaloTag expressed on top of the native copy visualized by Near-TIRF fluorescent microscopy.Images were taken every 10 seconds for 20 minutes.Video is 150x actual speed.Cyan is Salactin-HaloTag labeled with low concentrations of JF549 to generate speckles, and magenta is Salactin-HaloTag labeled with high concentrations of JF505 to label the whole filament.Scale bar = 2 µm.Movie corresponds to the montage in Figure 2F.SM3.Video of Salactin-HaloTag labeled with JF549 that was expressed on top of the native copy, visualized by Near-TIRF fluorescent microscopy Images were taken every 30 seconds for 30 minutes.Video is 600x actual speed.Scale bar = 4 µm.SM4.Video of Salactin-HaloTag expressed as a sole copy visualized by Near-TIRF fluorescent microscopy.Images were taken every 30 seconds for 1 hour.Video is 600x actual speed.Scale bar = 4 µm.SM5.Video of Salactin-msfGFP expressed as a sole copy visualized by Near-TIRF fluorescent microscopy.Images were taken every 2 minutes for 32 minutes.Video is 1,200x actual speed.Scale bar = 10 µm.SM6.Video of Salactin-msfGFP expressed on top of the native copy (hsJZ52).Cells were grown in low phosphate and visualized by Near-TIRF fluorescent microscopy.Images were taken every 5 seconds for 10 minutes.Video is 20x actual speed.

Table S2 .
Distribution of cells with filaments that are dynamic or not dynamic.

Table S4 .
Strains used in this study.

Table S5 .
Plasmids used in this study.