Salactin, a dynamically unstable actin homolog in Haloarchaea

Abstract

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.

Keywords: DNA segregation; actin; archaea; cytoskeleton; dynamic instability.

Fig 1

(A) Tree of the representative species in the Methanotecta clade in the Euryarchaeota phylum with MamK, a closely related homolog of Salactin. Phylogeny of salactin homologs in Haloarchaea and other Methanotecta; the topology indicates that salactin was likely already present in the common ancestor of Methanotecta. Branch supports are ultrafast bootstraps (39); only supports >90% are indicated. Branch length is proportional to the expected number of substitutions per site (indicated by the scale bar). All alignments and trees are available in File S1. (B) Alphafold2-predicted structure of Salactin colored by its surface electrostatic potential (available from Uniprot, ID: Q9HSN1). (C) Phase-contrast images of ∆ura3 parent strain (pink) and ∆salactin (blue) H. salinarum cells in rich media (CM + URA) (left) showing that both exhibit the same rod shape. Both images are on the same scale, and the scale bar on the left applies to both panels. Violin plot of the aspect ratio (mid) and width (right) of ∆salactin cells and ∆ura3 cells demonstrates no statistically significant difference between strains (width P = 0.0138, aspect ratio P = 0.4093). Data were taken across three biological replicates with a total N = 375 and 493 for ∆ura3 and ∆salactin cells, respectively.

Fig 2

Characterization of in vivo Salactin dynamics. (A) A montage of Salactin-msfGFP from a H. salinarum cell (strain hsJZ52). The two arrowheads indicate each end of the filament. A gamma filter correction of 1 was applied to improve visibility. Images are on the same scale, and the scale bar on the first panel applies to all panels. (B) A representative cell (left) is used to create a kymograph (right). Yellow arrowheads indicate the region used for drawing the corresponding kymograph. The kymograph was inverted to improve visibility. (C) Violin plot of measured in vivo polymerization rates (left, N = 184), time until catastrophe (mid, N = 152), and length at catastrophe (right, N = 181) obtained from analysis of 50 kymographs. (D) A montage of a speckle labeled Salactin-HaloTag filament in a H. salinarum cell (strain hsJZ86) (left). The entire Salactin-HaloTag polymer was labeled with JF505 (magenta) and also sparsely labeled with JF549 to generate speckles (cyan). Red arrowhead indicates the filament end, and blue arrowhead indicates a single molecule. Kymograph for the filament trace showing that monomers remain stationary within the growing filament (right). Images are on the same scale, and the scale bar on the first panel applies to all panels. (E) Two example kymographs showing multiple triangles (indicated by yellow arrowheads) arising from diffraction-limited filaments, indicating these structures may be composed of multiple filaments.

Fig 3

In vitro polymerization of Salactin. All assays were done with 2.9 M KCl unless otherwise noted. (A) Malachite green assay using 4 µM Salactin in different salt conditions (1.5, 1.9, 2.3, and 2.9 M). The higher ATPase activity implies polymerization is favored at higher salt concentrations. (B) Salactin polymerization only occurs in the presence of ATP and AMP-PNP. Salactin polymerization was assayed by pelleting, using 2.5 mM ATP, ATP analogs, ADP, GTP, or buffer alone. Gels were stained with SYPRO Orange. S, supernatant; P, pellet. (C) Critical concentrations of Salactin determined by pelleting (top) and light scattering (bottom). (D) Polymers of Salactin mixed with cy3B-conjugated Salactin-GSKCK are seen with TIRF microscopy in the presence of ATP but not ADP. Right panels are zoomed-in images of the left panel.

Fig 4

Cells lacking ∆salactin show defects in chromosomal partitioning and cell shape in low-phosphate media. Throughout, pink is ∆ura3, and blue is ∆salactin. (A) Synteny analysis of salactin (VNG_RS00630; VNG0153C) gene in closely related organisms to H. salinarum. (B) Growth curve showing the difference in the growth of ∆salactin cells in low-phosphate media taken from three technical replicates of three biological replicates, “∆sal + sal” indicates ∆salactin cells complemented by exogenous expression of Salactin. (C) Spot dilutions of ∆ura3 and ∆salactin cells grown on standard phosphate media and low-phosphate media indicating ∆salactin cells have a reduced viability in low-phosphate media relative to ∆ura3 cells (left). Representative phase images of ∆ura3 and ∆salactin cells from the spot dilution assay in low-phosphate media (right). Images are on the same scale, and the scale bar on the top right panel applies to the bottom right panel. (D) The bulk chromosomal number per cell by qPCR in standard phosphate and low phosphate reveals that there is no statistical difference (P > 0.05) in the average chromosomal number per cell regardless of media condition. Data were taken from three biological replicates. (E) Representative fluorescent images overlayed on the phase of ∆ura3 and ∆salactin cells stained with SYBR-safe DNA stain in standard (left) and low-phosphate media (right). Yellow arrowheads indicate cells lacking chromosomal material. Images are on the same scale, and the scale bar on the first panel applies to all panels. (F) Quantification of the fluorescent intensity of SYBR-safe stained ∆ura3 and ∆salactin cells in standard and low-phosphate media. (P < 0.0001 for both) Data were taken from three biological replicates. (G) SYBR fluorescence versus circularity of ura3 and ∆salactin cells in low-phosphate media at stationary phase. Data were taken from three biological replicates.