Tissue-like multicellular development triggered by mechanical compression in archaea

Abstract

The advent of clonal multicellularity is a critical evolutionary milestone, seen often in eukaryotes, rarely in bacteria, and only once in archaea. We show that uniaxial compression induces clonal multicellularity in haloarchaea, forming tissue-like structures. These archaeal tissues are mechanically and molecularly distinct from their unicellular lifestyle, mimicking several eukaryotic features. Archaeal tissues undergo a multinucleate stage followed by tubulin-independent cellularization, orchestrated by active membrane tension at a critical cell size. After cellularization, tissue junction elasticity becomes akin to that of animal tissues, giving rise to two cell types-peripheral (Per) and central scutoid (Scu) cells-with distinct actin and protein glycosylation polarity patterns. Our findings highlight the potential convergent evolution of a biophysical mechanism in the emergence of multicellular systems across domains of life.

Fig. 1. Uniaxial compression triggers multicellular development in Hfx. volcanii.

(A) Schematic of cells trapped and (B) phase-contrast time-lapses of cells growing in in the ArcCell microfluidic device. (C) Schematic of compressed cells under 2.5% agarose pads. (D) Phase-contrast (top row) and spinning-disk confocal (bottom row) time-lapses of compressed cells across ~6 generations. msfGFP-PCNA foci (blue) represent replication sites. (E) Stretched and compressed areas comprised in large monolayers of epithelia-like tissues. (F) 3D-SoRa microscopy images of a tissue (top) and unicells (bottom). (G) Laser ablation of tissue regions. False-colored overlays of tissues before (magenta) and 10 minutes after (green) ablation. Yellow areas indicate the ablated area. Directional motion from cells was calculated from MSD curves. (H) Laser ablation of cell membranes. False-colored overlays of tissues and cells before (magenta) and after (green) ablation. White arrowheads indicate the membrane recoil retraction. Unless specified, scale bars represent 2 μm.

Fig. 2. Hfx. mediterranei does not form tissues under compression.

(A) Cladogram depicting evolutionary relationships between compressed Haloferax species. Gray- and red-labeled species represent cells that form or do not form tissues, respectively. Hfx. prahovense is marked with an asterisk as it develops to considerably larger, deformed tissues. For a comprehensive phylogenetic tree, see Supplementary Data S1. (B) Phase-contrast time-lapses (left) of Hmed growth under 3% (top) and 5% (bottom) agarose pads. (right) SoRa microscopy of Hmed growth after 24 hours under 3% agarose pads. (C) Cartoon representation of microfabricated pillars used in the intermittent compression experiments. (D) Phase-contrast time-lapses of Hvo (top) and Hmed (bottom) under micropillar devices. The 24-hour datapoint is represented as a zoom inlet from the yellow area in the previous timepoint. (E) Viability of Hvo tissues compared to Hmed cells under micropillars measured by colony formation unit (CFU). Hvo and Hmed viabilities were normalized by their respective liquid cultures.

Fig. 3. Cellularization is independent of FtsZs and results in two cell types.

(A) 3D-STED super-resolution microscopy of the cellularization process. (B) (Left) iSIM of cells expressing cytoplasmic msfGFP and (right) 3D outline masks of scutoid cells segmented from 3D-STED highlights scutoid cells. Dashed arrows indicate the different scutoid surface neighbors across z planes. (C) Cartoon representation of top and side views showing Per and Scu cell types within tissues. (D-F) Area growth rate (D), lifespan (E) and deformation (F) measurements of unicells, Per, and Scu cells from phase-contrast time-lapses. (G) Epifluorescence microscopy of representative cell-division impaired ΔftsZ1ΔftsZ2 cells across different developmental stages. Early cellularization represents cells that just entered the cellularization stage. Peak cellularization represents cells at the onset of completing cellularization. (H) 3D-SoRa microscopy of representative cells expressing FtsZ1-mChartreuse across different developmental stages. White arrowheads indicate septation sites without FtsZ1 signal. Scale bars: 2 μm.

Fig. 4. Tissue cellularization is triggered by coenocytic size through a membrane tension threshold.

(A) Epifluorescence micrographs of wild-type (top) and ΔftsZ2 (bottom) cells from compression to cellularization. (B) Single-cell growth curves from compression to cellulation onset. (C) Time, cell area, and cell area are added at the onset of cellularization. (D) Phase-contrast images of unicell and coenocytes and bSpoJ single-molecule tracks overlay false-colored relative to their mean speed. Effective diffusion coefficients are calculated from MSD curves. (E) Live-cell Generalized Polarization measurements of wild-type and car⁻ stained with Laurdan. (F) Area at cellularization measurements of wild-type and car⁻ cells from phase-contrast time-lapses across temperatures. (G) Wild type and car⁻ membrane fluidity calculated by bSpoJ effective diffusion coefficients across developmental stages at 34°C. Correlation between cellularization areas and bSpoJ diffusion in wild-type and car⁻ cells at 34°C. Scale bars: 2 μm.

Fig. 5. Volactin and N-glycosylation are tissue-specific polarity markers.

(A) Volcano plot overlays from RNA-seq datasets collected across developmental stages and normalized by liquid unicellular cultures. Parentheses indicate the number of candidates above the arbitrary cutoff. (B) Normalized fluorescence by cell area of volA-msfGFP and constitutively expressed cytoplasmic msfGFP from confocal time-lapses. (C) Epifluorescence micrograph of a false-colored tissue relative to volA-msfGFP fluorescence (left) and dynamics of volA-msfGFP polymers represented by kymographs (right) from Per and Scu regions. (D) 3D-SoRa projections of representative cells expressing volA-msfGFP across developmental stages (left) and volA cable angle measurements relative to the coverslip plane (right). (E) Height measurements of wild-type and ΔvolA coenocytes from 3D-SoRa projections. (F) Representative epifluorescence micrograph of a ΔvolA cell stalled at cellularization. (G) 3D-confocal projections of cell surface N-glycosylated proteins in wild-type (top) and ΔaglB (bottom) tissues stained by ConA-Alexa488. Scale bars: 2 μm.