A geothermal amoeba sets a new upper temperature limit for eukaryotes

Abstract

The study of temperature limits has transformed our knowledge of the boundaries of life but has been largely focused on bacteria and archaea. We isolated a novel geothermal amoeba, Incendiamoeba cascadensis, that divides at 63°C (145.4°F), establishing a new record for the upper temperature limit across all eukaryotes. We demonstrated cellular proliferation with growth experiments and visualized mitosis via expansion microscopy. Using high-temperature live-cell imaging, we quantified movement up to 64°C. We assembled the genome of I. cascadensis and using comparative genomics found an enrichment of genes related to proteostasis, genome stability, and sensing the external environment. Taken together, our findings challenge the current paradigm of temperature constraints on eukaryotic cells and reshape our understanding of where and how eukaryotic life can persist.

Fig. 1.. Phylogenetic, morphological, and physiological data support establishment of Incendiamoeba cascadensis, a novel genus and species within Amoebozoa.

(A) Concatenated tree of 102 Amoebozoa genes, nodes labeled with filled circles have 100% bootstrap support, open circles have >90% bootstrap support. Novel amoeba sequences are highlighted in dark orange, Vermamoeba vermiformis in grey, and the species used for genome comparison are bolded. (B) DIC and corresponding RICM of representative I. cascadensis in vermiform state and (C) in amoebiform state. (D) SEM of I. cascadensis in amoebiform state and (E) SEM in vermiform state. (F) DIC and corresponding RICM of representative V. vermiformis in vermiform state and (G) in amoebiform state. (H) SEM of V. vermiformis in amoebiform state and (I) SEM in vermiform state. All scale bars A-H are 5μm. (J-O) TEM ultrastructure of I. cascadensis including: (J) Overview of fine structure of trophozoite, nucleus (n), mitochondria (m) (K) Lobate nucleus with dispersed chromatin aggregates. (L) Nucleus with chromatin aggregates beneath the nuclear envelope. (M) Bilayered cyst wall. (N) Cell surface of trophozoite. (O) Mitochondria with non-branching tubular cristae. Scale bars J-M are 1μm, N is 200nm, and O is 500nm.

Fig. 2.. Incendiamoeba cascadensis undergoes cellular replication at temperatures up to 63°C.

(A) Doubling time across temperature range of novel amoeba. Individual points represent average doubling time from the exponential growth period of a seven-day growth experiment. Gray shading represents a 95% CI. (B) Example of individual growth curves from 55°C and 63°C. (C-D) Ultrastructure expansion microscopy confocal images of mitotic cells fixed at 58°C (C) or 63°C (D), with tubulin channel (left), DNA (middle), composite (right) with tubulin (pink), DNA (blue), and Bodipy membrane (gray). Top row in D consistent with metaphase and in C and middle row of D, the mitotic spindle array is visible, and cells are arriving at the end of division. The bottom row in D displays late telophase. Scale bars 20μm, not adjusted for the expansion factor (approximately 4x expansion).

Fig. 3.. Temperature-controlled live cell imaging reveals multiple motility modes of Incendiamoeba up to 64°C.

(A) Box and whisker plots indicate the median velocity (μm/min) of tracked cell centroids where the percentiles are visualized as follows: 75th (top edge), 25th (bottom edge), and median (bold line); whiskers extend to 1.5×IQR from the box, excluding outliers. Points represent individual particle tracks. (B) Mean squared displacement over time of tracked cells across temperature. Error bars represent standard deviation. Asterisks represent significance values (**** = p < 0.0001) for comparisons of α against a reference value of 1 using two one-sided t-tests. Values greater than 1 indicate directed motion. (C) Representative contour traces of cells at one second intervals at 57°C and 70°C. Frame number is indicated by color. (D) Percentage of total time cells spent at given aspect ratios (>2.5 for vermiform, 1.2–2.5 for amoebiform, 1.0–1.2 for predominantly cysts). Bars represent each temperature recorded and exemplar cell outlines for each aspect ratio are provided along with corresponding DIC images at low, middle, and high aspect ratios. Boxes around A-D plots are colored by species (I. cascadensis in dark orange and V. vermiformis in gray).

Fig. 4.. Comparative analyses indicate enrichment of signaling pathways, increased protein stability, and distinct biophysical properties may support thermophilic homeostasis.

(A) Relationship between two draft I. cascadensis genomes and three mesophilic amoebae for comparison (V. vermiformis, A. terricola, and D. discoideum). Total predicted orthologs are below species name. Bars represent percent completeness and contamination. Heat map represents overlap of orthologs between I. cascadensis and other amoebae, with darker shading indicating more overlap. (B) plots of GC3S (GC content at silent third codon positions) versus effective codon number for I. cascadensis and V. vermiformis. (C) KEGG functions enriched in I. cascadensis relative to mesophilic amoebae from A. (D) Bar plots represent copies of three example genes enriched in I. cascadensis (Ic) relative to V. vermiformis (Vv), A. terricola (At), and D. discoideum (Dd). (E) T_m predictions of I. cascadensis compared to V. vermiformis. (F) Predicted changes in net surface charge, fraction of positively charged surface residues, and fraction of negatively charged residues between the protein sequences of I. cascadensis and V. vermiformis. Dots are analogous proteins, and each pair is connected by a line. Red and blue lines indicate a corresponding decrease or increase from I. cascadensis to V. vermiformis. (G) The surface of dihydropteridine reductase from I. cascadensis and V. vermiformis. Red and blue regions indicate negative and positive electrostatic surface potential, respectively.