A Complex Endomembrane System in the Archaeon Ignicoccus hospitalis Tapped by Nanoarchaeum equitans

Abstract

Based on serial sectioning, focused ion beam scanning electron microscopy (FIB/SEM), and electron tomography, we depict in detail the highly unusual anatomy of the marine hyperthermophilic crenarchaeon, Ignicoccus hospitalis. Our data support a complex and dynamic endomembrane system consisting of cytoplasmic protrusions, and with secretory function. Moreover, we reveal that the cytoplasm of the putative archaeal ectoparasite Nanoarchaeum equitans can get in direct contact with this endomembrane system, complementing and explaining recent proteomic, transcriptomic and metabolomic data on this inter-archaeal relationship. In addition, we identified a matrix of filamentous structures and/or tethers in the voluminous inter-membrane compartment (IMC) of I. hospitalis, which might be responsible for membrane dynamics. Overall, this unusual cellular compartmentalization, ultrastructure and dynamics in an archaeon that belongs to the recently proposed TACK superphylum prompts speculation that the eukaryotic endomembrane system might originate from Archaea.

Keywords: 3D; Archaea; FIB/SEM; electron tomography; eukaryogenesis; membranes; symbiosis; ultrastructure.

Figure 1

Ultrastructure of Ignicoccus. (A) 50 nm section of a high-pressure frozen, freeze-substituted I. hospitalis cell showing its characteristic constituents; CP, cytoplasm; IM, inner membrane; OCM, outer cellular membrane; IMC, inter-membrane compartment; V, vesicular structures; bar 0.5 μm; (B) 3D model of approximately half of an I. hospitalis cell based on 50 nm serial sections emphasizing the asymmetrical character of the cells; blue, OCM, orange cytoplasm and membrane surrounded structures in the IMC.

Figure 2

Cytoplasmic protrusions of I. hospitalis. “Voltex” (volume texture rendering) display of a I. hospitalis cell from the FIB/SEM data showing protrusions from the cytoplasm; arrows point to spherical swellings that might indicate constriction or fusion sites, respectively; additionally one slice of the image stack of the original data is shown, in which the OCM can be seen.

Figure 3

ATP synthase immunolabeling. Immunolabeling on 50 nm section of I. hospitalis against the A₁ subcomplex of its ATP synthase; the labeling pattern shows localization in the OCM as well as in cytoplasmic protrusions; CP, cytoplasm; OCM, outer cellular membrane; bar 0.5 μm.

Figure 4

Two examples of cylindrical macromolecular complexes underneath the OCM. (A,B) Showing details of single slices of the respective tomograms, arrow points to the ending of the cylindrical complex at the OCM; OCM, outer cellular membrane; CPP, cytoplasmic protrusion; bar 50 nm each; (C,D) combined view of the respective sub-volumes, showing segmented macromolecular complexes (blue and yellow) in a volume texture rendering display, additional filamentous structures are labeled in green; (E,F) show cross-sections of the subvolumes with “ObliqueSlice” tool; orthogonal to the xy plane an oblique plane was chosen that cuts through the structure “parallel” to the OCM; arrow points to the inner “ring” of the structure; (G,H) top view of segmented macromolecular complexes; note that the yellow and blue parts serve as a color coding for what is coming out of/going through the plane.

Figure 5

Matrix of filaments and/or thethers in the IMC. Three pairs of sections of tomograms each showing the original data (A,C,E) and the respective structures highlighted in green (B,D,F); (A,B) is taken from an edge region of a tomogram that shows a cytoplasmic protrusion and its connection via the branched matrix to the OCM; (C,D) as well as (E,F) show examples of interconnected protrusions and connection between the cytoplasm and protrusions, a connection between the protrusions and the outer cellular membrane can also be seen; OCM, outer cellular membrane; CP, cytoplasm; CPP, cytoplasmic protrusion; bars 100 nm, each.

Figure 6

V4R protein Igni_1332 immunolabeling. Immunolabeling on 50 nm sections against Igni_1332 showing (A) a whole I. hospitalis cell and (B–G) additional examples from different I. hospitalis cells; black arrows point to examples for localizations in the IM and the membrane of protrusions at putative fusing sites; white arrows point to labeling associated with the matrix of filaments and/or tethers in the IMC; bar 0.5 μm (A); 100 nm (B–G)

Figure 7

EDX analysis of dark contrasted inclusion in the cytoplasm. (A) Arrow points to the structure in a slice of a tomogram, bar 0.5 μm; (B) shows the 3D model of the 200 nm section; blue, OCM; orange, cytoplasm and cytoplasmic protrusions; black, inclusion in the cytoplasm; (C) linescan across the structure showing accumulation of phosphorus (green) inside the structure and a sulfur peak (red) at its edge

Figure 8

Contact site of N. equitans. (A–D) Show slices of tomograms in which the direct connection between the cytoplasms of both organisms can be followed; in each example the cytoplasm of I. hospitalis protrudes toward N. equitans, which appears to pierce both I. hospitalis membranes via a stalk like structure, the S-Layer appears to be disintegrated in the contact region; in (B) also N. equitans tethers can be seen (arrow), that were already described before; in (C) the arrow points to an electron dense structure of unknown composition and function in the cytoplasm of N. equitans at the basis of the stalk OCM, outer cellular membrane; SL, S-Layer; CP, cytoplasm; Iho, I. hospitalis; Neq, N. equitans; bar 100 nm, each; (E) cross section of the 3D model of the sub-volume shown in (A); purple, S-Layer of N. equitans; green, OCM of I. hospitalis; yellow, cytoplasms of both organisms; red, presumeably either filaments from I. hospitalis or parts of the disintegrated S-Layer of N. equitans.

Figure 9

Protein transfer from I. hospitalis to N. equitans. Micrographs show immunolabelings on 50 nm sections of I. hospitalis proteins (A) Vps4 (Igni_0994) and (B) a fatty acid coA ligase (Igni_0475); in both cases proteins are found to be distributed in the cytoplasm of both organisms, although no homologs are annotated for N. equitans; Iho, I. hospitalis; Neq, N. equitans; bars, 0.5 μm each.