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.

Idiopathic pulmonary fibrosis (IPF) is a lethal chronic lung disease characterized by aberrant intercellular communication, extracellular matrix deposition, and destruction of functional lung tissue. While extracellular vesicles (EVs) accumulate in the IPF lung, their cargo and biological effects remain unclear. We interrogated the proteome of EV and non-EV fractions during pulmonary fibrosis and characterized their contribution to fibrosis. EVs accumulated 14 days after bleomycin challenge, correlating with decreased lung function and initiated fibrogenesis in healthy precision-cut lung slices. Label-free proteomics of bronchoalveolar lavage fluid EVs (BALF-EVs) collected from mice challenged with bleomycin or control identified 107 proteins enriched in fibrotic vesicles. Multiomic analysis revealed fibroblasts as a major cellular source of BALF-EV cargo, which was enriched in secreted frizzled related protein 1 (SFRP1). Sfrp1 deficiency inhibited the activity of fibroblast-derived EVs to potentiate lung fibrosis in vivo. SFRP1 led to increased transitional cell markers, such as keratin 8, and WNT/β-catenin signaling in primary alveolar type 2 cells. SFRP1 was expressed within the IPF lung and localized at the surface of EVs from patient-derived fibroblasts and BALF. Our work reveals altered EV protein cargo in fibrotic EVs promoting fibrogenesis and identifies fibroblast-derived vesicular SFRP1 as a fibrotic mediator and potential therapeutic target for IPF.

Compartmentation is generally considered a feature of eukaryotic cells. One exception in the “prokaryotic world” is the marine hyperthermophilic Crenarchaeon Ignicococcus that exhibits a large inter-membrane compartment (IMC) between an inner (cytoplasmic) and an outer (cellular) membrane (CM and OCM) [1]. In addition, I. hospitalis supports the propagation of another archaeon - Nanoarchaeum equitans - on its surface [2]. Cryopreparation in combination with 3D methods (serial sectioning, FIB/SEM and electron tomography) enabled us to deliver a comprehensive insight into the anatomy of I. hopitalis and its contact to N. equitans. The 3D-models obtained, reveal a highly complex and dynamic endogenous membrane system with putative secretory function: The IMC makes up ~40% of the whole cell volume on average, but in few cells, it can reach an extent much larger than the volume of the cytoplasm. In the IMC, elongated protrusions of the cytoplasm are present. Apparently, these structures can constrict from or fuse with the CM or themselves. We also observed interactions of these protrusions with the OM via macromolecular, cylindrically shaped complexes. All interacting structures are connected via thin filaments (~3-6 nm in diameter), that span through the whole IMC and are presumably responsible for membrane dynamics. In addition, homologues in sequence and/or structure to eukaryotic proteins can be found in Ignicoccus, that are putatively involved in the system like their eukaryotic counterparts: small GTPases (Sar1/Arf like), Coatomer proteins, Sec61ß, proteins of the ESCRT-III system, a tethering complex component (Bet3) and the ATPase CDC48/p97 [3, 4]. Since Ignicoccus belongs to the recently proposed TACK superphylum, that is considered a sister group of eukaryotes [5], it is also tempting to speculate about a prokaryotic origin of the eukaryotic endogenous membrane system. Regarding the contact of N. equitans to I. hospitalis, a fusion of cytoplasms of both organisms was revealed, providing an important complement and explanation to recent proteomic [4], transcriptomic [6] and metabolomic [7] studies on this inter-archaeal system.

Early detection of acute lung injury (ALI) remains challenging, as conventional diagnostics rarely capture initial molecular changes. We therefore examined whether extracellular vesicles (EVs) in bronchoalveolar lavage fluid (BALF) can detect early regional injury and distinguish initial stress mechanisms in a porcine ALI model. Unilateral ALI was induced using Triton X-100, followed by six hours of mechanical ventilation with either fixed positive end-expiratory pressure (PEEP) at 5 cmH₂O or transpulmonary-pressure (TPP)-guided PEEP. Spatially separated BALF sampling allowed direct comparison between injured and contralateral mechanically stressed lungs. EV concentration and size distribution were quantified by nano-flow cytometry, while vesicular identity was confirmed by transmission electron microscopy and tetraspanin dot blot analyses. Furthermore, EV metrics were related to corresponding histologic injury scores. Across both PEEP strategies, EV concentration was markedly higher in both directly injured and contralateral mechanically stressed lungs compared with sham controls, indicating alveolar stress beyond the primary injury site. Median EV size differed significantly by injury type and correlated inversely with histologic injury, particularly with alveolar neutrophil infiltration. BALF-derived EV concentration and size may therefore provide complementary, spatially resolved molecular readouts of early ALI.