Figure S7.

Intercellular Communication Was Reduced upon Different Types of Stress, and SJs in Other Cyanobacteria Share Similar Architecture, Related to Figure 7

(A and B) Treatment with H₂O₂ was tested as an alternative stress. Calcein-stained cells were treated with 5 mM or 10 mM H₂O₂, followed by washing with fresh medium and further incubation for 3 h when indicated. Molecular exchange of the treated cells was analyzed by FRAP (A). Similar to CCCP treatment, wild-type cells showed a reduced level of communication upon H₂O₂ treatment, while the ΔfraC-ΔfraD mutant was impaired in ceasing molecular exchange upon H₂O₂ treatment. Shown are cumulated results from at least two independent experiments. Numbers within the bars indicate the number of analyzed cells n from different filaments. To check the viability of wild-type cells after H₂O₂ treatment, 10 μL spots were placed onto a Bg11 agar plate and incubated for three days (B). Cells treated with 5 mM H₂O₂ and washed afterward grew comparable to the untreated control.

(C–F) Incubation in the dark was tested as an alternative stress. Cells were incubated in the dark for 28 h prior to FRAP analyses (C). FRAP indicated that a significant fraction of wild-type cells ceased to communicate upon incubation in the dark, while the ΔfraC-ΔfraD mutant was less effective in ceasing molecular exchange. Shown are cumulated results from at least two independent FRAP experiments. Numbers within the bars indicate the number of analyzed cells n from different filaments. The color code is identical to (A). For ECT imaging (D-E), cells were incubated in the dark for 24 h prior to plunge freezing. Shown is a subtomogram average of SJs from Anabaena wild-type cells. The average shows SJs in the closed state (no individual arches are detectable). The dashed line in (D) indicates the position of the cross sectional view of the cap shown in (E). A surface representation of the subtomogram average is shown in (F). Bars, 10 nm.

(G and H) SJs in other cyanobacteria share a similar architecture. We also cryoFIB-milled and imaged two further cyanobacterial representatives, revealing the presence of SJs (arrowheads) without fundamentally different SJ architecture as compared to Anabaena. Importantly, SJ-related genes (amiC, fraC/fraD, sjcF1 and the C-terminal domain of sepJ) are also present in the genomes of these cyanobacteria. CM, cytoplasmic membrane; PG, septal peptidoglycan; TM, thylakoid membrane. Bars, 100 nm.