Structure and Function of a Bacterial Gap Junction Analog - PubMed (original) (raw)
Structure and Function of a Bacterial Gap Junction Analog
Gregor L Weiss et al. Cell. 2019.
Abstract
Multicellular lifestyle requires cell-cell connections. In multicellular cyanobacteria, septal junctions enable molecular exchange between sister cells and are required for cellular differentiation. The structure of septal junctions is poorly understood, and it is unknown whether they are capable of controlling intercellular communication. Here, we resolved the in situ architecture of septal junctions by electron cryotomography of cryo-focused ion beam-milled cyanobacterial filaments. Septal junctions consisted of a tube traversing the septal peptidoglycan. Each tube end comprised a FraD-containing plug, which was covered by a cytoplasmic cap. Fluorescence recovery after photobleaching showed that intercellular communication was blocked upon stress. Gating was accompanied by a reversible conformational change of the septal junction cap. We provide the mechanistic framework for a cell junction that predates eukaryotic gap junctions by a billion years. The conservation of a gated dynamic mechanism across different domains of life emphasizes the importance of controlling molecular exchange in multicellular organisms.
Keywords: cell-cell connections; cyanobacteria; electron cryotomography; fluorescence recovery after photobleaching; membrane trafficking; multicellularity; septal junctions; subtomogram averaging.
Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.
Conflict of interest statement
The authors declare no competing interests.
Figures
Graphical abstract
Figure S1
CryoFIB-Milling of Anabaena Filaments, Related to Figure 1 (A) Shown is a cryo-scanning electron microscopy (SEM) image of an EM grid with plunge-frozen Anabaena filaments. (B) Shown is one example for the preparation of a lamella through a filament. The target was identified in SEM view (SEM view, pre-milling). The focused ion beam (FIB) was used to inspect the same filament from a shallow angle (FIB view) and to choose a milling pattern (red box, upper panel). Material was then removed using the FIB and inspected again (FIB view, lower panel). The final lamella was inspected again by SEM (SEM view, post-milling). (C) The procedure was repeated for 9-24 lamellae. Shown is a SEM overview image of a grid area with seven lamellae. (D) Two examples of cryoFIB-milled lamellae through Anabaena filaments. Details like cell outline (arrowheads) or thylakoid membranes are already detectable.
Figure 1
In Situ Architecture of Septal Junctions Reveals Tube, Plug, and Cap Modules (A and B) Cryotomograms (magnified views in boxes) of a FIB-milled Anabaena filament. The two different slices at different Z-heights show the septum between adjacent vegetative cells. Multiple SJs were seen crossing the septum (arrowheads). The SJ lengths were precisely adjusted to the septum thickness. CM, cytoplasmic membrane; OM, outer membrane; PB, phycobilisomes; PG, septal peptidoglycan; TM, thylakoid membranes. Bars, 100 nm. Shown are projections of 13.5 nm-thick slices. (C–F) Subtomogram averaging of SJ ends revealed three structural modules: tube, cap, and plug. Shown is a 0.68 nm-thick tomographic slice through the average (C), a schematic representation of SJ modules (D; modules segmented in different shades of green), and oblique (E) and top (F) views of a surface representation (modules were segmented to match colors in D). The cap consisted of a ceiling that was held by five arches. Bars, 10 nm. See also Figures S1 and S2 and Videos S1 and S2.
Figure S2
Analysis of SJs in Anabaena Wild Type, Related to Figure 1 (A) Shown is a cryotomogram (9.45 nm-thick slice) of a SJ (arrowhead). The area indicated by the red box was used to calculate a density plot. CM, cytoplasmic membrane; PG, septal peptidoglycan. Bar, 10 nm. (B) The density plot of the area indicated in (A) revealed that the SJ tube had a higher density than the surrounding PG and the tube lumen had a lower density than the PG. (C) SJ length distribution in Anabaena PCC 7120 wild-type. SJ lengths were measured from plug to plug and their occurrence was plotted in the graph. n = 208 SJs (D) Measurements of SJ structural features. The indicated measurements were performed using the subtomogram average of the SJ end shown in Figure 1 C/E/F. CM, cytoplasmic membrane; PG, septal peptidoglycan. Bar, 10 nm. (E) The SJ cap module has 5-fold rotational symmetry. The cross-sectional view (position indicated in the longitudinal view by dashed line) of the non-symmetrized subtomogram average indicated a rotational 5-fold symmetry of the cap module. To further investigate this, different symmetries (indicated) were applied to the non-symmetrized subtomogram average (1.35 nm pixel size). The strongest reinforcement of densities was seen in the 5-fold symmetrized average. Bar, 10 nm.
Figure 2
Intercellular Communication Ceases upon Ionophore Treatment in a Reversible Manner (A) FRAP analysis of cells that were stained with fluorescent calcein. In the control experiment (control cells were treated with DMSO to exclude effects of the solvent), all cells showed full recovery of fluorescence after bleaching. After treatment with the ionophore CCCP (50 μM in DMSO), the bleached cells showed four different types of FRAP responses: “full recovery,” “slow increase” (delayed recovery to <50% of original fluorescence), “only two cells” (exchange of calcein only with one neighboring cell), and “no communication” (no recovery). For each FRAP response, representative images are shown at three time points (5 s before bleaching, ∼0.5 s after bleaching, 30–60 s after bleaching). Arrowheads point to the bleached cells. Anabaena was apparently able to control communication upon challenging the proton motif force, because the majority of CCCP-treated cells showed “no communication.” Bar, 5 μm. (B) Fluorescence recovery curves corresponding to the four FRAP responses that were observed in (A) (color scheme identical to A). Time point t = 0 shows the analyzed cell directly after bleaching. (C) Cell-cell communication after increasing the concentration of CCCP (color scheme identical to A). The effect of CCCP on cell-cell communication was concentration-dependent for CCCP concentrations between 0.5–50 μM. In the control experiment, cells were treated with 0.002% DMSO. Numbers within the bars indicate the number of analyzed cells (n) from different filaments and represent cumulated results from at least two independent cultures (except for 0.1 μM CCCP). (D) Recovery of cell-cell communication after incubation in fresh medium lacking CCCP and in the presence of chloramphenicol (color scheme identical to A). Regaining cell-cell communication was independent of de novo protein synthesis, “+” and “−” indicate the presence and absence of CCCP, washing in fresh medium, and chloramphenicol. Numbers in bars indicate number of analyzed cells (n). Shown are cumulated results from at least two independent cultures. See also Figure S3 and Videos S3, S4, and S5.
Figure S3
FRAP Response Distributions of Independent Cultures and Viability after CCCP Treatment, Related to Figure 2 (A and B) Shown are the distributions of FRAP responses of independent cultures (one bar represents one culture), to complement the cumulative results that are shown in Figure 2. Numbers within the bars indicate the number of analyzed cells n from different filaments. The shown graph in (A) corresponds to Figure 2C. The shown graph in (B) corresponds to Figure 2D. (C) Cells that were washed after CCCP treatment were viable. Anabaena was grown for three days on a Bg11 agar plate, resuspended to OD750 = 1.2, split and processed by different treatments (indicated on the left, details described in STAR Methods). Subsequently, 10 μL of different concentrations of the cultures were spotted onto a solid Bg11 agar plate and incubated for two days before taking a picture of the plate (shown). No difference in viability was observed between the control (no CCCP treatment) and CCCP-treated/washed cells. All spots were grown on the same plate, white lines were introduced for a clear view. (D) The ATP level of Anabaena cells is not affected by CCCP treatment. The ATP content of Anabaena cells was determined in cells treated with only DMSO (control to mimic the addition of calcein and CCCP), and cells treated with DMSO and different concentrations of CCCP for 90 min or 2 min as indicated in graph. The measured ATP level was normalized to OD750 = 0.6. Bars show the mean ± SD of two biological replicates with two technical replicates (one biological replicate for 2 min incubation with 50 μM CCCP). Differences between CCCP treated cells compared to DMSO control cells were not significant (Student’s t test).
Figure 3
Ceased Intercellular Communication after Ionophore Treatment Coincides with a Major Structural Rearrangement of the Septal Junction Cap (A) Shown is a 13.5 nm-thick slice through a cryotomogram (magnified view in box) of the septal area of a CCCP-treated Anabaena filament. SJs are indicated by arrowheads. CM, cytoplasmic membrane; PG, septal peptidoglycan; TM, thylakoid membranes. Bar, 100 nm. (B–E) Subtomogram averaging of SJs in the CCCP-treated non-communicating “closed” state (B–D) revealed major structural rearrangements in the cap module, compared to the “open” state (E). Shown are surface representations (B and C), and longitudinal and cross-sectional slices (0.68 nm) through the averages (D and E). Sliced positions are indicated in (D) and (E). Bars, 10 nm. See also Figure S4 and Videos S4 and S5.
Figure S4
SJ Gating Is Fast and Reversible, Related to Figure 3 (A) Shown are Fourier Shell Correlation (FSC) curves of two half-datasets of subtomogram averages of SJs in open (green) and closed (orange) states, respectively. The estimated resolution of the averages was ∼28 Å. (B–E) Speculative model of cap closure by arch rotations. (B) Perpendicular cross-section (0.68 nm thickness) of the subtomogram average of the SJ in the open conformation. The arches are represented by green ellipses. (C) Schematic indicating a 30° rotation of each arch around a rotation center (orange). (D) Schematic indicating the arches after the rotation indicated in (C). The result was a closed circle. (E) Overlay of the model in the closed state with a perpendicular cross-section (0.68 nm thickness) of the SJ in the closed conformation. Bars, 10 nm. (F–H) The structural rearrangement of SJs upon ionophore treatment is reversible. Wild-type Anabaena cells were incubated for 90 mins with 50 μM CCCP. Afterward, cells were washed three times, incubated in fresh medium for 2.5 h and plunge frozen. The subtomogram average revealed SJs in their open state (F/G). Dashed line in (F) indicates the position of the cross sectional view shown in (G). The five arches of the SJ cap, a hallmark for the SJ open state, are detectable. Surface representation (H) of the SJ cap generated from the subtomogram average shown in (F/G). Bars, 10 nm. (I–L) Intercellular communication is impaired within seconds upon CCCP treatment. Wild-type Anabaena cells were incubated with CCCP. FRAP analyses [shown in (I)] were performed either between 1.5 min and 4 min after the addition of CCCP, or more than 90 min after the addition of CCCP (data from Figure 2). Already after the short incubation time of ≤ 4 min, the majority of cells already ceased communication. Numbers within the bars indicate the number of analyzed cells n from different filaments. (J-L) show a subtomogram average (J/K) and surface representation (L) of SJs from Anabaena wild-type cells that were treated with CCCP (50 μM) for 45 s. The average [longitudinal view in (J); cross section in (K)] shows SJs in their closed state [dashed line indicates the position of the cross sectional view in (K)]. No individual arches are detectable. Bars, 10 nm.
Figure 4
AmiC1, SepJ, and SjcF1 Mutants Are Impaired in Intercellular Communication but Nevertheless Able to Control Molecular Exchange (A–C) Cryotomograms (shown are 13.5 nm-thick projections; bars, 100 nm) of different Anabaena mutants. Subtomogram averages of SJs (insets in A–C; bars, 10 nm) showed that neither the amiC1 mutant SR477 (A), nor Δ_sepJ_ (B), nor the sjcF1 mutant (C) were missing structural modules. CM, cytoplasmic membrane; PB, phycobilisomes; PG, septal peptidoglycan; TM, thylakoid membranes. (D) FRAP responses of the wild type and the mutants shown in (A)–(C). The amiC1 mutant SR477 and the sepJ mutant showed that compared to the wild type, a reduced fraction of cells was able to communicate already in the absence of CCCP (likely based on the lower total number of SJs). However, the open SJs of these mutants were able to close upon CCCP treatment, consistent with the unaltered SJ structure. The sjcF1 mutant was not impaired in closing its SJs upon CCCP treatment. “+” and “−” indicate the presence and absence of CCCP. Numbers within the bars indicate the number of analyzed cells (n) from different filaments. Results from at least two independent cultures were cumulated. See also Figure S5.
Figure S5
Details and Examples of Analyzed SJ Mutants and Their FRAP Response Distribution of Independent Cultures, Related to Figures 4 and 5 (A) SJ lengths in wild-type and mutant strains were measured from plug to plug and their frequency was plotted in the graphs. The data were also used to calculate the average SJ length. We also measured the septum thickness (as the shortest distance between the inner membranes within a tomogram of a septal region), showing an increase for all mutant strains. (B) Shown are the distributions of FRAP responses of independent cultures (one bar represents one culture), to complement the cumulative results that are shown in Figures 4 and 5. Numbers within the bars indicate the number of analyzed cells n from different filaments. +: 50 μM CCCP; ++: 200 μM CCCP (C) Further examples of cryotomograms showing SJs (black arrowheads) from different mutant strains. Shown are 13.5 nm thick slices. CM, cytoplasmic membrane; PG, septal peptidoglycan. Bars, 100 nm.
Figure 5
The Cap and Plug Modules Are Required to Control Intercellular Communication upon Ionophore Treatment (A–C) SJs from the Δ_fraC-Δ_fraD (A) and Δ_fraD_ (B) mutants were missing the cap and plug modules. SJs from Δ_fraC_ (C) showed a mixture of fully assembled and misassembled SJs (insets show magnified views; bars, 10 nm). Shown are 13.5 nm-thick sections through cryotomograms; bars, 100 nm. CM, cytoplasmic membrane; CP, cyanophycin; PB, phycobilisomes; PG, septal peptidoglycan; TM, thylakoid membranes. (D) FRAP experiments of the wild type and the mutants shown in (A)–(C). The CCCP-treated Δ_fraD_ and Δ_fraC_ single mutants showed a much smaller fraction of non-communicating cells than in the CCCP-treated wild type, indicating that the mutants were unable to gate communication. Strain CSVT2.768 is a complementation of the Δ_fraD_ mutant and showed wild type behavior. “+” and “−” indicate the presence and absence of CCCP. Numbers within the bars indicate the number of analyzed cells (n) from different filaments. Results from at least two independent cultures were cumulated (except for Δ_fraC-Δ_fraD treated with 200 μM CCCP). See also Figure S5.
Figure 6
FraD Localizes to the SJ Plug (A) Cryotomograms of Anabaena expressing gfp-fraD showed SJs with partially filled tubes (black arrowhead). Shown is a 13.5 nm-thick slice; bar, 100 nm. Subtomogram averaging (inset; bar, 10 nm) revealed an extra density (white arrowhead) situated in the SJ lumen and connected to the SJ plug (black arrow). CM, cytoplasmic membrane; PB, phycobilisomes; PG, septal peptidoglycan; TM, thylakoid membranes. (B) The difference map between subtomogram averages of septal junctions from Anabaena wild type and gfp-fraD expressing cells showed that the only major difference between these two structures was the extra density within the tube lumen, most likely corresponding to the GFP fusion. The cross sectional views (location indicated by dashed line) through the caps revealed that SJs from gfp-fraD expressing cells are in the open state. Bar, 10 nm. (C) Surface representation of the average of the SJ from the gfp-fraD_-expressing mutant showed that the cap was in the open state and similar to the wild type structure. Bar, 10 nm. (D) The Δ_fraD mutant CSVT2 complemented with gfp-fraD (CSVT2.779) or fraD (CSVT2.768) expressed from a plasmid was investigated by the FRAP assay under the stated conditions (“+” and “−” indicate the presence and absence of CCCP and washing in fresh medium). The gfp-fraD strain (CSVT2.779) was still able to control communication, even though reopening was less efficient. Numbers within the bars indicate the number of analyzed cells (n) from different filaments. Results from at least two independent cultures were cumulated. (E) The fluorescence recovery rate constant R was calculated from non-treated cells, showing a “full recovery” FRAP response. (n(WT) = 10, n(CSVT2.768) =14, n(CSVT2.779) = 13). The gfp_-fraD strain (CSVT2.779) showed a significantly reduced fluorescence recovery rate constant compared to wild type and the complemented Δ_fraD+fraD mutant (CSVT2.768). Significance was determined using Student’s t test in comparison to the wild type. A representative FRAP curve (bleaching at t = 0) for each strain is shown. See also Figure S6 and Video S6.
Figure S6
Examples of Cryotomograms Showing SJs of Anabaena Expressing gfp-fraD, Related to Figure 6 (A) Further examples of cryotomograms showing SJs (black arrowheads) from gfp-fraD expressing mutant. Shown are 13.5 nm thick slices. CM, cytoplasmic membrane; PG, septal peptidoglycan.Bars, 100 nm. (B) Shown is a Fourier Shell Correlation (FSC) curve of two half-datasets of the subtomogram average in Figure 6A, showing SJs from gfp-fraD expressing mutant.
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 Δ_fraC-Δ_fraD 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 Δ_fraC-Δ_fraD 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.
Figure 7
SJs Reversibly Gate Cell-Cell Communication by a Conformational Change SJs (green, open; orange, closed) of Anabaena are dynamic, gated cell-cell connections, which reversibly block intercellular molecular diffusion along the filament upon different types of stress. The Δ_fraD_ mutant was missing the cap and plug modules, consistent with the inability to close SJs upon stress. FraD was shown to localize to the plug module. See also Figure S7.
References
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