Fig 1.
Serial block-face scanning electron microscopy imaging of intact A. algerae spores.
(a) Representative data from SBFSEM imaging. Samples were serially sliced at 50 nm thickness (left), and a representative slice is shown (right). (b) Representative SBFSEM slice highlighting segmented organelles. Original micrograph is shown (left), as well as the same image with color overlays indicating segmented organelles (right): exospore (orange), endospore (yellow), PT (blue), vacuole (red), posterior polaroplast (purple), and anchoring disc (green). The anterior polaroplast is not segmented, as we do not clearly observe it. Magenta arrow indicates the thinnest part of the endospore layer where the anchoring disc is localized. (c) Representative 3D reconstruction of an A. algerae spore from SBFSEM data. Each color represents an individual organelle; color code as in (b).
Fig 2.
Configuration of the PT and other organelles in intact A. algerae spores.
(a) Representative 3D reconstruction of an A. algerae spore showing the relative orientations of the PT (blue), anchoring disc (green), posterior polaroplast (purple), and vacuole (red). (b) Measurement of the angle of PT coils relative to the anterior-posterior (A-P) axis of the spore. Schematic showing how the angle was measured between the A-P axis and the PT coil plane (left). Representative spore with the average angle and standard deviation annotated (right, n = 18). See also S1D Fig. (c) Chirality of the A. algerae PT. Red arrows indicate the right-handed helix of the PT. (d) Heterogeneity of PT ends observed in A. algerae PTs (n = 20). Black arrows indicate the location of the PT end. (e) Transmission electron microscopy (TEM) section of an A. algerae spore showing interdigitation between vacuole membrane (red) and the PT, and position of the nuclei (N, cyan) relative to the vacuole. Inset shows the region boxed in orange.
Fig 3.
Serial block-face scanning electron microscopy imaging of E. hellem spores and comparison with A. algerae.
(a) A representative slice from SBFSEM imaging of E. hellem spores. (b) Representative SBFSEM slice highlighting segmented organelles. Original micrograph is shown (left), as well as the same image with color overlays indicating segmented organelles (right): exospore (orange), endospore (yellow), PT (blue), anterior polaroplast (pink), and anchoring disc (green). Magenta arrow indicates the thinnest part of the endospore layer where the anchoring disc is localized. (c) Representative 3D reconstruction of an E. hellem spore from SBFSEM data. Each color represents an individual organelle; color code as in (b). We did not observe any tangled PT ends in E. hellem spores. The average angle of E. hellem PT coils relative to the A-P axis is annotated with a standard deviation (n = 20, See also S1D Fig). The angle measurement was performed as in Fig 2B. (d) Quantification of the distance between the PT coils in A. algerae and E. hellem. Error bars represent standard deviation (n = 18 for A. algerae and n = 20 for E. hellem), ****p<0.0001 (unpaired Student’s t-test). (e) Position of the anchoring disc (AD, green) relative to the spore coat. Black line indicates the anterior-posterior (A-P) axis of the spore. Coincidence of the AD and the A-P axis was scored as “center”, indicating that the AD is at the apical tip of the spore. Separation of the AD from the A-P axis was scored as “off-center”, indicating that the AD is not centered at the apical tip of the spore (n = 18 for A. algerae and n = 20 for E. hellem).
Fig 4.
Live-cell imaging of PT germination.
(a) Kymographs showing representative spore germination events from three microsporidian species: A. algerae, E. hellem, and E. intestinalis. Scale bars for distance and time intervals are shown on each kymograph. Three phases of the germination process are color-coded. The A. algerae PT appears to have a wavy pattern as it emerges from the spore, possibly forming a spiral, while the E. hellem and E. intestinalis PTs are less wavy in nature. As infectious cargo emerges from the distal end of the PT, the amplitude of the waveform is reduced, as has been previously noted[24]. After the A. algerae PT is fully extended, the distal end of the fired PT is curved and forms a hook-like structure, as previously described[25]. See also S3–S5 Videos. (b) Quantification of the PT length as a function of time for A. algerae (black), E. hellem (blue), and E. intestinalis (orange) (n = 20 for each species). The raw data are shown as faint lines with time points indicated as circles. The overall trends of each species were fitted (see Methods) and are represented as thick lines in the corresponding color. (c) Time taken for PT to extend to ≥ 90% (TEXT90) of its maximum length (n = 20 for each species). ****p<0.0001, **p = 0.0096 (unpaired Student’s t-test). (d) Average maximum velocity of PT extension calculated as described in Methods (n = 20 for each species). **p = 0.006; ns, not significant (unpaired Student’s t-test). (e) Average maximum acceleration of PT extension (n = 20 for each species). ****p<0.0001; ns, not significant (unpaired Student’s t-test). (f) Shortening of the E. hellem PT after the full extension (maximum length) has been reached and cargo ejected. Representative phase-contrast micrographs are shown, and orange lines represent the length of the PT quantified from the micrographs. (g) Reduction of PT length measured 3 seconds after sporoplasm ejection, as a percentage of the maximum PT length (n = 20 for each species, see other representations of these data in S9A and S9B Fig). The PT shortens by 3%, 24%, and 5% for A. algerae, E. hellem, and E. intestinalis, respectively. ****p<0.0001 (One sample t-test). (h) Comparison of the PT length for E. hellem, in dormant spores, obtained from SBFSEM (labeled “inside”) and the maximum PT length after germination, obtained from live-cell optical microscopy experiments (labeled “outside”) (n = 20, see Methods for how these measurements were made). ****p<0.0001 (unpaired Student’s t-test). All error bars in this figure represent standard deviations.
Fig 5.
Live-cell imaging of nuclear transport through the PT.
(a) Image of a fixed germinated A. algerae spore stained with a nuclear dye, NucBlue, overlaid with a phase-contrast image of the same spore. (b) Schematic diagrams show two possible hypotheses of how large nuclei may travel through the narrow PT. Nucleus is depicted in blue; in “hypothesis 1” it is drawn as an oval, to scale with the PT (nucleus diameter = ~0.7 μm; PT diameter = ~100 nm). (c) Time-lapse images of the two A. algerae nuclei inside the spore during PT germination, with a time interval of 36 ms. Nuclei are pre-stained with NucBlue, and white light was applied in order to observe the PT firing event simultaneously. Red arrow indicates the frame in which PT firing is initiated; black arrow indicates the frame in which the nuclei begin to leave the spore body. Inset highlights the two nuclei, labeled with numbers and color overlays, from the frame boxed in orange. (d) Kymograph of nuclear translocation through the PT, with a time interval of 50 ms. (e) Quantification of the aspect ratio of the nuclei during transport (inside the tube) and after being expelled (outside the tube), which represents the extent of nuclear deformation during the process. Error bars represent standard deviation (n = 7), ****p<0.0001 (paired Student’s t-test).
Fig 6.
Model for PT germination and nuclear transport through the PT.
(a) Model for 3-dimensional organization of a microsporidian spore. Combining our SBFSEM and 2D TEM data for both A. algerae and E. hellem, we generate a composite 3D model that shows spatial organization of organelles inside the microsporidian spore. Elements that were not observed in the A. algerae SBFSEM data (anterior polaroplast and nuclei) have been added manually and are in gray. The anterior part of the PT (blue) is straight and connected to the anchoring disc (green). This part of the PT is surrounded by the anterior (gray) and posterior (purple) polaroplast. The posterior part of the PT is coiled and packed at an angle relative to the A-P axis. The PT resembles a rib cage around the vacuole (red) and nuclei (gray). The vacuole is bowl shaped, and localized beneath the nuclei. (b) Model for PT germination and cargo transport in A. algerae. (1) The spore is triggered in the presence of a stimulus (in vivo triggers remain largely unknown; in vitro triggers are described in Methods). (2) PT firing is initiated at the thinnest part of the spore coat, where the anchoring disc is localized. (3) After initiation of PT firing and during Phase I (PT elongation), the nuclei, and presumably other organelles, are reoriented. (4) At the stage in which the PT is extended to just past 50%, the nuclei deform to fit into the PT and exit the spore. (5) Nuclei (and likely other cargo) are translocated through the PT, at a speed comparable to that of PT extension. (6) The nuclei exit the PT and regain a circular shape at the tip of the PT.