Figure 5. Hypotheses that can potentially explain the two-stage translocation of the cargo.

(A) Kymograph of nuclear transport inside the PT. Nuclei were stained with NucBlue prior to germination, and imaged using fluorescence microscopy. Previously deposited data from Jaroenlak et al., 2020 were used in this figure. A two-stage process is observed for nuclear translocation, with a long pause in the middle. The second stage of nuclear movement is overlaid with red, and the asterisk indicates the beginning of the second stage movement, in which the nuclei are expelled out of the PT. (B) Quantification of the nuclear position relative to spore coat over time (n=4). (C) 3D reconstructions of incompletely germinated and germinated spores from SBF-SEM data. 100% of spores in which the nuclei have been expelled are buckled (Supplementary file 6). The translocation of nuclei at the final stage can be explained by spore buckling. (D) Volumes of ungerminated and germinated spores calculated from SBF-SEM 3D reconstructions. Ungerminated: mean=8.78 μm³, std=1.41 μm³, n=19; Germinated: mean=5.52 μm³, std=1.03 μm³, n=14; p<0.0001. (E) Schematic model of an A. algerae spore used for calculating the spore wall buckling pressure, the relevant parameters used in the calculation and the formulae. Using the theory of elastic shell buckling (see text for detail), we showed that the pressure built up during the PT firing process is enough to buckle the spore wall, and the predicted buckling volume is enough to push cytoplasmic content in PT forward by 129–261 μm. (F) The predicted time series of pressure from Model 4 and Model 5 (n=54), overlaid with the critical pressure of spore wall buckling, water cavitation pressure and bubble nucleation. All three phenomena can cause volume displacement at the later stage of the germination process, and provide a driving force to push the cargo/nuclei forward. Model 5 is more compatible with experimental data than Model 4. The downward arrows indicate the mean time when the negative pressure first reaches the critical pressure. (detailed numbers mentioned in the main text.) (G) Theoretical predictions and experimental measurements from orthogonal approaches are compiled and are in agreement with each other. We obtained the prediction based on spore wall buckling theory and hydrodynamic energy dissipation theory, and we compiled the experimental observations from the SBF-SEM data. Symbols: ${\displaystyle R_{\text{spore}}}$: spore radius; ${\displaystyle \mathrm{\Delta }V}$: volume changes of spore after buckling; ${\displaystyle t}$: spore wall thickness; ${\displaystyle E}$: Young’s modulus of the spore wall; ${\displaystyle \nu }$: Poisson ratio of the spore wall; ${\displaystyle W}$: work; ${\displaystyle \mathrm{\Delta }x}$: predicted fluid displacement distance; ${\displaystyle L_{\text{PT}}}$: full length of the ejected PT.

Figure 5—figure supplement 1. Dependence of spore buckling probability on the threshold pressure of spore wall buckling.

The x-axis shows the buckling threshold we choose while the y-axis shows the predicted probability of buckling. The two curves are predictions from Model 4 and Model 5. The three vertical dashed lines show the minimum (51 atm), geometric averaged (141 atm), and maximum (390 atm) predicted buckling threshold.