Volume 110, Issue 7, Pages 1551-1562 (April 2016) Role of Inverse-Cone-Shape Lipids in Temperature-Controlled Self-Reproduction of Binary Vesicles  Takehiro Jimbo, Yuka Sakuma, Naohito Urakami, Primož Ziherl, Masayuki Imai  Biophysical Journal  Volume 110, Issue 7, Pages 1551-1562 (April 2016) DOI: 10.1016/j.bpj.2016.02.028 Copyright © 2016 Biophysical Society Terms and Conditions

Figure 1 Sequence of z-projection of 3D images of a DPPC/DLPE binary GUV in the vesicle division process driven by the heating-cooling cycle between 30 and 50°C. (A–E) Deformations of the first-generation vesicle (G1), where the mother produced daughters. (F–J) Second-generation deformations (G2), where the daughters produced granddaughter vesicles. Scale bar in (A) represents 10 μm. The extracted shapes obtained by relaxing the bending energy at fixed v and Δa are shown in (A′)–(J′). Biophysical Journal 2016 110, 1551-1562DOI: (10.1016/j.bpj.2016.02.028) Copyright © 2016 Biophysical Society Terms and Conditions

Figure 2 Pore formation of divided daughter GUV during the cooling process (another daughter vesicle is indicated by a triangle). The arrow in the 9.040 s frame indicates the pore (see also Movie S2). Biophysical Journal 2016 110, 1551-1562DOI: (10.1016/j.bpj.2016.02.028) Copyright © 2016 Biophysical Society Terms and Conditions

Figure 3 Time dependence of surface area (top panel), volume (middle panel), and temperature (bottom panel) of a binary GUV in the cyclic deformation-division process. (A–J) Top and middle panels correspond to vesicles shown in (A)–(J) in Fig. 1. (Solid and open symbols) Large and the small daughter formed after the first division, respectively, and their daughters; relative area and volume changes on heating and cooling are indicated in the top and middle panels. Biophysical Journal 2016 110, 1551-1562DOI: (10.1016/j.bpj.2016.02.028) Copyright © 2016 Biophysical Society Terms and Conditions

Figure 4 Deformation pathways of three DPPC/DLPE GUVs (V1, V2, and V3) in the (v, Δa) plane (middle panel). For vesicle V3, pathways for the first (G1), the second (G2a and G2b), and the third division (G3) are shown. Lines O, P, and L represent the oblate, prolate, and budded limiting shape branches, respectively. (Top panel) Micrographs of daughter (G1), granddaughter (G2a), and great-granddaughter (G3) of vesicle V3; scale bars, 10 μm. To see this figure in color, go online. Biophysical Journal 2016 110, 1551-1562DOI: (10.1016/j.bpj.2016.02.028) Copyright © 2016 Biophysical Society Terms and Conditions

Figure 5 (a) Phase diagram in the (v, Δa˜0 ) plane predicted by the ADE-SC model with the ratio of nonlocal and local bending constants of q = 3. (Solid lines) Discontinuous transitions between the stomatocyte and the oblate shape (Dsto/obl), the oblate and the prolate shape (Dobl/pro), and the prolate and the pear shape (Dpro/pea). The budded limiting shape line is labeled by L. The observed six deformation trajectories in Fig. 4 are shown by dashed lines connecting the oblate-prolate transition and the budded limiting shape (open circles). The legend also contains the radii of the spherical vesicles at the initial stage, R0. (Thick black line) Theoretical deformation trajectory obtained using our geometrical model. (Dotted line) Estimated deformation trajectory for a vesicle with a symmetric bilayer. (b) Phase diagram in the (v2/3, Δa˜0 v) plane to emphasize the relationship between Δa˜0 and v in Eq. 13. To see this figure in color, go online. Biophysical Journal 2016 110, 1551-1562DOI: (10.1016/j.bpj.2016.02.028) Copyright © 2016 Biophysical Society Terms and Conditions

Figure 6 Schematic representation of vesicle morphology changes from steps 1 to 4 including pore formation. During pore formation, lipids redistribute through the rim of pore. To see this figure in color, go online. Biophysical Journal 2016 110, 1551-1562DOI: (10.1016/j.bpj.2016.02.028) Copyright © 2016 Biophysical Society Terms and Conditions

Figure 7 Relationship between the vesicle radius R0 and the initial renormalized, preferred, reduced monolayer area difference, Δa˜0ini. To see this figure in color, go online. Biophysical Journal 2016 110, 1551-1562DOI: (10.1016/j.bpj.2016.02.028) Copyright © 2016 Biophysical Society Terms and Conditions

Figure 8 Deformation of a DPPC/DLPC/cholesterol = 8:2:2 ternary GUV caused by heating from 30 to 50°C. Scale bar, 10 μm. The extracted relaxed shapes obtained by 3D analysis are shown. Biophysical Journal 2016 110, 1551-1562DOI: (10.1016/j.bpj.2016.02.028) Copyright © 2016 Biophysical Society Terms and Conditions