Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 108 (5), 1104-13

Cytoskeletal Pinning Controls Phase Separation in Multicomponent Lipid Membranes

Affiliations

Cytoskeletal Pinning Controls Phase Separation in Multicomponent Lipid Membranes

Senthil Arumugam et al. Biophys J.

Abstract

We study the effect of a minimal cytoskeletal network formed on the surface of giant unilamellar vesicles by the prokaryotic tubulin homolog, FtsZ, on phase separation in freestanding lipid membranes. FtsZ has been modified to interact with the membrane through a membrane targeting sequence from the prokaryotic protein MinD. FtsZ with the attached membrane targeting sequence efficiently forms a highly interconnected network on membranes with a concentration-dependent mesh size, much similar to the eukaryotic cytoskeletal network underlying the plasma membrane. Using giant unilamellar vesicles formed from a quaternary lipid mixture, we demonstrate that the artificial membrane-associated cytoskeleton, on the one hand, suppresses large-scale phase separation below the phase transition temperature, and, on the other hand, preserves phase separation above the transition temperature. Our experimental observations support the ideas put forward in our previous simulation study: In particular, the picket fence effect on phase separation may explain why micrometer-scale membrane domains are observed in isolated, cytoskeleton-free giant plasma membrane vesicles, but not in intact cell membranes. The experimentally observed suppression of large-scale phase separation much below the transition temperatures also serves as an argument in favor of the cryoprotective role of the cytoskeleton.

Figures

Figure 1
Figure 1
FtsZ-YFP-MTS localizes to Ld domains. (A) Vesicles composed of DOPC/DOPG/eSM/Chol at 2.5:2.5:3:2 at various temperatures. At 37°C, the membrane is homogeneous. (B) Partitioning of Lo marker (DSPE-PEG-KK114) and Ld marker (Fast DiI). (C) Intensity profile across the GUV shown in (B). (D) FtsZ-YFP-MTS binds exclusively to the Ld phase marked by Fast DiI and avoids the Lo phase marked by DSPE-PEG-KK114. (E) Intensity profile across the GUV shown in (D). (F) FtsZ-YFP-MTS meshwork localizes to the Ld phase marked by Fast DiI. Temperature (B–F): 25°C. Scale bars: 10 μm. To see this figure in color, go online.
Figure 2
Figure 2
Quaternary lipid system with FtsZ-YFP-MTS binding to the Ld phase. To see this figure in color, go online.
Figure 3
Figure 3
Effect of FtsZ meshwork on phase separation: Presence of dense FtsZ meshwork increases the phase transition temperature and inhibits growth of larger domains at temperatures much below phase transition temperatures. Note that the GUV is not immobilized and moves and reorients between the images corresponding to the different temperatures. Scale bar: 5 μm. To see this figure in color, go online.
Figure 4
Figure 4
FtsZ meshwork increases the temperature range for membrane phase coexistence. Percentage of vesicles displaying phase separation in the presence and absence of FtsZ meshwork at different temperatures. Presence of cytoskeleton increases the temperature of miscibility for the membrane. Curves are drawn as a guide for the eye.
Figure 5
Figure 5
Presence of sparse FtsZ network has no effect on the phase transition temperature. Scale bar: 10 μm. To see this figure in color, go online.
Figure 6
Figure 6
Effect of the mesh size of the FtsZ network on the character of phase separation in the membrane. (A) Lo domain size as a function of the mesh size of the network formed by the FtsZ-YFP-MTS (symbols). The dependence y = x is shown as a guide for the eye (solid line). (B and C) Examples of the effect of dense (B) and sparse FtsZ network (C) on the domain sizes and shapes. Temperature: 25°C. Scale bars: 10 μm. To see this figure in color, go online.
Figure 7
Figure 7
The effect of meshwork geometry and filament stiffness on domain shape. (A) Elongated voids result in similarly shaped domains. (B) Upon addition of small amounts of MinC, ([MinC] = 0.4 μM, [MinC]/[FtsZ] ≈ 0.4), which reduces the stiffness of the filaments, domains attain a round shape (see text for discussion). The images in the rightmost column show the enlarged view of the region of interest marked with the white square. Temperature: 25°C. Scale bar: 10 μm. To see this figure in color, go online.
Figure 8
Figure 8
Effect of removal of the FtsZ filament network on phase separation in the membrane. (A) Confocal image of a pole of GUV showing assembled filaments as well as phase separation (top). On addition of MinC ([MinC] = 1 μM, [MinC]/[FtsZ] ≈ 1), a depolymerase for FtsZ, the filaments are removed from the GUV, as seen by the absence of fluorescence on the GUV. Immediately after the removal of the FtsZ network, the domains become mobile, but their size is initially the same as in the presence of filaments. Mobile domains then start to coalesce and 5 min later, large domains are formed on naked GUVs. (B) A time-lapse montage showing coalescence of domains upon removal of filaments by MinC. (C) Average domain size before and after addition of MinC. This graph represents data from 5–10 domains observed on each of 10 different vesicles. Scale bars, (A) 10 μm (B) 3 μm. To see this figure in color, go online.
Figure 9
Figure 9
MC simulations of a two-component lipid membrane in the absence and presence of a membrane skeleton: (A) Phase diagram of DMPC/DSPC lipid membrane (adopted from (6)). Lipid state binodal (solid blue curves), lipid state spinodal (dashed red curves), and lipid demixing curves (gray solid curves) (for definition and discussion, see (6)). The critical point is located at the critical composition DMPC/DSPC 20:80 and the critical temperature Tc = 320.5 K. Symbols on the graph denote the temperatures and lipid compositions for which MC simulations were carried out in this work and presented in (D) and (E). (B) Color codes representing the lipids and their conformational states on the lattice. (C) Geometry of the network of filaments interacting with the membrane in simulations. The fraction of sites along the filaments, which interact with the membrane (network pinning density) is 25%. (D) Effect of the cytoskeleton on the phase separation in the lipid membrane with the composition exhibiting an abrupt phase transition from the Ld (fluid) state to the Ld-solid ordered (fluid-gel) coexistence. The lipid composition is DMPC/DSPC 50:50. The corresponding transition temperature is Tt = 318.7 K. Images (af) and (gl) correspond to the free membrane and membrane interacting with the cytoskeleton, respectively. Simulations were carried out at the following temperatures: T = Tt (a, g), T = Tt – 1 K (b, h), T = Tt – 3 K (c, i), T = Tt – 5 K (d, j), T = Tt – 9 K (e, k), and T = Tt – 13 K (f, l). (E) Effect of the cytoskeleton on the phase separation in the lipid membrane with the composition exhibiting a continuous phase transition from the Ld (fluid) state to the Ld-solid ordered (fluid-gel) coexistence via a critical point. The lipid composition is DMPC/DSPC 20:80. The temperature is Tc = 320.5 K. Images (af) and (gl) correspond to the free membrane and membrane interacting with the cytoskeleton, respectively. Simulations were carried out at the following temperatures: T = Tc + 2 K (a, g), T = Tc + 1 K (b, h), T = Tc (c, i), T = Tc – 1 K (d, j), T = Tc – 2 K (e, k), and T = Tc – 3 K (f, l). To see this figure in color, go online.

Similar articles

See all similar articles

Cited by 15 articles

See all "Cited by" articles

Publication types

MeSH terms

LinkOut - more resources

Feedback