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. 2018 Sep:215:18-28.
doi: 10.1016/j.chemphyslip.2018.06.002. Epub 2018 Jul 22.

A simple supported tubulated bilayer system for evaluating protein-mediated membrane remodeling

Noah A Schenk  1 , Peter J Dahl  1 , Michael G Hanna 4th  2 , Anjon Audhya  2 , Gregory G Tall  1 , Jefferson D Knight  3 , Arun Anantharam  4
Affiliations

A simple supported tubulated bilayer system for evaluating protein-mediated membrane remodeling

Noah A Schenk et al. Chem Phys Lipids. 2018 Sep.

Abstract

Fusion and fission of cellular membranes involve dramatic, protein-mediated changes in membrane curvature. Many of the experimental methods useful for investigating curvature sensing or generation require specialized equipment. We have developed a system based on supported lipid bilayers (SLBs) in which lipid tubules are simple to produce and several types of membrane remodeling events can be readily imaged using widely available instrumentation (e.g., tubule fission and/or membrane budding). Briefly, high ionic strength during lipid bilayer deposition results in incorporation of excess lipids in the SLB. After sequentially washing with water and physiological ionic strength buffer solutions, lipid tubules form spontaneously. We find that tubule formation results from solution-dependent spreading of the SLB; washing from water into physiological ionic strength buffer solution leads to expansion of the bilayer and formation of tubules. Conversely, washing from physiological buffer into water results in contraction of the membrane and loss of tubules. We demonstrate the utility of these supported tubulated bilayers, termed "STuBs," with an investigation of Sar1B, a small Ras family G-protein known to influence membrane curvature. The addition of Sar1B to STuBs results in dramatic changes in tubule topology and eventual tubule fission. Overall, STuBs are a simple experimental system, useful for monitoring protein-mediated effects on membrane topology in real time, under physiologically relevant conditions.

Keywords: Endocytosis; Exocytosis; Membrane fission; Supported bilayer; Vesicle budding.

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Conflict of interest statement

Competing Interests:

The authors have no competing interests to declare, other than the grant support noted above.

Figures

Figure 1:
Figure 1:. Spontaneous formation of lipid tubules from SLBs.
(A) Bilayer of DOPC, 0.1% LRB-DOPE in water. (B) Same sample as A, after adding Buffer A. (C) Bilayer of 3:1 DOPC/DOPS, 0.1% LRB-DOPE in water. Some regions of the surface are depleted of lipid (dark spots). (D) Same sample as C, after adding Buffer A. Arrows highlight longer tubules. Focus is away from surface in order to highlight tubules that extend vertically away from the supported bilayer. (E) A second bilayer of 3:1 DOPC/DOPS, 0.1% LRB-DOPE in water. (F) Same sample as E, containing shorter, more abundant tubules. Each arrow indicates a tubule. Image intensities have been scaled to visualize tubules, including adjusting the contrast in panels D and F. There is no indication that LRB-DOPE preferentially partitions into tubules relative to the SLB. Movie S1 corresponding to panel D is available in the Supporting Information. Scale bars 10 μm.
Figure 2:
Figure 2:. Effect of NaCl concentration on tubulation.
Bilayers (3:1 DOPC/DOPS with 0.1% LRB-DOPE) were deposited using Buffer A plus NaCl at concentrations of (A) 0 M, (B) 0.50 M, or (C) 1 M, then washed thoroughly with water prior to imaging in Buffer A. Representative images are shown from triplicate samples, after background subtraction. Focus is adjusted up from the surface in order to capture tubules extending into solution. Scale bars, 10 μm. (D) When using 0 M NaCl in liposome deposition buffer tubules do not form. Increasing NaCl concentration from 0.5 M to 1 M significantly increases the number of tubules observed per field of view from 3.0 ± 0.9 to 16.6 ± 3.2. Error bars are ± SEM. ***: P < 0.001 using student’s t-test.
Figure 3:
Figure 3:. Tubulation is driven by expansion of the SLB.
(A-C) Images of a scratched SLB at various time points during exchange of the bathing solution by perfusion from water into Buffer A. Further expansion of the bilayer beyond that which was necessary to fill the scratched region results in tubule formation. Scale bars, 10 µm. (D) Changes in average fluorescence intensity per unit area upon exchange of the bathing solution from Buffer A into water (gray) or vice versa (black). SLB retraction is indicated by an increase in fluorescence (2.7 ± 0.3 percent increase exchanging from Buffer A into water). Expansion is indicated by decreased fluorescence (4.9 ± 0.9 percent decrease exchanging from water into Buffer A). All SLBs were 3:1 DOPC/DOPS with 0.1% LRB-DOPE. Error bars are ± SEM.
Figure 4:
Figure 4:. Sar1B membrane remodeling.
The addition of Sar1BGTP to STuBs alters the morphology of lipid tubules. (A) A flexible tubule. (B) Same tubule as in (A) following addition of Sar1BGTP, exhibiting a rigid morphology. (C) A pseudo-vesiculated tubule. (D) A rigid, bifurcated tubule and a tubule exhibiting a beads-on-a-string morphology. (E) As Sar1BGTP concentration increases, the number of flexible tubules per field of view significantly decreases. (F) The prevalence of Sar1BGTP-induced structures changes as a function of concentration. Statistics by 1-way ANOVA. * indicates comparison to 0-nM Sar condition, † indicates comparison to 50-nM Sar1B condition. Adjusted P Values; *, †: P < 0.05, **, †† P: < 0.01, ***, †††: P < 0.001. All are in Buffer A + 0.5 mM MgCl2 + 5 μM GTP. Scale bars 10 µm. Error bars ± SEM.
Figure 5:
Figure 5:. Fluorescently labeled Sar1B binds tubules.
(A) A flexible tubule with no bound Sar1B. (B) A rigid tubule with Sar1B decorating its length. (C) A tubule exhibiting a beads-on-a-string morphology with Sar1B bound at the “beads”. Conditions: 1 nM AF488-Sar1B, 10 nM unlabeled Sar1BGTP in Buffer A + 0.5 mM MgCl2 + 5 µM GTP. Scale bars 5 µm.
Figure 6:
Figure 6:. Two color imaging shows real time tubule fission at site of Sar1B binding.
Images are shown from a single image sequence of a tubule undergoing fission. Results show a tubule prior to significant AF488-Sar1BGTP binding (A) and after binding AF488-Sar1BGTP (B-C). After the AF488-Sar1BGTP localized to the tubule base (C), the tubule disassembled into a vesicle-like structure (D), presumably due to fission. The lipid bilayer was labeled with 0.1% LRB-DOPE and excited by 561-nm laser in an epifluorescence geometry. The AF488-Sar1BGTP was excited by a 488-nm laser in TIR. (E) Fluorescence intensity vs. time graph of tubule fission event shown in (A-D). (F) Average fluorescence intensity vs. time of 5 tubule fission events. Error bars ± SEM. Scale bars 10 μm.
Figure 7:
Figure 7:. Sar1B bends membranes in a GTP-dependent manner.
(A) Increases in DiD-labeled membrane curvature (P/S) and concentration (P + 2S) observed after 10 nM Sar1BGTP is added to STuBs. (B-C) Graphs corresponding to the event shown in panel A. Averaged P/S (D) and P+2S (E) changes for several membrane budding events observed in the presence of Sar1BGTP (n=5). Error bars ± SEM. No similar events were observed in otherwise identical samples with Sar1BGDP or lacking protein (n ≥ 3). (G) Predicted structure of the detected membrane indentations based on computer simulations (Anantharam et al., 2010). Scale bars 2 µm.
Figure 8:
Figure 8:. Sar1B drives tubule fission in a GTP-dependent manner.
Effect of wild-type Sar1B on tubule density in the presence of 5 µM GDP, 5 µM GTP, and 5 µM GMP-PNP. In these experiments, 50 nM Sar1B was titrated onto an SLB containing approximately 100 pre-formed tubules in each field of view sampled. *:P < 0.05, **: P < 0.01, ***:P < 0.001, ****:P < 0.0001 relative to the absence of protein. Error bars ± SEM. To test for significance, data were compared to 0 nM condition using Student’s t-test. All samples used 3:1 DOPC/DOPS with 0.1% LRB-DOPE as the fluorescent tracer.
Figure 9:
Figure 9:. Model for STuBs formation and effects.
(1) SLB deposition occurs via liposome fusion. In the presence of high salt, liposomes adsorb to the glass surface faster than they rupture and spread, resulting in a bilayer containing excess lipid. (2) Washing with water removes unincorporated liposomes; although the bilayer still contains excess lipid, it appears flat for reasons that could involve decreased area per lipid and/or stronger lipid-glass interaction in water (see text). (3) Following addition of physiological ionic strength buffer, the excess lipid is no longer accommodated in the planar bilayer and tubules form. (4a-b) Effects of Sar1B addition with various nucleotides.
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