1 Topological connection between vesicles and nanotubes in single - molecule lipid membranes driven by head -tail interactions

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Topological connection between vesicles and nanotubes in single-

molecule lipid membranes driven by head-tail interactions

Niki Baccile,a,* Cédric Lorthioir,a Abdoul Aziz Ba,a Patrick Le Griel,a Javier Perez,b Daniel

Hermida-Merino,c,d Wim Soetaert,e Sophie L. K. W. Roelantse

a Sorbonne Université, Centre National de la Recherche Scientifique, Laboratoire de Chimie de

la Matière Condensée de Paris, LCMCP, Paris, 75005, France

b Synchrotron Soleil, L’Orme des Merisiers, Saint-Aubin, BP48, Gif-sur-Yvette Cedex, 91192,

France

c Netherlands Organisation for Scientific Research (NWO), DUBBLE@ESRF BP CS40220,

Grenoble, 38043, France

d Departamento de Física Aplicada, CINBIO, Universidade de Vigo, Campus Lagoas-

Marcosende, Vigo, 36310, Spain

e InBio, Department of Biotechnology, Ghent University, Ghent, 9000, Belgium

* Corresponding author:

Dr. Niki Baccile

E-mail address: niki.baccile@sorbonne-universite.fr

Phone: +33 1 44 27 56 77

Abstract

Lipid nanotube-vesicle networks are important channels for intercellular communication and

transport of matter. Experimentally observed in neighboring mammalian cells, but also

reproduced in model membrane systems, a broad consensus exists on their formation and

stability. Lipid membranes must be composed of at least two molecular components, each

stabilizing low (generally a phospholipid) and high curvatures. Strong anisotropy or enhanced

conical shape of the second amphiphile is crucial for the formation of nanotunnels. Anisotropic

driving forces generally favor nanotube protrusions from vesicles. In the present work, we

report the unique case of topologically-connected nanotubes-vesicles obtained in the absence

of directional forces, in single-molecule membranes, composed of an anisotropic bolaform

glucolipid, above its melting temperature, Tm. Cryo-TEM and fluorescence confocal

microscopy show the interconnection between vesicles and nanotubes in a single-phase region,

between 60° and 90°C under diluted conditions. Solid-state NMR demonstrates that the

glucolipid can assume two distinct configurations, head-head and head-tail. These

arrangements, seemingly of comparable energy above the Tm, could explain the existence and

stability of the topologically-connected vesicles and nanotubes, which are generally not

observed for classical single-molecule phospholipid-based membranes above their Tm.

Keywords: Nanotube vesicle networks; Tunnelling nanotubes; Block liposomes; Liposomes;

Lipid nanotubes; Biosurfactants; Microbial glycolipids;

Introduction

Topological connections between closed lipidic compartments through nanotubes1–3

have been shown to play a crucial role in the transfer of matter and communication in

neighboring mammalian cells.4 These singular nanosystems, observed since the 1990s as

spontaneous non-equilibrium structures in electroformed model liposome membranes,5,6 have

since been largely studied, both experimentally and theoretically.7–9 Addressed in the literature

by different terms, tunnelling nanotubes (TNT),1,4,9 block liposomes10–12 or nanotube-vesicle

networks,13–16 (instead of tubes, some work speaks of tethers6,17) all refer to a similar

phenomenon, driven by various internal or external forces. The latter must overcome the energy

barrier needed to bend a phospholipid bilayer from low positive mean and Gaussian curvatures

(vesicle) to a high mean and zero Gaussian (tube) curvatures.

A large body of both experimental and theoretical work has shown that budding and

eventual nanotube formation from an existing membrane can only occur spontaneously for

membranes of at least two molecular components9–12,18–26 and below phase transition event

(corresponding to Tm for lipids or glass transition temperature, Tg, for block copolymers).27–29

If internal driving forces, like intra-membrane polymer-polymer phase separation,30 can trigger

nanotube protrusion, external anisotropic forces like electroformation,5–8,31,32 osmotic pressure,6

laser “tweezers”33 or electrodynamics13–16 must generally be employed with, in some cases, an

impressive degree of 2D and 3D organization.13–16 The origin of spontaneous nanotube

formation has been shown to be related to a nanoscale phase separation between two membrane

molecules, stabilizing low and high curvatures respectively,10–12,34 with at least one molecule

being highly anisotropic.24,35,36 Theoretically, this behavior has been explained by deviations in

the elastic properties of membranes due to in-plane orientational ordering of membrane

inclusions composed of anisotropic amphiphiles, these referring to a non-symmetrical shape

upon a 90° tilt along the amphiphile axis.8,9,19,20,23,36

In this work, we show unexpected nanotubing of membranes prepared from a single-

molecule lipid, in the absence of external directional forces and above the lipid’s Tm. This

phenomenon is observed for a novel anisotropic double amphiphile (bolaform amphiphile, or

bolaamphiphile), a glucolipid composed of β-D-glucose and a C18:1-cis fatty alcohol (G-

C18:1-OH, Figure 1). This compound is obtained by microbial fermentation of a genetically-

modified S. bombicola yeast in the presence of oleyl alcohol37 and is developed in the broader

context of extending the library of new biobased surfactants and lipids, in view of replacing

petrochemical low molecular weight amphiphiles.38–44 The structure of G-C18:1-OH is

analogous to that of other microbial glycolipids developed through genetic engineering.45,46

Topological connections between nanotubes and vesicles are observed by means of

cryogenic transmission electron microscopy (cryo-TEM), fluorescence microscopy and

temperature-resolved in situ small and wide angle X-ray scattering (SAXS-WAXS) above the

melting temperatures, Tm= 48.3°C. Spin diffusion 2D solid-state nuclear magnetic resonance

(ssNMR) spectroscopy under magic angle spinning (MAS) help understanding the vesicle-

nanotube coexistence. The bolaform glucolipids could be in a head-head/tail-tail configuration

in the vesicles and in a head-tail configuration in the nanotubes. These facts could explain the

stability of nanotubes, while the following hypothesis is formulated for their formation:

membrane inclusions with different orientational ordering13–16 possibly driven by inter-vesicle

collisions.

Synthesis path

Figure 1 – Non-acetylated C18:1 alcohol glucoside, G-C18:1-OH, is obtained by a bioprocess performed

with modified S. bombicola yeast.

Experimental Section

Synthesis of non-acetylated C18:1 alcohol glucosides (G-C18:1-OH). G-C18:1-OH (Mw=

418.56 g.mol-1) was produced by aerobic whole cell bioprocess with a modified S. bombicola

strain as described by Van Renterghem et al. (Fig. S4 in Ref. 37). The molecule was purchased

from the Bio Base Europe Pilot Plant (Gent, Belgium) and has the generalized chemical

structure given in figure Figure 1. The HPLC and 1H NMR spectrum (MeOD-d4) with peak

assignment are shown in Figure S 1. High purity levels (99%) and high degree of uniformity

were obtained, as can be derived from HPLC-ELSD chromatogram, 1H NMR and table of

contaminant given in Figure S 1.

1H solution Nuclear Magnetic Resonance (NMR). 1H solution NMR experiments were

performed on a Bruker Avance III 300 spectrometer using a 5 mm 1H-X BBFO probe using

methanol-d4 as solvent. The number of transients is 8 with 3 s recycling delay, an acquisition

time of 5.46 s and a receiver gain of 362. The 1H NMR spectrum and relative assignment are

shown in Figure S 1. 13C solution NMR were performed on the same probe using DMSO-d6.

Referencing is done with respect to TMS. δ1H= 0 ppm, δ13C= 0 ppm.

Sample preparation. The sample was dissolved in milliQ-grade water at the concentration of 5

mg/mL (0.5 wt%), although specific experiments involving SAXS, solid-state NMR or DSC

have been performed on hydrated but more concentrated samples (50 wt%). Lack of pH-

sensitive probes (e.g., COOH groups) in G-C18:1-OH, as otherwise found in other microbial

amphiphiles,47 but also the will to avoid ion-specific effects,48 exclude the use of buffer. In all

experiments, the sample is heat above 110°C and analyzed during cooling. On the bench

qualitative experiments are performed using a CH3-150 Combitherm-2 dry block heating

device.

Differential Scanning Calorimetry (DSC). DSC was performed using a DSC Q20 apparatus

from TA Instruments equipped with the Advantage for Q Series Version acquisition software

(v5.4.0). Acquisition was performed on both hydrated and dry powder sample (~ 3-5 mg) sealed

in a classical aluminium cup and using an immediate sequence of heating and cooling ramps at

a rate of 10°C.min-1. Melting temperatures, Tm, 1 and 2, Tm1 and Tm2, were taken at the minimum

of the endothermic peak.

Small and Wide Angle Scattering experiments. Small angle neutron scattering (SANS)

experiments were performed at the D11 beamline of Institut Laue Langevin (Grenoble, France)

during the run No. 9-13-778. Four q-ranges have been explored and merged using the following

wavelengths, λ, and sample-to-detector (StD) distances. 1) ultra-low q: λ= 13.5Å, StD= 39 m;

2) low-q: λ= 5.3Å, StD= 39 m; 3) mid-q: λ= 5.3Å, StD= 8 m; 4) high-q: λ= 5.3Å, StD= 1.4 m.

The sample (C= 5 mg/mL) was prepared in 99.9% D2O to limit the incoherent background

scattering. The sample solution was analyzed in standard 1 mm quartz cells. The direct beam,

an empty quartz cell, and H2O (incoherent scatterer) within the quartz cell were recorded and

boron carbide (B4C) was used as neutron absorber. The sample acquisition was measured at

90°C, where temperature was controlled through the controller thermalized sample holder

available at the beamline. The background sample (D2O) signal was subtracted from the

experimental data. Absolute values of the scattering intensity were obtained from the direct

determination of the number of neutrons in the incident beam and the detector cell solid angle.

The 2D raw data were corrected for the ambient background and empty cell scattering and

normalized to yield an absolute scale (cross section per unit volume) by the neutron flux on the

samples. The data were then circularly averaged to yield the 1D intensity distribution, I(q). The

software package Grasp (developed at ILL and available free of charge) was used to integrate

the data, while the software package SAXSUtilities (developed at ESRF and available free of

charge) was used to merge the data acquired at all configurations and subtract the background.

Wide-angle X-ray scattering (WAXS) was performed under temperature control at the

SWING beamline of SOLEIL synchrotron facility (Saint-Aubin, France) during the run number

20201747 (energy: 14 keV, sample-to-detector distance: 0.5 m). Sample concentration in H2O

was C= 5 mg/mL. The 2D data were integrated azimuthally at the beamline using the software

Foxtrot and in order to obtain the I(q) vs. q spectrum after masking the beam stop shadow.

Silver behenate (d(001)= 58.38 Å) was used as a standard to calibrate the q-scale. Sample

solutions were inserted in borosilicate capillaries of 1.5 mm in diameter. Capillaries were flame-

sealed. A capillary oven with controlled temperature (± 0.5°C), provided at the beamline, was

used to control the sample temperature. Experiments are recorded between 111°C and 25°C.

Data were normalized by the transmission and calibrated to the SAXS signal of H2O at large q-

values (I= 0.0163 cm-1) in order to obtain an absolute intensity scale. The water signal was

measured by subtracting the signal of the empty capillary from the signal of a water-filled

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1Topologicalconnectionbetweenvesiclesandnanotubesinsingle-moleculelipidmembranesdrivenbyhead-tailinteractionsNikiBaccile,a,*CédricLorthioir,aAbdoulAzizBa,aPatrickLeGriel,aJavierPerez,bDanielHermida-Merino,c,dWimSoetaert,eSophieL.K.W.RoelantseaSorbonneUniversité,CentreNationaldelaRechercheScientifiqu...

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1 Topological connection between vesicles and nanotubes in single - molecule lipid membranes driven by head -tail interactions

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