ATOMIC FORCE MICROSCOPY AS A TOOL TO STUDY CELL MEMBRANE REMODELING PROCESSES

Lorena Redondo-Morata

INSERM U1019, Institut Pasteur de Lille - CMPI-CIIL, CNRS UMR 8204 Univ. Lille, Lille, France

Atomic Force Microscopy (AFM)-based studies constitute today a fairly established methodology to observe the structure of biomolecules and to measure their mechanical properties. However, biomolecules are dynamic in nature; hence, to understand how they work we need to increase the spatiotemporal resolution of conventional AFM. In the last decade, High-Speed AFM (HS-AFM) was developed [1] and successfully applied to several cellular machineries, either cytoplasmic or bound to membranes [2]. The molecular movies obtained by this method provide insights otherwise not accessible by other means to date.

Recently, we have succeeded to visualize by these means the molecular mechanism of Snf7 assembly formation. Snf7 is the major polymerization component of the Endosomal Sorting Complex Required for Transport-III (ESCRT-III). We observed the formation of spiraling filaments on negatively charged membranes and estimated that these filaments can store sufficient elastic energy to drive membrane deformation [3]. In our most recent work, we studied the molecular role of Vps4, an ATPase that it is known to drive the disassembly of persisting filaments of ESCRT-III [4]. Surprisingly, in the presence of a soluble Snf7 pool, ESCRT-III assemblies shrink under the action of Vps4, liberating free space in the membrane were new ESCRT-III assemblies are growing simultaneously. This results in a high exchange and lateral mobility of ESCRT-III assemblies on membranes. Dynamic exchange provides an explanation for how ESCRT-III filaments gradually adapt their shape during membrane constriction, which has broad implications in diverse cellular processes, differing in size, shape and duration –such as plasma membrane repair, cytokinesis or viral budding.

We are also interested in the dynamic nanomechanical changes of the lipid membrane in the conversion of sphingomyelin to ceramide, particularly the impact of chain length and unsaturation of sphingomyelin and ceramide in the overall membrane nanomechanical properties. Atomic Force Microscopy (AFM)-based Force Spectroscopy is an ideal technique to investigate the mechanical properties of lipid bilayers at the nanoscale, their elastic constants [5] but also their plastic deformation and rupture [6]. However, the viscoelastic parameters of lipid membranes have been less explored by these means. In this work, we systematically studied the enzymatic conversion of sphingomyelin-containing supported lipid bilayers to ceramide by adding sphingomyelinase in situ. The local production of ceramide induces, in turn, local changes in the membrane mechanics that depend on the chain length and degree of unsaturation of the original sphingomyelin (Figure 1). We assess here the elasticity directly from the AFM force-distance curves and discuss possible approaches to evaluate the viscoelasticity of lipid membranes [7], i.e. using fast mapping with bimodal AFM [9]. The different ceramide localization in the membrane and mechanical properties is relevant in several biological contexts as apoptosis or viral infection.

[1] Kodera, N.; Yamamoto, D.; Ishikawa, R.; Ando, T. Nature, 2010, 468:72-76.

[2] Casuso, I.; Khao, J.; Chami, M.; Paul-Gilloteaux, P.; Husain, M.; Duneau, J.-P.; Stahlberg, H.; Sturgis, J. N.; Scheuring, S. Nat. Nanotech., 2012, 7:525-529.

[3] Chiaruttini*, N.; Redondo-Morata*, L.; Colom, A.; Humbert, F.; Lenz, M.; Scheuring, S.; Roux, A. Cell, 2015, 163:866-879.

[4] Mierzwa*, B.E.; Chiaruttini*, N.; Redondo-Morata*, L.; Moser von Filseck, J.; König, J.: Larios, J.; Poser, I.; Müller-Reichert, T.; Scheuring, S.; Roux, A.; Gerlich, D.W. Nat. Cell Biol., 2017,19:787-798.

[5] Redondo-Morata, L.; Sanford, R. L.; Andersen, O.S.; Scheuring, S.; Biophys. J., 2016, 111, 363-372.

[6] Redondo-Morata, L.; Giannotti, M.I.; Sanz, F.; Langmuir, 2012, 28, 12851-12860.

[7] Al-Rekabi, Z. and Contera, S.; Proc. Natl. Ac. Sci. USA, 2018, 115, 2658-2663.

[8] Benaglia, S.  Amo, C.A.; Garcia, R.; Nanoscale, 2019, 11, 15289-15297.