Lorena Redondo-Morata

Chargée de Recherche Inserm U1019

ResearchGateGoogle Scholar

CURRICULUM

→ Education

  • 2012 – PhD in Biotechnology, University of Barcelona, Spain. Thesis dissertation entitled “The Stability of Lipid Bilayers as Model Membranes: Atomic Force Microscopy and Spectroscopy Approach”; director: Prof. Fausto Sanz
  • 2008 – Master in Molecular Biotechnology, University of Barcelona, Spain
  • 2007 – Degree in Pharmacy, University of Barcelona, Spain

→ Working experience

  • January 2013-present: Postdoc contract, Institut National de la Santé et de la Recherche Médicale, Simon Scheuring’s lab (Marseille, France).
  • October 2011-January 2013: Assistant professor, Physical Chemistry Department, University of Barcelona, Barcelona, Spain.
  • January 2012-December 2012: Research contract, Institute for Bioengineering of Catalonia (IBEC), Barcelona, Spain.
  • October 2011-December2011: Scientific stage, Experimental Biophysics Department (Prof. Dr. Anselmetti’s lab), Physics Faculty, University of Bielefeld, Germany.
  • December 2007-November2011: Research grant of the Institute for Bioengineering of Catalonia (IBEC), Barcelona, Spain.
  • September 2006-November 2007: Collaboration grant with the Safety, Health and Environment Office, University of Barcelona, Barcelona, Spain.
  • January 2006-June 2007: Undergraduate student in Dr. Hernandez-Borrell lab, Pharmacy Faculty, University of Barcelona, Barcelona, Spain.
  • May 2004-December 2004: Collaboration grant with Nanometric Techniques Unit, Scientific-technical Services, University of Barcelona, Barcelona, Spain.

→ Awards and honors

  • SBE-33 award, (Spanish Biophysical Society award for young Biophysicist with age under 33), 2016.
  • Journal of Molecular Recognition award, 2013.
  • Cum laude, University of Barcelona, 2012.
  • Institute for Bioengineering of Catalonia Predoctoral Fellowship, 2007-2011.
  • Pregraduate Fellowship from Spanish Ministry of Education and Science, 2007.
  • Collaboration grant with the Safety, Health and Environment Office, University of Barcelona, 2006-2007.
  • Collaboration grant with Nanometric Techniques Unit, Scientific-technical Services, University of Barcelona, 2004.

RESEARCH – POSTDOCTORAL PERIOD (2013-2017)

→ ESCRT-III membrane remodeling

The main line of my postdoc research is to study the ESCRT-III (Endosomal Sorting Complex Required for Transport). ESCRT-III is required for lipid membrane remodeling in many cellular processes, from abscission to viral budding and multi-vesicular body biogenesis. However, how ESCRT-III polymerization generates membrane curvature remains debated. In this work we showed that Snf7, the main component of ESCRT-III, polymerized into spirals at the surface of lipid bilayers.

We reasoned that Snf7 spirals could function as spiral springs. By measuring the polymerization energy and the rigidity of Snf7 filaments, we showed that they were deformed while growing in a confined area. Furthermore, we showed that the elastic expansion of compressed Snf7 spirals could stretch the lipids they are bound to, generating an area difference between the membrane leaflets and thus curvature. This spring-like activity underlies the driving force by which ESCRT-III could mediate membrane remodeling. This property is in my opinion, new to the field of membrane remodeling, and further investigations will uncover, without any doubt, other unexpected properties for ESCRT-III proteins. This study was a collaboration with Aurélien Roux laboratory at the University of Genève (Switzerland). In Scheuring’s team, I used High-Speed Atomic Force Microscopy (HS-AFM) to study the ESCRT machinery. HS-AFM allows simultaneous observation of structure, dynamics and function of biological assemblies, with nanometer spatial and sub-second temporal resolution.

We showed HS-AFM movies of the Snf7 complex formation and its dynamics from filament to the maturated circular assembly around the membrane constriction site. We observed interfilament dynamics that provide a basis for a mechanistic explanation how the machinery creates tension for membrane fission. Furthermore, I could show that Snf7 assemblies compress the inner diameter during maturation, direct evidence for force generation during the assembly process. Related to this work, there is paper published in Cell (12) and another manuscript in preparation.

Besides, we are also studying in collaboration with Daniel Gerlich’s lab (Insitute of Molecular Biotechnology, Viena) the molecular role of Vps4, an ATPase that is known to drive the disassembly of persisting ESCRT-III filaments. We observed that in the presence of Vps4, ESCRT-III polymers disassemble partially, remaining the innermost part of the ring-like structure refractory to the action of the enzyme. Surprisingly, in the presence of a pool of soluble Snf7, ESCRT-III assemblies shrink under the action of Vps4, liberating free space on the membrane where new ESCRT-III assemblies are growing simultaneously. This results in turnover and high lateral mobility of ESCRT-III assemblies on membranes. Dynamic turnover 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.

→ Elastic properties of lipid bilayers

Topography image of a multicomponent lipid bilayer and the corresponding elasticity maps top) before and down) upon the addition of a hypolipidemic drug.

Many drugs and other xenobiotics may reach systemic concentrations where they interact not only with the proteins that are their therapeutic targets but also modify the physicochemical properties of the cell membrane, which may lead to altered function of many transmembrane proteins beyond the intended targets. These changes in bilayer properties may contribute to nonspecific, promiscuous changes in membrane protein and cell function because membrane proteins are energetically coupled to their host lipid bilayer. It is thus important, for both pharmaceutical and biophysical reasons, to understand the bilayer-modifying effect of amphiphiles (including therapeutic agents). Here I used AFM topography imaging and nanomechanical mapping to monitor the effect of statins, a family of hypolipidemic drugs, on synthetic lipid membranes. Our results reveal that statins alter the nanomechanical stability of the bilayers and increase their elastic moduli depending on the lipid bilayer order. Our results also suggest that statins increase bilayer heterogeneity, which may indicate that statins form nanometer-sized aggregates in the membrane. This is further evidence that changes in bilayer nanoscale mechanical properties may be a signature of lipid bilayer-mediated effects of amphiphilic drugs. Related publication (13).

→ Dynamin mediated membrane constriction

Dynamin is a dimeric GTPase that assembles into a helix around the neck of endocytic buds. Upon GTP-hydrolysis, dynamin breaks these necks, a reaction called membrane fission. Fission requires dynamin to first constrict the membrane. It is unclear however, how dynamin helix constriction works. Here we undertook a direct high-speed atomic force microscopy imaging analysis to visualize the constriction of single dynamin-coated membrane tubules. We show GTP-induced dynamic rearrangements of the dynamin helix-turns: the average distances between turns and between dimers along the polymer reduce with GTP-hydrolysis.

However, these distances vary over time, as helical turns were observed to transiently pair and dissociate. At fission sites, these cycles of association and dissociation were correlated with relative sliding of the turns and constriction. Our findings support a model in which conformational changes at the dimer level drive relative sliding of helical turns, and constriction by torsion. Our manuscript is currently under revision.

→ Identification of membrane-bound prepore species in Pore Forming Toxins

Pore-forming toxins (PFTs) are cytolytic proteins belonging to the molecular warfare apparatus of living organisms. The assembly of the functional transmembrane pore requires several intermediate steps ranging from a water-soluble monomeric species to the multimeric ensemble inserted in the cell membrane. The non-lytic oligomeric intermediate known as prepore plays an essential role in the mechanism of insertion of the class of β-PFTs. However, in the class of α-PFTs evidence of membrane-bound prepores is still lacking. We have employed AFM to contribute to the identification, for the first time, of a prepore species of the α-PFT fragaceatoxin C bound to lipid bilayers (related publication (14)).

In the presence of supported lipid bilayers containing sphingomyelin, fragaceatoxin self-assembles in a dense array of closely packed oligomers. These oligomers, presumably corresponding to pore particles, cover the sphingomyelin-rich domains. The dimensions from the high resolution AFM images indicate that the prepore is made of eight protein subunits. This work is a collaboration with Jose M. M. Caaveiro’s lab at the University of Tokyo).

→ Technical development: temperature-controlled High-Speed AFM

Under certain conditions some lipid bilayers adopt a so-called ripple phase, a structure where solid and fluid phase domains alternate with constant periodicity. Because of its narrow regime of existence and heterogeneity ripple phase and its transition dynamics remain poorly understood. We developed and integrated a temperature control device to a high-speed AFM to observe dynamics of phase transition from ripple phase to fluid phase reversibly in real time. Based on High-Speed AFM imaging, the phase transition processes from ripple phase to fluid phase and from ripple phase to metastable ripple phase to fluid phase could be reversibly, phenomenologically, and quantitatively studied. The results here show phase transition hysteresis in fast cooling and heating processes, while both melting and condensation occur at 24.2 °C in quasi-steady state situation. A second metastable ripple phase with larger periodicity is formed at the ripple phase to fluid phase transition when the buffer contains Ca2+. The presented temperature-controlled HS-AFM is a new unique experimental system to observe dynamics of temperature-sensitive processes at the nanoscopic level. Related publication (15).


RESEARCH – PREDOCTORAL PERIOD (2006-2012)

→ Force as a molecular fingerprint of the membrane mechanical stability

→ Use of the AFM tip as a nano-indentor to measure the onset of the plastic deformation of model lipid membranes.

One of the main lines of my PhD was to understand the effect of mechanical stress on membranes, which is of primary importance in biophysics. It was used AFM-based force spectroscopy to quantitatively characterize the nano-mechanical stability of supported lipid bilayers as a function of their chemical composition. The onset of plastic deformation reveals itself as a repetitive jump in the approaching force curve, which represents a molecular fingerprint for the bilayer mechanical stability. By systematically probing a set of chemically distinct supported lipid bilayers, I showed a quantitative nano-mechanical effect in the different molecular determinants that compose the phospholipids. Besides, the results were discussed in the framework of the continuum nucleation model.

This mechanical fingerprint was also used to explore how metal cations affect stability and structure of phospholipid bilayers, which role ion binding plays in the insertion of proteins and in the overall mechanical stability of biological membranes. I gained experience in lipid systems, Z-potential measurements, dynamic light scattering, scanning electron microscopy and acquired strong skills in AFM and force measurements. (Related publications (4, 5, 8, 11)).

→ Phase segregated lipid bilayers

Another work as a predoctoral fellow was to study the mechanics of phase coexisting lipid bilayers. Cholesterol (Chol) plays the essential function of regulating the physical properties of the cell membrane. I explored a lipid model system by means of temperature-controlled AFM imaging and force mapping to assess the influence of Chol on the membrane ordering and stability. I proved how Chol influences in the phase segregation of lipid bilayers and enhances the mechanical stability of the membrane, demonstrating that temperature-controlled force spectroscopy (that I implemented in the group) is capable of identifying a thermal transition for lipid bilayers and differentiate phase coexistence, bridging the gap between the results obtained by traditional methods for bulk analysis, the theoretical predictions and the behavior of these systems at the nanoscale. During these studies, I acquired experience in lipid mixtures preparation and implementation of accessories to the microscope. (Related publications (7, 9) and awarded by the Journal of Molecular Recognition (Wiley)).

→ Kinetics of lipid bilayers

AFM-based force clamp technique applied to lipid bilayer rupture was conceived, developed by myself and used in my PhD group, for the first time, to evaluate how lipid membranes respond when compressed under an external constant force in the range of nN. Using this method I was able to directly quantify the kinetics of the membrane rupture event and the associated energy barriers, both for single supported bilayers and multibilayers. Moreover, the affected area of the membrane during the rupture process was calculated using an elastic deformation model. This project was my own initiative to acquire skills in force-clamp measurements and data analysis. (Related publications (6, 10)).

→ Bioinspired nano-pores for DNA translocation

I carried out a project in collaboration with Dario Anselmetti’s group in the University of Bielefeld, Germany. The project involved the controlled translocation of DNA through bioinspired nanopores with optical tweezers mediated by an electric field under buffer conditions. Upon threading dsDNA complexed by single proteins through solid-state nano-pores, we found out that changes in force signals can be correlated to biophysical parameters such us the dsDNA elasticity. During this project I acquired experience in Optical Tweezers and Patch-Clamp measurements. (Related publication (3)).

→ Specific adsorption of cytochrome c in lipid monolayers and bilayers

During my undergraduate research, I studied the adsorption of cytochrome c on monolayers, supported bilayers and liposomes. The studies were a compendium of different techniques from which I benefited to gain experience: AFM of extracted Langmuir−Blodgett films, Langmuir isotherms, fluorescence anisotropy and differential scanning calorimetry. Based on my work, we proposed mechanisms of the particular penetration of the protein into the lipid bilayer, relevant for the apoptotic processes. I also acquired skills in lipid-based systems preparation. (Related publications (1, 2).


PUBLICATIONS

18.Gumi-Audenis, B., Costa, L., Redondo-Morata, L., Milhiet, P-E., Sanz-Carrasco, F., Felici, R., Gianotti, M.I.¶ and Carla, F.¶. (2017). In-plane molecular organization of hydrated single lipid bilayers: DPPC:cholesterol. Nanoscale (accepted), DOI: 10.1039/C7NR07510C. (¶co-corresponding author).

17. Mierzwa, B.E.*; Chiaruttini, N.*; Redondo-Morata, L.*; von Filseck, J.M.; König, J.; Larios, J.; Poser, I.; Müller-Reichert, T.; Scheuring, S.; Roux, A.¶; Gerlich, D.W.¶, Dynamic subunit turnover in ESCRT-III assemblies is regulated by Vps4 to mediate membrane remodelling during cytokinesis. Nature Cell Biology, doi:10.1038/ncb3559. (*equal contribution, ¶co-corresponding author). And News and views by Henri G. Franchelim & Petra Schwille.

16. Colom, A., Redondo-Morata, L., Chiaruttini, N., Roux, A¶. and Scheuring, S¶. (2017). Dynamic remodeling of the dynamin helix during membrane constriction. Proceedings of the Natural Academy of Sciences 2017, 114 (21), 5449-5454. (¶co-corresponding author).

15. Takahashi, H. *; Miyagi, A.*; Redondo-Morata, L.; Scheuring, S.; Temperature-controlled high-speed AFM: Real time observation of ripple-phase transitions, Small, 2016, 12 (44), 6106-6113, (*equal contribution).

14. Morante, K.; Bellomio, A.; Gil-Cartón, D.; Sot, J.; Redondo-Morata, L.; Sot, J.; Scheuring, S.; Valle, M.; González-Mañas, J.M.; Tsumoto, K.; Caaveiro, J. M. M. ; Identification of a membrane-bound prepore species clarifies the lytic mechanism of actinoporins, Journal of Biological Chemistry, 2016, 291 (37):19210-19.

  • Paper of the week & Featured in journal cover (Issue 2016 September 9)

13. Redondo-Morata, L.; Sanford, R.L., Andersen, O.S. and Scheuring, S.; Effect of statins on the nanomechanical properties of supported lipid bilayers, Biophysical Journal, 2016, 111 (2): 363-72.

12. Chiaruttini, N.*; Redondo-Morata, L.*; Colom, A.; Humbert, F.; Lenz, M.; Scheuring, S.; Roux, A.; Relaxation of loaded ESCRT-III spiral springs drives membrane deformation, Cell, 2015, 163 (4): 866-79 (*equal contribution).

  • Featured in journal cover (Volume 163, Issue 4 2016 November 5)
  • Preview in L. A. Carlson, Q. T. Shen, M. R. Pavlin, J. H. Hurley; ESCRT as spiral springs, Developmental Cell, 2015, 35 (4):397-398.

11. Redondo-Morata, L.; Giannotti, M.I.; Sanz, Structural impact of metallic cations on lipid bilayer model membranes, Molecular membrane biology, 2014; 31(1), 17-28.

10. Redondo-Morata, L.; Giannotti, M.I.; Sanz, F.; AFM-Based Force-clamp Indentation: Force-Clamp Monitors the Lipid Bilayer Failure Kinetics, Imaging & Microscopy, 2013; 15(4): 25-27.

9. Lima, L. M.C.; Giannotti, M. I.; Redondo-Morata, L.; Vale, M.L.C; Marquesa, R. F.; Sanz, F., Morphological and nanomechanical behavior of supported lipid bilayers on addition of cationic surfactants, Langmuir, 2013, 29 (30), 9352-9361.

8. Redondo-Morata, L.; Giannotti, M.I.; Sanz, F.; “Stability of Lipid Bilayers as Model Membranes: Atomic Force Microscopy and Spectroscopy Approach” in Atomic Force Microscopy in Liquid. Ed. A.M. Baró and R.G. Reifenberger, 2012, Wiley-VCH Verlag.

7. Redondo-Morata, L.; Giannotti, M.I.; Sanz, Influence of cholesterol on the phase transition of lipid bilayers: A temperature-controlled force spectroscopy study, Langmuir, 2012, 28 (35), 15851-12860.

6. Redondo-Morata, L.; Giannotti, M.I.; Sanz, Lipid bilayer response under AFM-based force-clamp, Langmuir, 2012, 28 (15), 6403-6410.

5. Garcia-Manyes, S.; Redondo-Morata, L.; Oncins, G., Sanz, F.; Nanomechanics of lipid bilayers: Heads or tails?, Journal of the American Chemical Society, 2010, 132, 12874-12886.

4. Redondo-Morata, L.; Oncins, G.; Sanz, F.; Force Spectroscopy reveals the effect of different ions in the nanomechanical behavior of phospholipid model membranes: the case of potassium cation, Biophysical Journal, 2012, 102, 66-74.

3. Spiering, S. Knust, S. Getfert, A. Beyer, K. Rott, L. Redondo, K. Tönsing, P. Reimann, A. Sischka and D. Anselmetti; Single-molecule DNA translocation through Si3N4 and graphene solid-state nanopores, Nanopores-Zing Conferences Royal Society of Chemistry.

2. Domènech, Ò.; Redondo, L., Montero, M.T.; Hernández-Borrell, J., Specific adsorption of cytochrome c on cardiolipin-glycerophospholipid monolayers and bilayers, Langmuir, 2007, 23, 5651-5656.

1. Domènech, Ò.; Redondo, L., Picas, L., Morros, A., Montero, M.T., Hernández-Borrell, J. Atomic force microscopy characterization of supported planar bilayers that mimic the mitochondrial inner membrane, Journal of Molecular Recognition, 2007, 20, 546-553

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