Dr. Ignacio Casuso

My scientific goal: the cell membrane studied by high speed AFM.

The membranes of the cell enclose the different organelles and the cell themselves. All the signaling, nutritional and waste exchange between cells and organelles passes through or is generated in the membranes of cells, the membranes actively participate in the regulation of the metabolic biochemical pathways of the cells. The membrane mass is composed 50% of the lipid bilayer and 50% of the associated proteins, being the associated membrane proteins (8% of all human protein-coding genes code for membrane proteins) the responsible of carrying out specific functions. Their medical relevance is highlighted by the fact that 70% of drugs work on the membranes of cells. The membrane biological function is regulated by a continuous dynamic and reversible reorganization of cell membranes structure including temporary association of protein or lipids and the formation of domains. Reorganization mechanisms are (i) inherent related to the membrane itself and (ii) related to the interaction of the membrane with the cytoskeletal and extracellular structures.

Among the techniques to experimentally obtain information about the membrane architecture only the Atomic Force Microscope (AFM) provides imaging at sub-protein resolution of local membrane structure in physiological conditions, and enables the study of the dynamics of individual protein and membrane re-arrangements. The AFM does not only image the sample´s topography it uniquely allows the characterization by the touch of AFM tip and sample, variations of rigidity and viscosity of the lipids and proteins forming the membrane are characterized.

The AFM can characterize the topography of the plasma membrane at molecular resolution. In 2013, I achieved molecular High Speed AFM imaging of an eukaryotic plasma membrane of the Lens Cells, Nevertheless most cells are not rigid enough for AFM imaging, or the extracellular space does not allow the AFM tip to touch the plasma membrane. For AFM observation, most membranes must be placed on top of solid supports, where model membranes or native membrane extraction are absorbed.

The High Speed AFM (HS-AFM), a technique present in a limited number of labs in the world., is capable of x1000 times faster imaging than conventional AFM. It can observe molecular dynamics at sub-second framerates. The HS-AFM constitutes the central experimental tool of my research that I use for visualizing the molecular dynamics of the cell membranes.

My teaching. I teach the AFM technology and its biological applications at PhD and master courses both at the Aix-Marseille Université (http://biologie.univ-mrs.fr/masterBBSG/ ), and sporadically at other educative schools organisms.

My Future Research Perspective (Fall 2017). In recent years the HS-AFM has created new capabilities for the observation of the behavior of molecules on the cell membrane, the HS-AFM uniquely allows structural observation of the totality of the molecules in a defined area of the membrane, together with parallel simultaneous characterization of the dynamics on the area. In the last ten years the HS-AFM has gone from a prototype-state technology that only of labs in the world had access to (including ours) to an established commercialized technology that is present in tens of labs around the world. In the next five years, the HS-AFM use will spread, new light will be shed from this new tool and experimental perspective, some of the open issues in the functioning of biology at the molecular scale will benefit from this approach. I ascribe my research line along this trend. My scientific objectives are listed below, but I will maintain open eyes on unexpected observations which my experience has shown can be of scientific significance. Additionally, to keep the top-level research I will pursue technological improvement of the HS-AFM machine, it will provide exclusive access new types of observations of the cell membranes. In the future, the developed technology will be potentially transferable to the industry. This is an aspect to which I pay attention; I hold two patents on HS-AFM technology, I am ‘chargé de valorisation’ and I perform my work in collaboration with spin-off companies.

+ + + +

Project #1A. Development of a module to implement mechanical mapping in the HS-AFM

Protein fold, the membranes deform and membrane domains constantly renew and modify their mechanical properties. Mechanics at the molecular scale of life is as important as it is at larger scales. In order to sample the mechanics of the membrane at high AFM speed, x1000 faster than current possible AFM mechanical mapping, I am developing in collaboration with the Department of Optics and Quantum Electronics of the University of Szeged in Hungary, the spin-off company Hinstra, Hungary, and the ForceTool Group of the Institut of Material Science of Madrid Spain, a new Bimodal-type of mechanical mapping for the HS-AFM. The required custom-made electronics are now under optimization installed in the HS-AFM set-up, and could be ready for operation over the next 1-2 years depending on the funding. Once finished it will allow obtaining rigidity and viscosity mapping simultaneously to the conventional topography channel of the HS-AFM at sub-second framerates.

Project #1B. Application of the mechanical mapping HS-AFM to study the action of the cell membrane (thematic under evaluation)

+ + + +

Project #2A. Development of a module for the stimulation of biological activity under electric field application

A module for the stimulation of biological activity under electric field application is in development. Recently, I have obtained a proof of concept of the electric field application module using a set-up based on a High Voltage application (3kV). A transmembrane voltage of 10mV across a supported lipid bilayer on mica was measured using state of the art voltage-sensitive dyes inserted in a supported lipid membrane (dyes provided by Evan Miller Lab, Berkeley University USA). Further improvement of the module is required to achieve larger voltages to the level of the transmembrane resting potential (70mV).

I note that this project has been awarded the Seal of Excellence of the European Commission following a Postdoc MCSA grant application. If funding obtained, the setup should be ready over the next 1-2 years.

Project #2B. Application of module for electrical stimulation to the study of Voltage Sensing Domains

The new module will be first applied to the study of Voltage Sensing Domains of the Voltage Gated Channels. Nowadays, data on the structure of Voltage Gated Ion Channels (VIC) superfamily is missing for the state of applied voltage (resting state), this is due to the technical difficulty of the measurement with applied voltage. The new module could provide information on the structure of the channels in the resting state (voltage applied), and additionally monitor its dynamics and interactions, in particular between the Voltage Sensing Domain and the Channel Domain.

+ + + +

Project #3 Complexity in the membrane; its creation and its function: (i) Regulation of toxin activity by membrane crowding related phenomena, (ii) pH role of Lysenin toxin structural change during membrane permeabilization).

· ´Lysenin toxin membrane insertion is pH-dependent and non-cooperative´ Munguira, I. Casuso (co-first autor), H. Takahashi, S. Scheuring 2017 Biophysical Journal accepted (IF: 3.6)

· ´Glasslike Membrane Protein Diffusion in a Crowded Membrane´ I. Munguira, I. Casuso (co-first autor), H. Takahashi, F. Rico, A. Miyagi, M. Chami, S. Scheuring 2016 ACS nano 10 (2), 2584-2590 (IF: 12.8)

+ + + +

Project #4 Action of antimicrobial peptide antibiotics on membranes. starting...

+ + + +

Project #5 Dynamics of fusion of glycoproteins in Vesicular Stomatitis Virus.

This line of research is associated to the ANR project MoBaRhE (Bases moléculaires de l'entrée des rhabdovirus dans la cellule hôte). Project coordinator Monsieur Yves GAUDIN (Institut de Biologie Cellulaire Intégrative). Ignacio Casuso is the local PI of the project.

- In order to enter the host cell, enveloped viruses require fusion between the viral and the cellular membrane. Fusion is driven by conformational changes of the viral glycoprotein-G (G). Previous studies based on electron microscopy (EM) using negative staining (NS) show that these changes are driven by a difference of pH. In NS, the negative charges of heavy metal may distort the sample and produce undesirable outcomes such as aggregation and molecular dissociations. In collaboration with the team of Yves GAUDIN from the Institut de Biologie Intégrative de la Cellule à Gif-sur-Yvette, coordinator of the MoBaRHe project, I use the HS-AFM to determine the structure, the dynamics and the oligomerization of Gs in physiological conditions. At slightly basic and neutral pH, I observe a majority of monomers with rare pre-fusion trimers present. By pH decrease to acidic conditions, I observe a transition to post-fusion trimers which are abundant on the VSV surface. At acidic pH, I observe the aggregation of post-fusion trimers into trimers-of-trimers that diffuse and rotate on the VSV surface. The results confirm previous oligomerization states observed by EM and mass spectrometry, furthermore we provide previously unseen dynamics of the Gs correlated to the structural transitions driven by pH changes. Currently, more assays are being performed at low pH to confirm previous structural observations at higher pH. A better understanding of the molecular machinery enabling the entry in the host cell of the Rhabdovirus will help the development of new antiviral molecules and vaccines

+ + + +

Project #6, the study of the formation of tubulations in the pathogenic action of Salmonella, project started on January 2017 and associated to the ANR Salmotubes project performed in collaboration with Stéphane Meresse of the Centre d’Immunologie de Marseille-Luminy.

The research shown on TV

Professional experience

· May 2008 – today. PostDoc + Permanent researcher Inserm CR1. Institut Curie, Paris and Inserm Marseille, France. /+/ Teaching for Master and PhD courses at Aix-Marseille Université

Scientific Output: 13 publications and 2 patents

· Nov. 2002 – Fall 2007. PhD. (PhD Supervisor: Gabriel GOMILA, Barcelona University)

Dept. of Electronics of the Barcelona University, Institute of Bioengineering of Catalonia

Scientific Output: 11 publications and 2 book chapters

· May 2001 – July 2002. Scientific Consultant (Company owners: Antonio CORREIA, Tim HARPER) CMP-CIENTIFICA S.L. Madrid, Spain

· Sept. 2000 – Feb. 2001. Intership (Team leader: Guy PEREZ) Centre National d'Études Spatiales, Toulouse, France

· June 1998 – Sept. 1998. Intership. Cirent Semiconductor, Bell Labs Innovations, Orlando, USA

Education

· Nov. 2002 – December 2007. PhD on Physics. Barcelona University. Barcelona, Spain

PhD title: ‘Electrical characterization of biological elements by Atomic Force Microscopy’,

· March 2000 - August 2000. MSc on databases and web services. Info 93, Madrid, Spain

· Sept 1994 - Feb 2000. MSc and BSc on Physics and Materials. U. Complutense, Madrid, Spain

· Sept 1993- June 1994. High School Graduation. Berkeley High School Berkeley, USA

· Sept 1990- June 1994. High School Graduation. Instituto Carlos Bousoño, Majadahonda, Spain

Direction

Direction of 2 PostDoc researchers (2017) and co-direction of 3 PhD thesis (2009-2012 Mohamed Husain; 2009-2012 Adai Colom; 2014-2017 Ignacio Munguira)

Patents

2009. The high-resolution contact mode AFM imaging robot. (use of the vertical and lateral cantilever vibration signal for ideal, imaging force determination and force drift compensation). European patent EP09305150.

2011. Optical component for integrating the optical microscopy into the atomic force microscopy maintaining maximal performance of the atomic force microscopy. European patent EP11305031

Selected Refereed journals, h-index=17

· ´Lysenin toxin membrane insertion is pH-dependent and non-cooperative´ Munguira, I. Casuso (co-first autor), H. Takahashi, S. Scheuring 2017 Biophysical Journal accepted (IF: 3.6)

· ´Glasslike Membrane Protein Diffusion in a Crowded Membrane´ I. Munguira, I. Casuso (co-first autor), H. Takahashi, F. Rico, A. Miyagi, M. Chami, S. Scheuring 2016 ACS nano 10 (2), 2584-2590 (IF: 12.8)

· ´High-speed force spectroscopy unfolds titin at the velocity of molecular dynamics simulations´ F. Rico, L. Gonzalez, I. Casuso, M. Puig-Vidal, S. Scheuring Science 2013 342 (6159), 741-743 (IF: 33.6)

· ´A hybrid high-speed atomic force–optical microscope for visualizing single membrane proteins on eukaryotic cells´ A. Colom, I. Casuso (co-first autor), F. Rico, S. Scheuring 2013 Nature communications 4, 2155 (IF: 11.5)

· ´High-speed atomic force microscopy: cooperative adhesion and dynamic equilibrium of junctional microdomain membrane proteins´ A. Colom, I. Casuso, T. Boudier, S. Scheuring 2012 Journal of molecular biology 423 (2), 249-256 (IF: 4.3)

· ´Characterization of the motion of membrane proteins using high-speed atomic force microscopy´ I. Casuso, J. Khao, M. Chami, P. Paul-Gilloteaux, M. Husain, JP. Duneau, H. Stahlberg, JN. Sturgis, S.Scheuring 2012 Nature nanotechnology 7 (8), 525-529 (IF: 34.0)

· ‘Experimental evidence for membrane-mediated protein-protein interaction’ I. Casuso, P. Sens, F. Rico, S. Scheuring, 2010 Biophysical J. Letters 99, L47-49 (IF: 4.4) // Paper highlighted in the News and Notable sections of Biophysical Journal (October 2010) and Nature Methods (November 2010).

· ‘Automated setpoint adjustment for biological contact mode atomic force microscopy imaging’ I. Casuso, S. Scheuring, 2010 Nanotechnology 21, 035104-035112 (IF: 3.1)

· ‘Contact-mode high-resolution high-speed atomic force microscopy movies of the purple membrane’ I. Casuso, N. Kodera, C. Le Grimellec, T. Ando, S. Scheuring 2009 Biophys J. 97(5), 1354-1361 (IF: 4.4)

· ‘Quantitative dielectric constant measurement of thin films by DC electrostatic force microscopy’ G. Gramse, I Casuso, J. Toset, L. Fumagalli, G. Gomila. 2009 Nanotechnology 20, 395702-395710 (IF: 3.1)



Professional honors and grants

· 2015. French Embassy in Spain Grant for organizing the Microsecond AFM project panel of experts

· 2014. Awarded Excellence INSERM Prime

· 2012. AXA PostDoctoral Grant (https://www.axa-research.org/en/projects/ignacio-casuso)

· 2010. Best poster at AFM Biomed 2010 Conference. Croatia.

· 2005. Travel grant for stay at Autonomous University of Madrid, Spanish government Funding.

· 2004. Travel grant for stay at Purdue University, Spanish government Funding.

· 2004. Grant for PhD studies, Spanish government Funding.

· 2003. Grant for assisting Trends in Nanotechnology (TNT) 2003, TNT Funding.

· 2003. Grant for PhD studies, University of Barcelona Funding

· 2001. Leonardo da Vinci grant for the internship at CNES, EU Funding.

· 1998. Bells Labs summer scholarship, EU Funding.