The journals Nature Structure and Molecular Biology, Nature Communications and Nano Letters published works that are the result of a collaboration that have taken years between researchers from IBioBA and CIBION.

One more example that interdiscipline allows building new horizons of knowledge that are achieved with combined skills and experience, in this case, between biology and physicochemistry.

The research groups of the IBioBA and the CIBION (Center for Research in Bionanosciences), located in the same building at the Technological Scientific Pole, work daily to reinforce the institutional link from a fruitful academic complementarity, an important value in the philosophy of both institutions. Such is the case of the Molecular Neurobiology group led by Dr. Damián Refojo (IBioBA) and the Applied Nanophysics group led by Dr. Fernando Stefani (CIBION).

The disciplinary crossover between both groups occurs midway between the formulation of biological questions and the development of new technologies in microscopy. While Dr. Refojo’s group studies molecular mechanisms present during neuronal development and that needs to be visualized with the highest possible resolution, Dr. Stefani’s group develops tools for visualizing biological systems with super-resolution fluorescence microscopies that allow the visualization of cellular structures with a level of detail between 5 and 100 times higher, reaching molecular resolution.

On the other hand, a third equally essential part in this interaction is the group of Dr. Alfredo Cáceres from the Cordoba University Institute of Biomedical Sciences (IUCBC). Cáceres is a pioneer and international benchmark in the study of the neuronal cytoskeleton and its role in axodendritic development, and his contributions have also been essential in order to accurately visualize neuronal cytoskeletal structures and adequately interpret the images obtained.

PhD Damián Refojo (IBioBA) and PhD Fernando Stefani (CIBION).
PhD Damián Refojo (IBioBA) and PhD Fernando Stefani (CIBION).

The three studies in detail

The first publication was made in the journal Nature Structure and Molecular Biology in February 2020. There was presented a method developed by the Refojo´s Group that detects proteins regulated by Nedd8, a molecule necessary for both cell proliferation and normal development of synaptic connections between neurons and memory and learning processes. This development represents a variant of a methodology called mass spectrometry, by means of which they were able to obtain the first catalog with hundreds of nedylated proteins, that is, modified by the Nedd8 paste. This traking showed that the neddilation in neurons mainly affected proteins of the cytoskeleton, fascicles that fulfill essential structural and transport functions for neuronal development.

(Left) Image of a normal developing neuron that begins to emit prologations (in green) that will then allow it to form synaptic connections. These extensions arise from growth cones formed by actin networks (in red) controlled, in turn, by the neddilated Cofilin protein. (Right) When cofilin protein cannot be neddylated, the neuron loses the organization of its actin networks and develops in an abnormal way.

“With this we propose a substantial change in the way this molecular modification was thought up to today”, highlights Damián Refojo and adds: “Until now it was believed that Nedd8 bound to a single type of proteins called Cullinas, which control cell proliferation . In this work we discovered that Nedd8 actually binds to hundreds of other proteins to control their function, their intracellular location, their stability, or their ability to form other molecular complexes. Among them we found many proteins of the cytoskeleton”.

To visualize the microscopic bundles that make up the cytoskeleton in neurons, they used super-resolution microscopy from Fernando Stefani’s laboratory. It was with this technology that they verified that the inhibition of neddilation altered the formation of these fascicles and, as a consequence, neuronal maturation and dendrite formation.

Super-resolution images of microtubules in cells obtained using SIMPLER-DNA-PAINT method. The resolution achieved in these images is 10 nm. The figure on the left shows the top view of the microtubules; the figures on the right show a cross section that allows the interior cavity of the microtubules to be visualized.

The following article was published in the prestigious journal Nature Communications in January 2021. There the SIMPLER visualization method was presented, which allows observing biological systems with a level of detail in 3D greater than that of conventional nanoscopies. Thanks to this technology they were able to visualize spectrin rings, the center of microtubules in neurons of just 20nm and nuclear pores in other cell lines, different macromolecular complexes that are studied in the laboratories of Drs. Refojo and Cáceres.

This new gateway to 3D molecular study allows the internal structure of neurons to be studied in maximum detail in normal conditions and in various degenerative diseases.

On the other hand, this method does not require modifications in the hardware of conventional microscopes, so it promises to be widely used in any laboratory that carries out super-resolution experiments.


The third advance was featured in March 2021 in Nano Letters magazine. There, a new method is detailed to locate the position of two molecules interacting with a precision five times greater than that available until now. The most used method to visualize molecular interactions is based on a phenomenon of energy transfer between two molecules called FRET (Förster Resonance Energy Transfer). There are countless protocols for FRET imaging that report interactions between various molecules or between molecules and their environment. However, until now, there was no generally applicable method for obtaining super-resolution FRET images.

This is exactly what Fernando Stefani’s team achieved with the support of the IBioBA group, opening the way for a huge range of possibilities to investigate how and where molecular interactions occur within cells.

Figura que combina los métodos de medición STED y FRET de superresolución. Muestra la distribución de las proteínas actina y aducina en el esqueleto de las neuronas y sus puntos de interacción marcados en color turquesa. El esqueleto se conforma de anillos separados por 190 nm de distancia, dentro de los cuales se distribuyen ambas proteínas.
Jerónimo Lukin (PhD student IBioBA), Alan Szalai (PhD student CIBION) and PhD Sebastián Giusti (Researcher IBioBA).

Work between teams

For the two leading researchers of these three developments, Damián Refojo and Fernando Stefani, the possibility of working closely is a unique opportunity that leads them to multiple research questions that are possible thanks to this open and frequent dialogue between laboratories and disciplines: “Interdisciplinary interaction is challenging and fun, but it is also the path we must follow to maintain our high scientific standards,” says Damián Refojo. On his part, Stefani remarks that “both techniques will allow us to do studies that were previously imposible to do. We will be able to see how proteins are organized and interact within cells and this gives us a fundamental competitive advantage for our future studies”, concludes Fernando Stefani.


Nature Structure and Molecular Biology:
IBIOBA: Raquel Becerra, Sebastian Giusti and Damian Refojo
CIBION: Martin Bordenave and Fernando Stefani.

Nature Communications:
IBioBA: Jeronimo Lukin and Damian Refojo.
CIBION: Alan Szalai, Bruno Siarry and Fernando Stefani

Nano Letters:
IBioBA: Jeronimo Lukin, Sebastian Giusti and Damian Refojo
CIBION: Alan Szalai, Bruno Siarry and Fernando Stefani