Intermediate filaments: from single filament to dynamic network – SiFi2Net

This project used innovative and multidisciplinary methods to provide a quantitative description of IFs at several scales, from the single, isolated filament to the entire network present in the cell. To do this, we used two complementary approaches: (i) a «top-down« approach in which, starting from a living cell, we removed the components involved one by one to study their influence on the organization and dynamics of the IFs, and (ii) a «bottom-up« approach, which aimed to reconstitute the assembly of the IFs step by step, starting from purified components placed in a simple and well-controlled environment. We used astrocytes, the most abundant glial cells in the brain, as a study model for migration, as well as glioma lines as a cancer cell model. The development of advanced microscopy tools such as super-resolution imaging has allowed significant improvements in spatial and temporal resolutions, which has been essential to elucidate the role of IFs in migration. For the in vitro experiments, we used single molecule detection methods to study filament elongation in situ.

In the cell, we have shown that, during cell polarization, retrograde transport of IFs is inhibited, indicating that polarity-associated signaling regulates IF dynamics. We also developed a method to characterize the IF structural organization which allowed us to understand the link between their mechanical and structural properties. Finally, we showed that IFs were not under tension in the cell, which was surprising given their mechanical function of protecting the cell. In vitro reconstitution experiments showed that the IF assembly is reversible, which had been neglected until now.

The project perspectives concern the study of the mutual regulation of the different types of cytoskeleton: actin, microtubules and intermediate filaments. The aim is to understand how the dynamic morphology of composite cytoskeleton networks is determined by its molecular components and how they regulate each other's properties, either directly or through specific proteins linking them, and how these interactions are modulated by external constraints.

Our work has resulted in the publication of 9 papers in leading international peer-reviewed journals, plus 3 papers in publication, and 2 in progress.

Intermediate filaments (IF) are one of the three major components of the cytoskeleton in animal cells along with actin microfilaments and microtubules, but they remain the least understood of the three despite numerous studies. Cytoplasmic IF form an extensive and elaborate network which connects the cell surface with the nucleus and the other organelles. For a long time, they were considered to be static biopolymers involved in the maintenance of the structural integrity of cells. Only recent discoveries indicate that IF also display dynamical and motile properties involved in cellular processes as diverse as cytosol organization, mechano-transduction or signaling. More than 95 diseases have been related to mutations in IF coding sequences. In some cases, the diseases result from alteration of their mechanical role (e.g. in muscular dystrophy or premature aging), but many diseases also result from defects in non-mechanical IF functions (e.g. in Parkinson or Alexander disease). The expression level of IF proteins has also been shown to dramatically increase in glioblastoma, the most frequent and aggressive brain tumors. Interestingly, this high expression level correlates with high migration and invasion capacities of these tumors. Overall, it is crucial to reach a better understanding of IF properties and functions and find how they are altered in pathological conditions, especially in view of further applications in therapeutic strategies.

The goal of this project is to gain insights into the physical mechanisms responsible for IF dynamical properties and how they are regulated during cell migration. At the interface between physics and biology, this project will use multidisciplinary and innovative methods for providing quantitative studies from the scale of isolated filaments to the whole network inside a cell. To achieve this goal, we will develop complementary approaches: (i) a top-down approach in which, starting from a living cell, we will remove the components related to IF one by one and investigate their influence individually, and (ii) a bottom-up approach, which aims at rebuilding the functional IF assembly step by step, starting from purified components reconstituted in vitro in a simple and well controlled environment. Both experimental approaches will be combined with theoretical modeling. From the live cell experiments, we expect to gain understanding of IF assembly and motility, and to elucidate how these properties are interconnected with the other cytoskeleton systems to achieve their cellular functions. The development of cutting-edge microscopy techniques including super-resolution imaging will improve the temporal and spatial resolutions, which are crucial parameters to better understand the fine tuning of the IF functional roles in the cell. For the in vitro experiments, we will develop a microscopy system combining single molecule detection and a microfluidics set up able to manipulate single filaments. With this set up, we will study in details the elongation of single filaments in situ and how it is impacted by IF composition, IF associated proteins and the interaction with the other cytoskeleton filaments.

The ultimate objective will be to bridge the gap between in vitro and live cell measurements. Confrontation of in vitro and in vivo data will allow a clear understanding of the IF dynamical properties and the mechanisms responsible for the IF network assembly and reorganization. Because IF functions are directly related to their dynamics, we believe that our findings will pave the way for a fundamental knowledge of the physiological functions of IF in live cells. A better comprehension of the mechanisms involved in IF alterations in diseases is also crucial to improve diagnosis and therapeutic treatments, and more specifically for glioblastoma and neurodegenerative diseases.