FORFUNIGO

Fungi are the cause of a range of infections, some leading to death, and are part of an emerging global microbial threat. Fungal filamentous growth is critical for pathogenicity, in particular ability to invade and evade host cells. Little is known with respect to the physical forces generated by fungal (hyphal) filaments, the importance of such physical forces in cell and tissue penetration or escape, nor how external resistive forces affect hyphal filaments, i.e. perturb the cell wall, intracellular organization, etc. This international and interdisciplinary project is the result of extensive interactions between biologists and physicists with complementary expertise in live cell imaging, correlative large volume electron tomography, molecular genetics, physical measurements, microfabrication and mathematical modeling. The primary aim of this project is to elucidate the interplay between physical forces, fungal tip growth and intracellular organization, specifically determining how external (resistive) forces are responded to by the human fungal pathogen, Candida albicans. We will take advantage of single cell force sensors, using microfabricated wells comprised of elastomer, such as PDMS, to quantitate the physical forces important for C. albicans filamentous growth. Using such size-constrained microchambers of varying stiffness, we will determine the importance of the cell wall, cytoskeleton, secretory pathway, GTPase signaling and lipid signaling in hyphal force generation during substrate contact and invasion. Specifically, we will quantitate hyphal cell deformation and chamber deformation as well as the limit of rigidity in which penetration of substrate is observed, together with the hyphal extension rate during growth on and in substrate of different stiffness. These analyses will be carried out in wild-type and mutant strains, in the presence and absence of a range of chemical perturbants. We will also determine the shape of the filament apex upon hyphal contact and penetration into different stiffness PDMS as well as thickness of the cell wall during these processes, using a novel live-cell method. Furthermore, we will quantitate the distribution of activated Rho GTPases, providing a read out for polarized growth, upon contact and invasion into the PDMS substrate. In a complementary approach, we will quantify the impact on the force parameters of perturbing apex-dependent invasive growth, using optogenetics to generate new sites of growth. We will also assess mutants altered for force generation during invasive growth in in vitro and in vivo mucosal infection models. We will determine overall 3D organization, focusing on endo-/exocytic compartments, in the hyphal filament at the nm scale and how it is altered in response to mechanical perturbations. This will be accomplished by live cell microscopy, correlative large volume tomography (focused ion beam/scanning electron tomography). Furthermore the function of the Spitzenkörper will be probed using laser nanosurgery and cryo-tomography. Lastly, we will establish physical and mathematical models of hyphal invasive growth, in which a range of parameters that mimic the environment of an infection will be varied. This combination of physical and mathematical models will be critical to define the important steps or processes (both mechanical and biological), key parameters and scales for invasive filamentous growth and ultimately to predict behavior upon chemical, genetic and physical perturbations. This ambitious project is only possible as an interdisciplinary collaborative venture and the complementary expertise coupled with the common interests of the partners will contribute to its success. Our preliminary data provide proof of principle for this exciting project at the interface of biology and physics, which has a high potential for uncovering basic principles of morphogenesis, biomechanics and fungal pathogenesis as well as identifying novel antifungal targets.