Understanding the Machinery Cutting One Cell into Two
The mechanism of cell division is conserved in many eukaryotes, ranging from yeast to man. A contractile ring of filamentous actin and myosin II motors generates the force to bisect a mother cell into two daughters. The cytokinetic actomyosin ring is among the most complex cellular machines, comprising over 150 proteins. Understanding how these proteins organize themselves into a functional ring with appropriate contractile properties remains one of the great challenges in cell biology. Using supported lipid bilayers as a two-dimensional fluid scaffold and total internal reflection fluorescence microscopy, we test and characterize the function of isolated, actin binding proteins and protein complexes involved in cytokinetic ring formation.
Role of Caveolae in Membrane Tension Regulation
How are cells membranes protected from sudden stresses?
Caveolae can unfold under sudden mechanical stress to protect the cell plasma membrane from ruptures.
My PhD at the Institut Curie (Paris, France) investigated the role of caveolae for the mechanical properties of the cell plasma membrane and its reactions to different kinds of mechanical stresses. Though caveolae, which are specialized invaginations of the plasma membrane, were discovered in the 1940s, their role in membrane biology was debatable. My work demonstrated that caveolae are important for maintaining the integrity of the plasma membrane and act as a membrane tension buffer upon sudden mechanical stresses. This work changed the view of the role of caveolae in cell membranes, was published in Cell (Sinha, Köster et al., Cell, 2011) and was recommended by Faculty of 1000.
This project was supervised by Dr. Pierre Nassoy and was a close collaboration between the groups of Dr. Patricia Bassereau and Dr. Ludger Johannes/Dr. Christophe Lamaze. It combined various biophysical techniques with cell biology strategies. Optical tweezers were used to extract membrane tethers from adherent cells at different osmotic conditions, since the force required would be directly proportional to the effective cell membrane tension. For this, I adapted our optical tweezers setup to work with adherent cells and implemented a system to change the osmotic pressure while holding a membrane tether with the tweezers. Together with my colleague Bidisha Sinha, we also designed a PDMS based cell stretcher device which aids the study of single cells under a uni-axial stretch with TIRF microscopy. This device allowed us to confirm the disappearance of single caveolae under defined stress levels.
Thirdly, I studied plasma membrane spheres (PMS) obtained from cells expressing labeled caveola markers. Controlling the PMS membrane tension with a micropipette while measuring the force to pull a membrane tube with optical tweezers and observing the presence of functional caveolae with a confocal microscope, I could confirm the proposed role of caveolae in plasma membranes.
Based on these findings, I collaborated with Dr. Gillian Butler-Brown at the Institut de Myologie - Hopital Pitie-Salpetriere (Paris), an expert in muscular dystrophy. Muscular dystrophy is a severe debilitating disease, several forms of which are linked to mutations in the caveolin gene. This collaboration allowed me to obtain muscle cells from patients showing a gamut of phenotypes. After establishing a protocol to probe differentiated myotubes with optical tweezers, I was able to show that myotubes with a defect in caveolin show increased levels of membrane tension and have higher probabilities of rupturing under hypo-osmotic shocks, as compared to wild type myotubes (Sinha et al, Cell, 2011; DeWulf et al, biorxiv, 2018).