Membrane nanotube formation from endosomes by cooperating motors: physical regulation and quantitative characterization

Organization of cells depends strongly on transport between different organelles and correct transport is crucial for the healthy functioning of cells. In this project we have studied interactions in teh endosomal pathway and found that late endosomes influence the properties of early endosoms. Furhtermore we find that by silencing a late endosomal coat, Rab7 we can influence the endoplasmic reticulum (ER) from tubules to sheets. New data states that ER can influence endosomes and we now show for the first time that the opposite is also true. In another part of the projects we have established the optical tweezers to test the interaction of different nanoparticles on different types of cells (macrophages and cancer cell lines) and, for the first time ever, micromanipulated microinjected nanoparticles inside whole living organism (zebrafish). This is important for understanding how and why nanoparticles (used for disease treatment and delivery of drugs) interact with different structures inside a living organism. The new approach increases the efficiency of experiments and opens up for analyzing quantitatively interaction strengths, which is not possible using traditional approaches.

The aim of this project is to quantitatively characterize the cooperativity of motor proteins during membrane nanotube formation from endosomes and to determine in living cells the regulatory role of physical properties on tubulation. Membrane nanotubes ( typically 50 nm diameter) are ubiquitous in cells and play an important role in trafficking and sorting. To form these highly curved membrane shapes significant forces need to be exerted. Cytoskeletal motor proteins (such as kinesins or dyneins) have been shown to be important for membrane nanotube formation. It is now clear however that single motors are not strong and persistent enough, hence they need to join forces and cooperate. We aim to unravel the details of the mechanisms through which they clust er together and to characterize the balance between the motor protein cluster size and the forces required to form tubes, which are set by physical properties of the membrane. In order to do this, we will develop new techniques and apply live cell imaging with high temporal and spatial resolution. We will use reconstitution with giant unilamellar vesicles and purified endosomal proteins and other factors relevant for endosome tubulation. In parallel, we will directly quantify and control the endosomal me mbrane tension and tube forces using optical tweezers and micropipette aspiration techniques.