Search

Transport of cargos not only plays a critical role on the meter scale in our daily life when we travel from A to B but also plays an essential role for cellular processes on the nanometer scale without which we would not exist. This cellular transport is carried out by motor proteins which walk on cellular highways and are responsible for almost all directed transport in cells. Moreover, these motor proteins play key roles in other cellular processes including mitosis and cilia motility. One of these motor proteins is the microtubule-based motor dynein. Dynein is a complex, flexible, and large machine that has to coordinate it’s two engines and feet in order to achieve directed and continuous motility. Recent structural and biochemical studies uncovered key molecular mechanisms contributing to dynein motility. However, a comprehensive understanding of how dynein steps along its microtubule track, and how its different domains are coordinated to achieve this movement were lacking in the field. Therefore, I first set out to determine how dynein’s ATPase activity and mechanics are coupled among the motor domain of dynein and showed that the ~15 nm long coiled-coil linking the catalytic AAA ring and its microtubule-binding domain is indispensable in regulating motor activity. Moreover, I found that the length rather than the sequence of this coiled-coil is remarkably well conserved and that the length conservation is paramount for directional motility. Integrating these observations allowed us to generate an updated model for the internal regulation of dynein.Our understanding of how the different domains of dynein move relative to each other has been limited by insufficient high spatiotemporal resolution. To overcome this, I first created a method that enables three-color image registration and distance measurements with one nanometer accuracy and second, I developed DNA FluoroCubes that enabled me to track the position of multiple domains of dynein for a prolonged time with nanometer precision. Combining both of these methods enabled me to gain insights into the conformational changes of dynein’s domains while moving along microtubules. I found that the motor domain of dynein is very flexible and that this flexibility is important for dynein motility and enables dynein to adopt a large variety of conformations. Together, these findings revealed a new model for dynein stepping that defines the minimal requirements to facilitate directed and continuous motility.

This project is described in:High accuracy measurements of nanometer-scale distances between fluorophores at the single-molecule level (2018): https://www.biorxiv.org/content/10.1101/234740v2 Here, we provide raw datasets to test the software, to create and test registration maps, and to measure distances of samples used in this study. Furthermore, we provide the raw negative-stain EM micrographs used in this study. The data is supplied in zip format and contains an html file with additional information. Thus, please first read the README.html file.

This project is described in: High accuracy measurements of nanometer-scale distances between fluorophores at the single-molecule level (2018): https://www.biorxiv.org/content/10.1101/234740v2 Here, we provide raw datasets to test the software, to create and test registration maps, and to measure distances of samples used in this study. Furthermore, we provide the raw negative-stain EM micrographs used in this study. The data is supplied in zip format and contains an html file with additional information. Thus, please first read the README.html file.

Polycomb repressive complexes (PRC) play a key role in gene repression and are indispensable for proper development. Canonical PRC1 forms condensates in vitro and in cells and the ability of PRC1 to form condensates has been proposed to contribute to maintenance of repression. However, how chromatin and the various subunits of PRC1 contribute to condensation is largely unexplored. Using single-molecule imaging, we demonstrate that nucleosomal arrays and PRC1 act synergistically, reducing the critical concentration required for condensation by more than 20-fold. By reconstituting and imaging PRC1 with various subunit compositions, we find that the exact combination of PHC and CBX subunits determine the initiation, morphology, stability, and dynamics of condensates. In particular, the polymerization activity of PHC2 strongly influences condensate dynamics to promote formation of structures with distinct domains that adhere to each other but do not coalesce. Using live cell imaging, we confirmed that CBX properties are critical for condensate initiation and that PHC polymerization is important to maintain stable condensates. Together, we propose that PRC1 can fine-tune the degree and type of condensation by altering its composition which might offer important flexibility of regulatory function during different stages of development.