Mechanism of Cytoplasmic DyNein
Cytoplasmic dynein is a AAA motor that transports a variety of intracellular cargo towards the microtubule minus-end in eukaryotic cells. Compared to other cytoskeletal motors (kinesin and myosin), the mechanism of dynein motility is significantly less understood due to its large size and complexity. We showed that dynein motility differs significantly from kinesin and myosin in many aspects. By using single molecule high resolution tracking, FRET and optical trap microscopy, we are aiming to dissect the detailed mechanism of dynein processivity, directionality and force production. In addition, we introduce specific mutations in the motor domain and test the role of each subunit in dynein mechanism.
IMAGING GENOMIC Loci with cas9
Imaging chromatin dynamics is crucial to understand genome organization and its role in transcriptional regulation. Recently, the RNA-guidable feature of CRISPR-Cas9 has been utilized for imaging of chromatin within live cells. However, these methods are mostly applicable to highly repetitive regions, whereas imaging regions with low or no repeats remains as a challenge. To address this challenge, we use single-guide RNAs (sgRNAs) integrated with up to 16 MS2 binding motifs to enable robust fluorescent signal amplification. These engineered sgRNAs enable multicolour labelling of low-repeat-containing regions using a single sgRNA and of non-repetitive regions with as few as four unique sgRNAs. Using this tool, we can track the locations of native chromatin loci throughout the cell cycle and determine differential positioning of transcriptionally active and inactive regions in the nucleus.
Intraflagellar Transport and Cell Motility
Intraflagellar transport (IFT) is a critical protein-transport system that builds and maintains the flagella present on almost all eukaryotic cells. IFT trains carry the various building blocks of the flagellum to the site of assembly at the tip and shuttle proteins out the flagellum. We use IFT in Chlamydomonas as a model system to study how motor protein assemblies transport cargo in vivo. Using live-cell imaging and optical trapping techniques, we have shown that Chlamydomonas uses the forces generated by IFT motors to power gliding motility along surfaces. Using the Photogate to observe IFT proteins at the single-particle level, we have elucidated how these trains mix, remodel, and change direction at the ciliary tip. Moreover, we have shown how kinesin-II and dynein-1b motors share transport duties to allow for efficient transport. We are also interested in how the dynamics of IFT motors and proteins may play a part in regulating the length of flagella.
Protection of Telomeres
The gradual shortening of telomeres upon each cell division ultimately triggers cell cycle arrest and apoptosis. Hence telomeres serve as an internal clock in the aging process of an organism. Understanding the details of how this clock works and how it can be reset has broad implications for developmental biology, aging, cancer, and stem cell biology. Telomeres are protected by a large nucleoprotein complex, shelterin. Currently, we are investigating the effect of morphological changes brought about by the shelterin proteins on telomeres and their influence on protection against DNA damage response pathways by using super resolution microscopy and single-molecule FRET. We are also developing assays to assemble the shelterin complex to gain a better understanding on the interplay between the six core components and their in role in restructuring and maintenance of telomeres.