Spatiotemporal analysis of the assembly and motility of dynein transport complexes on dynamic microtubules

• Applicant: Dr. Janina Baumbach
• Subject Area: Cell Biology ; Biochemistry
• Term: from 2016 to 2018
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 313854929

Current state of research: Cytoplasmic dynein is a large cytoskeletal motor that actively transports a variety of cargoes including organelles, mRNA and viruses inside cells. This process is particularly important in extended neuronal cells and defects in transport by dynein lead to neurological diseases in humans. The tracks that dynein walks along, microtubules, are highly dynamic structures that alternate between bouts of growth and shrinkage. There is evidence that a group of proteins that specifically bind to the growing microtubule plus end (plus end-tracking proteins) are important for recruiting dynein and its cargo to the microtubule track. This process has been suggested to serve as a search and capture mechanism by which dynamic microtubules probe the cellular space and efficiently capture cargoes. Recruitment of dynein to plus ends is difficult to study in cells due to the challenges of visualizing single molecules in vivo and a seemingly complex network of protein interactions. Thus, we still have a poor understanding of how cargo-dynein complexes are assembled and how transport is initiated and executed along dynamic microtubules. Research objective: The aim of this project is to reconstitute dynein transport initiation and movement on dynamic microtubules in vitro with purified proteins and thus dissect how this process is orchestrated. Research plan: I will purify and fluorescently label recombinant protein components required to reconstitute motile human dynein complexes. I will also produce labelled versions of several microtubule plus end-tracking proteins that have been implicated in transport initiation in vivo. I will use these proteins to reconstitute moving dynein along dynamic microtubules and visualize this using a fluorescent microscopy technique able to resolve single molecules. I will then add different proteins that bind to growing microtubule plus ends to identify the minimal sufficient system for dynein transport initiation from these structures. Next I will dissect the sequence of events that control transport initiation by filming proteins labelled with different fluorophores to see in which order they get recruited to the microtubule. Finally, I will purify protein complexes of dynein and plus end-tracking proteins that are formed during transport initiation by gelfiltration chromatography and, in collaboration, study their structure using negative stain electron microscopy and cryo-electron microscopy. The proposed project will allow us to analyse how dynein transport is initiated in time and space and how it goes awry in neurological diseases.

• DFG Programme: Research Fellowships
• International Connection: United Kingdom
• Host: Dr. Simon Bullock, Ph.D.