Project 1: The structural basis of viral transport by dynein/dynactin.

Viruses are too large to move inside cells unless they hijack cytoskeletal motors such as the microtubule motor dynein. Remarkably dynein is co-opted by all classes of virus. Examples include members of the herpes family which travel along neurons and influenza viruses which use dynein for unpackaging their genomes. However, the connections that link these viruses to dynein are unknown.

The goal of this PhD project is to determine the network of proteins that link viral components to the motor. We will use cell biology techniques to identify host:virus interactions. Single molecule fluorescence microscopy assays will be used to reconstitute the movement of these proteins with dynein and cryo-electron microscopy (cryoEM) to determine structures of the transport machinery.

References

Reck-Peterson SL, Redwine WB, Vale RD, Carter AP. (2018)
The cytoplasmic dynein transport machinery and its many cargoes.
Nat Rev Mol Cell Biol. 19:382-398.

Banerjee I, Miyake Y, Nobs SP, Schneider C, Horvath P, Kopf M, Matthias P, Helenius A, Yamauchi Y. (2014)
Influenza A virus uses the aggresome processing machinery for host cell entry. Science. 346:473-477

Urnavicius L, Lau CK, Elshenawy MM, Morales-Rios E, Motz C, Yildiz A, Carter AP. (2018)
Cryo-EM shows how dynactin recruits two dyneins for faster movement.
Nature. 554:202-206.

Zhang, K., Foster, H.E., Rondelet, A., Lacey, S.E., Bahi-Buisson, N., Bird, A.W. and Carter, A.P. (2017)
Cryo-EM Reveals How Human Cytoplasmic Dynein Is Auto-inhibited and Activated.
Cell. 169:1303-1314

Urnavicius, L., Zhang, K., Diamant, A.G., Motz, C., Schlager, M.A., Yu, M., Patel, N.A., Robinson, C.V. and Carter, A.P. (2015)
The structure of the dynactin complex and its interaction with dynein.
Science. 347:1441-6.

Project 2: Reconstitution of a ciliary rootlet for cryoEM structure determination.

Cilia are found on all cells and have functions ranging from receiving signals to moving mucus out of the lungs. At their feet are basal bodies and below these are long striated filaments known as rootlets. This cytoskeletal filament is hardly mentioned in textbooks and little is known about its function and yet mutations in them cause ciliopathies (a class of genetic diseases related to loss of cilia function).  In some algae the rootlets can act as mini-muscles that contract and pull the nucleus towards the base of the cilia. It is not known if any human rootlets have the same function or what the role of this contraction is.

This PhD project will involve reconstitution of rootlets from its known components.  The structure of these reconstituted rootlets will be solved by cryoEM with the goal of understanding how a filament can contract with enough force to move the cell’s largest organelle. The project will provide training in biochemistry and structural biology. It will provide the opportunity to improve our understanding of one of the most enigmatic structures in the cell.

Salisbury JL, Floyd GL. (1978)
Calcium-induced contraction of the rhizoplast of a quadriflagellate green alga.
Science. 202:975-7.

Chen JV, et al. (2015)
Rootletin organizes the ciliary rootlet to achieve neuron sensory function in Drosophila.
J Cell Biol. 211:435-53.

Vlijm R, et al. (2018)
STED nanoscopy of the centrosome linker reveals a CEP68-organized, periodic rootletin network anchored to a C-Nap1 ring at centrioles.
Proc Natl Acad Sci U S A. 115:E2246-E2253.

Urnavicius L, Lau CK, Elshenawy MM, Morales-Rios E, Motz C, Yildiz A, Carter AP. (2018)
Cryo-EM shows how dynactin recruits two dyneins for faster movement.
Nature. 554:202-206.

Zhang, K., Foster, H.E., Rondelet, A., Lacey, S.E., Bahi-Buisson, N., Bird, A.W. and Carter, A.P. (2017)
Cryo-EM Reveals How Human Cytoplasmic Dynein Is Auto-inhibited and Activated.
Cell. 169:1303-1314