Using viruses to study kinesin-1 recruitment, regulation and function

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Cells need to sort and transport cargo to distinct parts of the cell and this is fundamental for cell survival and for determining cell structure and function. To do this cells possess a skeleton (cytoskeleton) that is composed of microtubules. Microtubules are associated with motors that bind and transport cargo in a highly specific and regulated manner. One type of motor, called kinesin-1, is responsible for transporting cargos to specific locations within the cell. Defects in kinesin-1 transport cause human diseases, ranging from age-related macular degeneration and cataracts, to invasive cancers and neurological disorders. Kinesin-1 transport is very important for moving cargos longer distances along nerve axons and defects in kinesin-1 cause several neurodegenerative disorders, including Parkinson's, Huntington's, Alzheimer's and inherited spastic paraplegia. How kinesin-1 binds to and moves along microtubules is quite well studied. In contrast, how kinesin-1 recruits specific cargos and how kinesin-1 activity and target destination are regulated remain poorly understood.Viruses replicate inside cells and have evolved mechanisms to hijack cellular machinery to enhance their replication and spread. Several viruses exploit kinesin-1 during their replication cycle, for instance to help export newly formed virus particles out of the cell. An example is vaccinia virus (VACV), the prototype poxvirus and the vaccine used to eradicate smallpox. Our lab uses the recruitment of kinesin-1 by VACV as a model to study the mechanisms that control the binding of cargo to kinesin-1 and activation of the motor.The kinesin-1 complex is made up of two kinesin heavy chains (KHC) and two kinesin light chains (KLC). Its activity is regulated by autoinhibition. Activation involves multiple binding events by cargo proteins that induce a structural change in the complex enabling association with microtubules. Mammalian cells express multiple kinesin-1 types (isoforms), each with slightly different biochemical properties that may bind different cargos or move to different destinations.Three VACV proteins, A36 and the F12/E2 complex, are involved in recruiting kinesin-1 to transport virus particles to the cell surface. To understand the role of each of these proteins, their interaction with each other and with components of the kinesin-1 complex will be characterised biochemically and using structural biology approaches. We have shown that F12/E2 and A36 associate with different domains of KLC and preferentially bind to different KLC isoforms. Gene knock-down using RNA interference and gene knock-out using CRISPR/Cas9-mediated gene editing will be used to determine the importance of different KLC isoforms for virus transport and for normal cellular function.The roles of various KLC isoforms in cell function remain largely unknown. Cells in which individual KLCs or combinations of KLC have been knocked-out will be characterised by microscopy. Additionally, the cellular functions of individual KLCs will be determined by identifying and comparing the proteins that associate with them. Proteins that are also found to associate with the viral transport complex will be studied to determine if they are required for virus export. The contribution of viral proteins, individual KLC isoforms and cellular proteins to the activation of kinesin-1, and their possible role in its modulation will be measured using a combination of in vitro and in cell assays.The proposed research project aims to investigate the mechanisms used by VACV to recruit and activate kinesin-1 by characterising the virus and cellular proteins involved. This research will also help our understanding of the normal cellular processes and how disease ensues if these dysfunction. Structural information about the interaction of the virus with kinesin-1 will aid the development of compounds that inhibit this process and prevent disease caused by poxviruses.