Abstract

A fundamental question in cell biology is: How do cells adapt their intracellular transport networks in response to internal and external cues to meet functional demands? The microtubule motor kinesin-1 is a central player in this, controlling both organelle dynamics and cytoskeletal organisation. Its dysregulation causes and contributes to many pathologies including neurological disease and viral infection. Our recent work has defined key pathways that mediate cargo recognition by and activation of kinesin-1. We have shown that this enzyme can be manipulated in cells, by both small molecules and designed peptides, to obtain new insights into the molecular function of transport systems. This opens the door to targeting intracellular transport in disease states. Here, we propose to explore how kinesin-1 is controlled by pH changes within cells. We hypothesise that kinesin-1 is a pH sensor and a pH-dependent transport effector that operates at the organelle-cytoskeleton interface. We integrate cell biology, chemical biology and protein design to test this proposition. The proposal stems from a successful collaboration between the Dodding and Woolfson groups in Biochemistry and Chemistry at the University of Bristol. In three integrated work packages we will: determine the biophysical and structural basis for pH sensing by the kinesin-1 heterotetramer (WP1); combine chemical and cell biology to explore the role of pH-sensing in organelle transport and cytoskeletal organisation (WP2); and apply structural and mechanistic understanding of this interface to garner design principles and rules to deliver dynamic, switchable coiled coils for novel architectures (WP3). Collectively, these WPs and objectives will develop an advanced understanding of kinesin-1 structure; uncover the molecular basis for the pH dependence of intracellular transport and our newly discovered microtubule targeting agent; and deliver new principles and tools for synthetic biology.