Mechanism and design of a pH sensor at the organelle-cytoskeleton interface

Cells possess many specialised components that must be in the right place at the right time to fulfil their functions. After their use, these components must be transported away for recycling or degradation. In addition, cells must be able to adapt their organisation to meet functional demands or respond to changes in their environment. Mis-regulation or disruption of these transport processes can contribute to human diseases ranging from neurodegenerative conditions such as Alzheimer's disease to cancer. Also, the natural transport systems of the cell can be 'hijacked' during viral infections by HIV-1 or bacterial infections such as Salmonella. Therefore, interrogating these transport systems and processes is key to understanding the natural workings of cells, diseases and infections.

To move components around, cells use a transport system composed of a network of cables known as microtubules. Much like a railway network, these cables link together regions of the cell. Cells also possess 'vehicles' that travel along this network known as motor proteins. On of the most important of these motors, kinesin-1, is the subject of our proposed study. Motor proteins can selectively attach to cellular components and move them on the microtubule network. They can also control the shape and organisation of the network itself by sliding the cables against one another. Despite the importance of motor proteins across many areas of cell biology, we lack a proper understanding of how complex machines like kinesin-1 are controlled. This proposal is all about understanding and exploiting this control within cells.

We have made preliminary observations that suggest a new and unexplored factor in the control mechanism. This is the balance between acidity and alkalinity of the cell (its pH), which appears to control activity of the kinesin-1 motor, and so controls cellular organisation. This is important because changes in pH occur when cells gain or lack nutrients, cellular components are damaged, and, in cancer, where tumours generate a local acidic environment. We will also explore whether this 'pH-dependence' can be exploited to develop drug-like molecules that disrupt cells in these contexts.

Our approach is unique. This joint proposal stems from a new and successful collaboration between the Dodding group in the School of Biochemistry and the Woolfson Group in the School of Chemistry of the University of Bristol. It combines two disciplines of cell biology and protein design, with each informing the other. Our fruitful collaborative work has highlighted important aspects of how kinesin-1 attaches to the cellular components it carries and how protein design and engineering can be used to obtain new insights into natural transport systems.

Through this proposal, we ask how kinesin-1 senses pH, how this is translated into transport activities, and whether this can be manipulated using drug-like molecules. We seek to apply our knowledge emerging from this natural system to develop new protein-design or synthetic-biology approaches that will test our understanding and lead to the development of synthetic transport machines that function in living cells. The outcomes of the proposal will lead to a deeper understanding of protein motors and the cellular processes that they orchestrate. In turn, this may lead to advances in more-applied fields such as synthetic biology, biotechnology and medicine.

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.