Dissecting the Molecular Mechanism of Intraflagellar Transport Motors

The 30 trillion cells that make up the human body fall into ~200 major types. Virtually all of these cell types grow an antenna-like structure called the cilium, which projects from the cell surface into the environment. Distantly related organisms such as protozoa and green algae also possess cilia, suggesting that they are ancient organelles that pre-date the last common ancestor of all eukaryotes. Cilia serve vital sensory roles, detecting and processing environmental stimuli such as light, olfactants, morphogens and fluid flow. A subset of cilia actively beat with a wave-like motion to generate cell propulsion, for example in sperm cells, or movement of fluid over the epithelia lining the airways and oviduct. Defects in the architecture and composition of cilia cause a plethora of disorders collectively known as ciliopathies. Hence, deciphering the mechanisms by which cilia are assembled and function is fundamental to understanding a wide array of biological processes and the molecular basis of disease states. We are particularly interested in the central mechanism underpinning cilia formation and function. This constitutes a transport system that moves building blocks from the cytoplasm to the tip the cilium and returns products to the cell body. It is powered by two types of ATP-fueled motor protein, which move in opposite directions along the cilium. We wish to uncover the unique mechanisms through which the motor proteins generate force and movement, coordinate round trips of transportation, and avoid colliding with each other as they traverse the cilium. To achieve this, we will use new tools to produce and manipulate the motors for detailed study.

We seek to gain insight into the molecular mechanism of intraflagellar transport (IFT), the motor-protein driven process essential for the construction and maintenance of cilia. We will focus on the microtubule-based motors Kif3, which moves building blocks and functional components from the cell body to the tip of the cilium, and dynein-2, which powers transport in the opposite direction. A striking feature of IFT is that Kif3 and dynein-2 operate in long linear arrays containing multiple copies of both motors, but how these multi-megadalton motor assemblies coordinate IFT is not well understood. In Aim 1, we will exploit our newly developed recombinant expression systems for human Kif3 and dynein-2 to determine their motile behaviour and force production at the single molecule level, and probe their regulation using site directed mutagenesis. In Aim 2 we will systematically explore the impact and functional consequences of multiple motors in IFT, developing tools to link together Kif3 and dynein-2 with exquisite control over the number, position, and type of motor per assembly. In Aim 3, we will discover if Kif3 and dynein-2 use post-translational modifications on their microtubule tracks in order to avoid colliding with one another as they move in opposite directions along the cilium. Such ciliary kinesin and dynein navigation using post-translational modifications would represent a landmark result in the 'tubulin code'. Overall, this combination of biochemical, biophysical and synthetic biology approaches will shed new light on motor protein mechanisms and the assembly and maintenance of cilia, while developing new biotechnological tools that may be applied to arrays of other molecular machines.

This research will make a direct impact in understanding mechanisms responsible for the formation and function of cilia, influencing understanding of motor protein action, protein trafficking, the tubulin code, and organelle biogenesis, as well as ciliary functions required for numerous essential cellular and developmental processes. It will produce a postdoctoral trainee equipped with sought-after skills in challenging eukaryotic multi-protein complex production, nanotechnology and advanced fluorescence microscopy, as well as professional experience that is valuable across different economic sectors. It will foster an international collaboration that will enrich UK science. It will develop nanotechnology tools for discovery-based research, which could also aid similar technologies being used to advance drug delivery and manufacturing. Defects in intraflagellar transport are associated with vision impairment, craniofacial abnormalities, skeletal abnormalities, cystic kidneys, extra or partially fused fingers and toes, sterility, childhood obesity and developmental delay among other conditions, thus severely affecting the quality of life for sufferers and in many cases proving fatal. This research is focussed on the fundamental mechanisms of cilia assembly and function rather than characterisation of disease states, but it may help patients and their families gain a better understanding of cilia and the origin of ciliopathies. Drugs acting on cytoskeletal-motor systems involved in cell division and cardiac muscle contraction are in development for treatment of cancer and heart failure respectively, and improved understanding of intraflagellar transport may contribute to new avenues for therapeutic intervention in cilia in the long term.