No abstract

No abstract

The cilium is an antenna-like organelle that performs numerous cellular functions, including motility, sensing, and signaling. The base of the cilium contains a selective barrier that regulates the entry of large intraflagellar transport (IFT) trains, which carry cargo proteins required for ciliary assembly and maintenance. However, the native architecture of the ciliary base and the process of IFT train assembly remain unresolved. In this work, we used in situ cryo–electron tomography to reveal native structures of the transition zone region and assembling IFT trains at the ciliary base in Chlamydomonas . We combined this direct cellular visualization with ultrastructure expansion microscopy to describe the front-to-back stepwise assembly of IFT trains: IFT-B forms the backbone, onto which bind IFT-A, dynein-1b, and finally kinesin-2 before entry into the cilium.

The intraflagellar transport (IFT) system is a remarkable molecular machine used by cells to assemble and maintain the cilium, a long organelle extending from eukaryotic cells that gives rise to motility, sensing and signaling. IFT plays a critical role in building the cilium by shuttling structural components and signaling receptors between the ciliary base and tip. To provide effective transport, IFT-A and IFT-B adaptor protein complexes assemble into highly repetitive polymers, called IFT trains, that are powered by the motors kinesin-2 and IFT-dynein to move bidirectionally along the microtubules. This dynamic system must be precisely regulated to shuttle different cargo proteins between the ciliary tip and base. In this Cell Science at a Glance article and the accompanying poster, we discuss the current structural and mechanistic understanding of IFT trains and how they function as macromolecular machines to assemble the structure of the cilium.

Intraflagellar transport (IFT) is a conserved process of cargo transport in cilia that is essential for development and homeostasis in organisms ranging from algae to vertebrates. In humans, variants in genes encoding subunits of the cargo-adapting IFT-A and IFT-B protein complexes are a common cause of genetic diseases known as ciliopathies. While recent progress has been made in determining the atomic structure of IFT-B, little is known of the structural biology of IFT-A. Here, we combined chemical cross-linking mass spectrometry and cryo-electron tomography with AlphaFold2-based prediction of both protein structures and interaction interfaces to model the overall architecture of the monomeric six-subunit IFT-A complex, as well as its polymeric assembly within cilia. We define monomer-monomer contacts and membrane-associated regions available for association with transported cargo, and we also use this model to provide insights into the pleiotropic nature of human ciliopathy-associated genetic variants in genes encoding IFT-A subunits. Our work demonstrates the power of integration of experimental and computational strategies both for multi-protein structure determination and for understanding the etiology of human genetic disease.