Abstract

Understanding the organizing principles driving the assembly of cellular organelles and of protein machines is a fundamental pursuit in biology. The centriole is a microtubule-based organelle that is essential for the formation of flagella, cilia and centrosomes. The centriole has a signature nine-fold symmetrical arrangement of microtubules organized around a likewise symmetrical cartwheel. The cartwheel comprises a stack of ring-containing elements that each accommodates 9 homodimers of SAS -6 proteins. Although important advances have been made in recent years, understanding of centriole assembly remains incomplete, partly due to the lack of cell-free assays to probe the dynamics of the process at nanometric scale. To fill this gap, we have developed a high-speed atomic force microscopy (AFM) to reconstitute and monitor the assembly dynamics of SAS -6 proteins. Moreover, to develop tools to dissect and modulate centriole assembly, we have identified monobodies that recognize SAS -6 proteins with high affinity. Our analysis established that some of these monobodies prevent the assembly of ring-containing elements in vitro and of the centriole organelle in cellulo. Together, these approaches provide novel insights into the mechanisms governing the assembly of the fundamental centriole organelle.