Uncovering new roles of actin cytoskeleton using correlative light and electron microscopy

Abstract text (incl. figure legends and references)

Sterols are key lipids for biological membranes, thereby their abundance and distribution must be tightly regulated. At subcellular level, organelles such as endosomes are central hubs for sterol distribution, mediating sterol transfer between the diverse subcellular destinations. Sterols reach and leave endosomes via small vesicular carriers or via sterol transfer proteins at short-range membrane contact sites. How endosomal sterol transfer is orchestrated is poorly understood. Using a genetically encoded sterol biosensor (D4H) in the fission yeast Schizosaccharomyces pombe, we have found that sterol steady-state distribution can be re-routed from the plasma membrane to endosomal structures upon pharmacological inhibition of branched actin assembly. In yeast, branched actin plays major roles in plasma membrane reshaping, whereas in complex eukaryotes branched actin also facilitates membrane trafficking in endosomes, however the role of actin at endosomes remains unclear. We show that upon inhibition of Arp2/3 complex, essential for actin branching, endosomes undergo ultrastructural remodeling from elongated to round organelles and become sterol rich. Upon reactivation of Arp2/3 complex, sterols rapidly redistribute to the plasma membrane and endosomes regain their elongated ultrastructure. Using correlative light and electron tomography (CLEM) we have found that under Arp2/3 inhibition, endosomes are smooth ellipsoidal organelles with no evidence of active membrane remodeling. Processing cells for CLEM immediately after Arp2/3 complex re-activation revealed that endosomes undergo intense ultrastructural remodeling into morphologically complex organelles with tubulations, fenestrations and budding events. Altogether our findings suggest a novel role for branched actin facilitating membrane remodeling at early endosomes. We investigate the mode of activation of Arp2/3 at endosomes and the role of endosomal ultrastructure on sterol trafficking. We propose that branched actin is necessary for endosome tubulation, facilitating membrane remodeling by trafficking complexes and thus enabling sterol trafficking through the endosomal system.