FerriTag is a new genetically-encoded inducible tag for correlative light-electron microscopy

Abstract

A current challenge is to develop tags to precisely visualize proteins in cells by light and electron microscopy. Here, we introduce FerriTag, a genetically-encoded chemically-inducible tag for correlative light-electron microscopy. FerriTag is a fluorescent recombinant electron-dense ferritin particle that can be attached to a protein-of-interest using rapamycin-induced heterodimerization. We demonstrate the utility of FerriTag for correlative light-electron microscopy by labeling proteins associated with various intracellular structures including mitochondria, plasma membrane, and clathrin-coated pits and vesicles. FerriTagging has a good signal-to-noise ratio and a labeling resolution of approximately 10 nm. We demonstrate how FerriTagging allows nanoscale mapping of protein location relative to a subcellular structure, and use it to detail the distribution and conformation of huntingtin-interacting protein 1 related (HIP1R) in and around clathrin-coated pits.

Principles of self-organization and load adaptation by the actin cytoskeleton during clathrin-mediated endocytosis

“…As a result, upon increasing membrane tension, the overall endocytic energy efficiency increased ( Figure 7I). We conclude that sufficient Hip1R coverage around the pit (Clarke and Royle, 2018;Sochacki et al, 2017) allows endocytic actin filaments to orient in such a way that they can encounter more Arp2/3 complexes at the base of the pit to nucleate more actin filaments. This spatial organization allows the actin network to adapt to sustain force production under a range of opposing loads ( Figure 7J).…”

“…Second, the effective anchoring of actin filaments to the surface of the pit depends on the distribution of linker proteins on the pit surface. Since these linker proteins are embedded within the clathrin coat (Clarke and Royle, 2018;Engqvist-Goldstein et al, 2001;Sochacki et al, 2017) , their surface coverage is directly proportional to the coat coverage on the endocytic pit. This observation suggests that one possible function of a large coat is for the actin-linking proteins Hip1, Hip1R and Epsin to cover enough surface area to provide leverage for internalization.…”

“…Filament growth decreases with load according to the Brownian ratchet mechanism (Mogilner and Oster, 1996;Peskin et al, 1993) . Growth of the actin network is coupled to internalization of the endocytic pit by an actin-linking protein (Hip1/Hip1R/Epsin, simplified here as Hip1R), which is embedded in the coated pit and binds to actin filaments (Clarke and Royle, 2018;Engqvist-Goldstein et al, 1999, 2001Sochacki et al, 2017) . Importantly, most of the parameters in this model have been determined with measurements in vitro or in vivo , including the dimensions of the endocytic pit, its resistance to internalization (modeled as a spring, Figure 1D), rates of association and dissociation of different proteins, branching angles, capping rates, filament persistence length, and stall force (Table S3 and Methods).…”

“…Our finding that self-organized endocytic actin networks grow toward the base of the pit prompted us to explore the molecular mechanism by which actin filaments self-organize. Actin dynamics in association with the endocytic machinery can be thought of as a polymerization engine constrained by two spatial boundary conditions --active Arp2/3 complex at the base of the pit (Almeida-Souza et al, 2018;Idrissi et al, 2008;Kaksonen et al, 2003;Mund et al, 2018;Picco et al, 2015 ;Kaplan et al, in preparation) and Hip1R/actin attachments on the curved pit surface (Clarke and Royle, 2018;Engqvist-Goldstein et al, 1999, 2001Sochacki et al, 2017) ( Figure 4A). Given that such spatial boundary conditions confer unique mechanical properties and adaptation to loads under flat geometries in vitro (Bieling et al, 2016) , we aimed to understand how the boundary conditions corresponding to the curved endocytic pit affect endocytic actin organization and internalization.…”

“…Using this framework, we sought to answer the following questions: How do branched actin networks assemble, organize, and produce force around an endocytic pit? How does the spatial segregation of Arp2/3 complex activators (Almeida-Souza et al, 2018;Mund et al, 2018) and actin-binding proteins associated with endocytic coats (Clarke and Royle, 2018;Engqvist-Goldstein et al, 2001;Sochacki et al, 2017) influence this organization? And finally, how do endocytic actin networks adapt to changing loads due to the stochastic environment and changes in membrane tension?…”