Recruitment of clathrin to intracellular membranes is sufficient for vesicle formation

Abstract

The formation of a clathrin-coated vesicle is a major membrane remodeling process that is crucial for membrane traffic in cells. Besides clathrin, these vesicles contain at least 100 different proteins although it is unclear how many are essential for the formation of the vesicle. Here, we show that intracellular clathrin-coated formation can be induced in living cells using minimal machinery and that it can be achieved on various membranes, including the mitochondrial outer membrane. Chemical heterodimerization was used to inducibly attach a clathrin-binding fragment "hook" to an "anchor" protein targeted to a specific membrane. Endogenous clathrin assembled to form coated pits on the mitochondria, termed MitoPits, within seconds of induction. MitoPits are double-membraned invaginations that form preferentially on high curvature regions of the mitochondrion. Upon induction, all stages of CCV formation - initiation, invagination, and even fission - were faithfully reconstituted. We found no evidence for the functional involvement of accessory proteins in this process. In addition, fission of MitoPit-derived vesicles was independent of known scission factors including dynamins and dynamin-related protein 1 (Drp1), suggesting that the clathrin cage generates sufficient force to bud intracellular vesicles. Our results suggest that, following its recruitment, clathrin alone is sufficient for intracellular clathrin-coated vesicle formation.

Excitation Spectral Microscopy for Highly Multiplexed Intracellular Imaging via Quasi-Blind Unmixing

“…Intriguingly, some large and bright clathrin structures colocalized with mitochondria (yellow arrowheads in Figure 6d), suggesting a newly discovered role of mitochondria in forming clathrin-coated vesicles. 29 Together, Re-MMI unraveled complex spatial organizations and potential interactions between intracellular structures in fixed cells, which are increasingly recognized as pivotal for diverse cellular functions. Experimental Validation of Re-MMI in Live-Cell Imaging.…”

Mitochondrial signalling and homeostasis: from cell biology to neurological disease

“…Recent proteomic analyses have provided the first detailed mechanistic insights into machinery required for generation of TOM20 + MDVs, revealing that tubulation of mitochondrial membranes during MDV biogenesis is driven by the mitochondrial Rho GTPases MIRO1 and MIRO2 along microtubules prior to DRP1 recruitment and scission [41]. Reconstituting clathrin delivery to mitochondria also promotes mitochondrial protrusions (which the authors termed MitoPits), suggesting an alternative mechanism of MDV production [42]. Early studies revealed that MDVs Glossary AAA proteases: ubiquitous family of ATP-dependent proteases embedded in the IMM, characterised by the structural conservation of a central ATPase domain called the AAA module.…”

Clathrin: Bender or bystander?

“…Interestingly, a second study, which has recently appeared on bioRxiv ( 9 ), takes an entirely different experimental approach to produce data that ultimately supports a similar set of conclusions. The investigative team led by Küey and Royle recruited clathrin artificially to the surfaces of mitochondria, which have high curvatures, similar to the ridges used in the study by Cail et al ( 8 ).…”

“…Collectively, the studies by Cail et al ( 8 ) and Küey et al ( 9 ) suggest that both clathrin and its adaptor network play important roles in generating membrane curvature. When clathrin-coated vesicles bud from the relatively flat plasma membrane, both the adaptors and clathrin are required to complete the process.…”

“…When clathrin-coated vesicles bud from the relatively flat plasma membrane, both the adaptors and clathrin are required to complete the process. In contrast, when vesicles are derived from a curved substrate, either the adaptor network, in the case of Cail et al ( 8 ), or the clathrin lattice, in the case of Küey et al ( 9 ), will suffice. The unified picture that emerges from these disparate studies shows clearly that clathrin in an important driver of membrane bending, bringing us closer to resolving a controversy that has existed since the early days of the field.…”