By Sandy Schmid
Living cells face a dilemma; in order to prevent the unregulated influx and efflux of molecules they need a plasma membrane that is literally water-tight, but they also need to be able to take up specific molecules such as proteins from their environment. One of the ways in which they solve this problem is by the use of clathrin-mediated endocytosis.
This complex and highly regulated process involves a tightly orchestrated sequence of steps that entails the formation of pits in the plasma membrane, coated with a basket-like array of clathrin (known as clathrin-coated pits, or CCPs), followed by invagination, constriction, and pinching-off to form clathrin-coated vesicles (CCVs).
This process requires not only the major coat proteins, clathrin and adaptor protein complexes (AP2) and the GTPase dynamin, but a myriad of endocytic accessory proteins (EAPs), whose exact functions are still not clearly defined. Although studied for almost 50 years, the true complexity of clathrin-mediated endocytosis was only recently revealed through the advent of live cell microscopy to image the dynamics of CCPs.
For the PLOS Biology 15-Year Anniversary I’ve chosen to highlight this paper by Christien Merrifield and co-workers (Taylor et al., 2011) as it described a sophisticated and highly precise microscopy-based method to detect the scission event that leads to CCV formation and maps the temporal hierarchy of EAP recruitment to CCPs.
The method involved the use of transferrin receptors externally tagged with a pH-sensitive GFP (TfR-phl); these were imaged by total internal reflection fluorescence microscopy in a perfusion chamber in which the media is periodically cycled between pH 5 and pH 7. Imaging was coordinated at 2-second intervals with each pH change, so that the appearance of pH 5-insensitive TfR-phl precisely marked the point of scission.
This elegant approach was used to comprehensively analyze the temporal hierarchy of recruitment of 34 EAPs relative to the scission event, which allowed their classification into functional modules temporally linked to CCP initiation and maturation, actin dynamics, scission, uncoating, and post-scission vesicle motility.
The paper was and remains the highest resolution temporal map of the molecular events governing the clathrin-mediated endocytosis process to date. In addition to the hierarchical classification of EAPs, the paper also revealed other key principles of CCP dynamics.
- First, the kinetics, extent and frequency of EAP recruitment to CCPs, as well as the dynamics of CCPs themselves were highly heterogenous. Merrifield reported that short-lived CCPs often failed to sequester TfR-phl and thus unambiguously established that short-lived CCPs were abortive events.
- Second, in several cases Merrifield detected multiple, sequential fission events associated with continuously detected clathrin structures. These were classified as ‘non-terminal events’ that likely reflect pinching-off of CCVs from the periphery of larger clathrin-coated structures. Importantly, the recruitment signatures of EAPs to terminal vs non-terminal events were indistinguishable, suggesting a common mechanism governing CCP maturation and scission.
- Third, the work established a remarkable coordination between scission and uncoating of the released vesicle.
This seminal paper represents a significant leap in our understanding of vesicle trafficking and questions raised by the study continue to be addressed by researchers today. What is the significance and molecular basis for the dynamic and compositional heterogeneity of CCPs? How is uncoating so tightly coupled with membrane scission, what prevents the uncoating of deeply invaginated CCPs? What determines the productive maturation vs. early abortion of CCPs?
Taylor MJ, Perrais D, Merrifield CJ (2011) A High Precision Survey of the Molecular Dynamics of Mammalian Clathrin-Mediated Endocytosis. PLoS Biol 9(3): e1000604. https://doi.org/10.1371/journal.pbio.1000604
Sandy Schmid works at UT Southwestern and is a member of the PLOS Biology Editorial Board and Advisory Board. Find out how she’s leading the movement away from impact factors and towards better assessment of scientists.
Featured image credit: John Heuser, via pbio.1001037