It has been known for decades that clathrin- and dynamin-mediated endocytosis is the major pathway for recycling the components of vesicle membranes after strong stimulation and high rates of exocytosis in secretory cells. This pathway occurs over tens of seconds to minutes after fusion of the secretory vesicle membrane with the plasma membrane. It resembles classical receptor-mediated endocytosis, but it has a trigger that is unique to secretion: the sudden appearance of the secretory vesicle membrane on the surface of the cell. However, the spatial localization, the relationship to individual fusion events, the nature of the cargo, and the timing and nature of nucleation events have been unknown. An elegant study by Mary Bittner, Rachel Aikman, and Ronald Holz has addressed these issues [Reference Bitner, Aikman and Holz1].
Bittner et al. used a combination of total internal reflection fluorescence microscopy of transiently expressed proteins and time-resolved quantitative confocal imaging of endogenous proteins on chromaffin cells from the bovine adrenal medulla. These are the cells that secrete epinephrine (also know as adrenaline) into the bloodstream. They demonstrated that (1) chromaffin vesicle membrane proteins remain as a distinct punctate entity in the plasma membrane for minutes after fusion, (2) clathrin and dynamin begin to accumulate within seconds at sites of fusion, (3) endogenous chromaffin vesicle membrane proteins are gradually removed from individual surface puncta by a “nibbling” mechanism (Figure 1) and appear intracellularly over a time course of minutes and are dependent on dynamin, and (4) stimulated endocytosis adds a new population of internalized vesicles distinct from those reflecting constituative endocytosis.
Figure 1: Model of a nibbling mechanism for clathrin-mediated endocytosis. Upon stimulation, the membrane of fused chromaffin vesicles inserts into the plasma membrane. Clathrin adapters are then rapidly recruited to sites of fusion by phosphatidylinositol-4,5-diphosphare (PIP2) and vesicle membrane proteins (for example, synaptotagmin-1, VMAT2) with adapter binding motifs. Subsequent binding of clathrin permits the formation of clathrin-coated vesicles whose separation requires dynamin GTPase activity. Because the typical clathrin-coated vesicle (90 nm diameter, 0.025 square microns) is too small to internalize the entire patch of chromaffin vesicle membrane (0.282 square microns) as a single unit, the process is repeated (nibbled) and the patch is incrementally internalized.
Dopamine-β-hydroxylase (DBH) is the enzyme that converts dopamine to norepinephrine (which in turn is converted to epinephrine by another enzyme). DBH is both a soluble protein within chromaffin vesicles and bound on the internal surface of the vesicle. After the vesicle membrane fuses with the plasma membrane, the membrane-bound DBH is exposed to the external environment. It was demonstrated with immunocytochemistry that DBH (and other vesicle membrane proteins) remained associated as puncta on the plasma membrane for many minutes after fusion with clathrin and dynamin joining the crowd, often within 20 seconds of fusion. Thus, the vesicle membrane itself is the likely nucleation site for the endocytosis that follows fusion. In a large series of well-controlled experiments, Bittner et al. showed, using quantitative techniques, that the vesicle membrane was internalized bit-by-bit over seconds to minutes, a process they dubbed “nibbling.” The retrieval by nibbling was completely inhibited by the dynamin inhibitor dynogo4a.
A nibbling mechanism is a surprisingly logical explanation for the area mismatch between the clathrin-coated vesicles observed by electron microscopy and the fused vesicle membrane. The studies of Bittner et al. convincingly demonstrated that a nibbling mechanism is the major pathway for retrieval of the fused vesicle membrane from the cell surface after a strong stimulus to secrete.
