Biomedical subjects
Publications and source records attributed to David Zenisek.
In this issue of Neuron, Voglmaier et al. provide new evidence that the retrieval of synaptic vesicle transporters after exocytosis proceeds along at least two different endocytic pathways. This work provides new insight into the mechanisms of sorting synaptic vesicle cargo at the cell surface.
During clathrin-mediated endocytosis, membrane scission marks the isolation of a cargo-laden clathrin-coated pit (CCP) from the cell exterior. Here we used live-cell imaging of a pH-sensitive cargo to visualize the formation of clathrin-coated vesicles (CCVs) at single CCPs with a time resolution of seconds. We show that CCPs are highly dynamic and can produce multiple vesicles in succession. Using alternating evanescent field and epifluorescence illumination, we show that CCP invagination and scission are tightly coupled, with scission coinciding with maximal displacement of CCPs from the plasma membrane and with peak recruitment of cortactin-DsRed, a dynamin and F-actin binding protein. Finally, perturbing actin polymerization with latrunculin-B drastically reduces the efficiency of membrane scission and affects many aspects of CCP dynamics. We propose that CCP invagination, actin polymerization, and CCV formation are highly coordinated for efficient endocytosis.
Neurons of the visual, auditory and vestibular systems that signal through graded changes in membrane potential rely upon synaptic ribbons for the exquisite control of neurotransmitter release. Although clearly important for tonic neurotransmission, the precise role of synaptic ribbons remains elusive. In recent years, several genetic, biochemical, electrophysiological and optical approaches have begun to shed light on the functions of these enigmatic organelles.
Visual and auditory information is encoded by sensory neurons that tonically release neurotransmitter at high rates. The synaptic ribbon is an essential organelle in nerve terminals of these neurons. Its precise function is unknown, but if the ribbon could be visualized in a living terminal, both its own dynamics and its relation to calcium and vesicle dynamics could be studied. We designed a short fluorescent peptide with affinity for a known binding domain of RIBEYE, a protein unique to the ribbon. When introduced via a whole-cell patch pipette, the peptide labeled structures at the presynaptic plasma membrane of ribbon-type terminals. The fluorescent spots match in size, location, number, and distribution the known features of synaptic ribbons. Furthermore, fluorescent spots mapped by confocal microscopy directly match the ribbons identified by electron microscopy in the same cell. Clearly the peptide binds to the synaptic ribbon, but even at saturating concentrations it affects neither the morphology of the ribbon nor its tethering of synaptic vesicles. It also does not inhibit exocytosis. Using the peptide label, we observed that the ribbon is immobile over minutes and that calcium influx is concentrated at the ribbon. Finally, we find that each ribbon in a retinal bipolar cell contains approximately 4000 molecules of RIBEYE, indicating that it is the major component of the synaptic ribbon.
Neurons communicate with one another through the release of molecules from synaptic vesicles and large dense core granules through the process of exocytosis. During exocytosis, molecules are released to the extracellular space through a fusion pore, which can either dilate, resulting in full fusion, or close, resulting in incomplete exocytosis, often referred to as 'kiss and run' exocytosis. Recently, there has been much interest in the regulation of this process in both neurons and neuroendocrine cells. There has been much recent work that addresses the existence of incomplete exocytosis in neurons and neuroendocrine cells, as well as recent work probing the molecular components and modulation of the fusion pore.
We investigated the location of calcium entry sites and synaptic ribbons in the type-Mb goldfish bipolar neuron and the bullfrog saccular hair cell. Cells were loaded with a fast calcium indicator (Fluo-3 or Fluo-5F) and an excess of a high-affinity but slow Ca buffer (EGTA). The cell surface was imaged by evanescent field microscopy. Small fluorescent "hot spots" representing calcium entry sites appeared abruptly when a voltage step opened Ca channels and disappeared or dimmed abruptly when Ca channels closed. In bipolar cells, the fluorescence of hot spots tracked the calcium influx. Hair cells showed similar Ca hot spots. Synaptic ribbons or dense bodies were labeled by immunofluorescence with an antibody that recognizes the ribbon protein ribeye. The antibody labeled punctate structures beneath the plasma membrane. In both bipolar neurons and hair cells, the number of Ca entry sites was similar or identical to that of ribbons or dense bodies, consistent with the idea that calcium-channel clusters reside near ribbons, and that both mark active zones. In bipolar cells, the number of Ca entry sites and ribeye-positive fluorescent spots is also strikingly similar to that of exocytic active zones but significantly less than the number of total exocytic sites including solitary fusion events outside active zones. We suggest that in bipolar terminals, active zones, Ca entry sites, and synaptic ribbons all colocalize, but also that a significant number of vesicles can fuse outside active zones and, hence, independently of synaptic ribbons.
Perhaps synaptic vesicles can recycle so rapidly because they avoid complete exocytosis, and release transmitter through a fusion pore that opens transiently. This view emerges from imaging whole terminals where the fluorescent lipid FM1-43 seems unable to leave vesicles during transmitter release. Here we imaged single, FM1-43-stained synaptic vesicles by evanescent field fluorescence microscopy, and tracked the escape of dye from single vesicles by watching the increase in fluorescence after exocytosis. Dye left rapidly and completely during most or all exocytic events. We conclude that vesicles at this terminal allow lipid exchange soon after exocytosis, and lose their dye even if they connected with the plasma membrane only briefly. At the level of single vesicles, therefore, observations with FM1-43 provide no evidence that exocytosis of synaptic vesicles is incomplete.