Evidence for a primary endocytic vesicle involved in synaptic vesicle biogenesis.

The regulated release of neurotransmitters at synapses is mediated by the fusion of neurotransmitter-filled synaptic vesicles with the plasma membrane. Continuous synaptic activity relies on the constant recycling of synaptic vesicle proteins into newly formed synaptic vesicles. At least two different mechanisms are presumed to mediate synaptic vesicle biogenesis at the synapse as follows: direct retrieval of synaptic vesicle proteins and lipids from the plasma membrane, and indirect passage of synaptic vesicle proteins through an endosomal intermediate. We have identified a vesicle population with the characteristics of a primary endocytic vesicle responsible for the recycling of synaptic vesicle proteins through the indirect pathway. We find that synaptic vesicle proteins colocalize in this vesicle with a variety of proteins known to recycle from the plasma membrane through the endocytic pathway, including three different glucose transporters, GLUT1, GLUT3, and GLUT4, and the transferrin receptor. These vesicles differ from "classical" synaptic vesicles in their size and their generic protein content, indicating that they do not discriminate between synaptic vesicle-specific proteins and other recycling proteins. We propose that these vesicles deliver synaptic vesicle proteins that have escaped internalization by the direct pathway to endosomes, where they are sorted from other recycling proteins and packaged into synaptic vesicles.

Regulation of neuronal function by protein trafficking: a role for the endosomal pathway.

Protein trafficking plays a central role in many aspects of neuronal function, from the release of neurotransmitters by exocytosis and the recycling of synaptic vesicle proteins to the regulation of receptor signalling. Synaptic function can be significantly modified on a short time scale by alterations in the levels of receptors, ion channels and transporters both pre- and postsynaptically. In many cases, these alterations appear to be mediated by acute changes in the rates at which the proteins are endocytosed from and exocytosed to the cell surface from intracellular pools. While our current understanding of the signalling mechanisms and the intracellular pathways responsible for these acute changes is still in its infancy, intriguing details are beginning to emerge from a number of systems.

Involvement of programmed cell death in morphogenesis of the vertebrate inner ear.

An outstanding challenge in developmental biology is to reveal the mechanisms underlying the morphogenesis of complex organs. A striking example is the developing inner ear of the vertebrate, which acquires a precise three-dimensional arrangement of its constituent epithelial cells to form three semicircular canals, a central vestibule and a coiled cochlea (in mammals). In generating a semicircular canal, epithelial cells seem to 'disappear' from the center of each canal. This phenomenon has been variously explained as (i) transdifferentiation of epithelium into mesenchyme, (ii) absorption of cells into the expanding canal or (iii) programmed cell death. In this study, an in situ DNA-end labeling technique (the TUNEL protocol) was used to map regions of cell death during inner ear morphogenesis in the chicken embryo from embryonic days 3.5-10. Regions of cell death previously identified in vertebrate ears have been confirmed, including the ventromedial otic vesicle, the base of the endolymphatic duct and the fusion plates of the semicircular canals. New regions of cell death are also described in and around the sensory organs. Reducing normal death using retrovirus-mediated overexpression of human bcl-2 causes abnormalities in ear morphogenesis: hollowing of the center of each canal is either delayed or fails entirely. These data provide new evidence to explain the role of cell death in morphogenesis of the semicircular canals.