Ultrastructural localization of calcium homeostasis modulator 1 in mouse taste buds.

Taste receptor cells are morphologically classified as types II and III. Type II cells form a unique type of synapses referred to as channel synapses where calcium homeostasis modulator 1 (CALHM1) together with CALHM3 forms voltage-gated channels that release the neurotransmitter, adenosine triphosphate (ATP). To validate the proposed structural model of channel synapses, the ultrastructural localization of CALHM1 in type II cells of both fungiform and circumvallate taste buds was examined. A monoclonal antibody against CALHM1 was developed and its localization was evaluated via immunofluorescence and immunoelectron microscopy using the immunogold-silver labeling technique. CALHM1 was detected as puncta using immunofluorescence and along the presynaptic membrane of channel synapses facing atypical mitochondria, which provide ATP, by immunoelectron microscopy. In addition, it was detected along the plasma membrane lined by subsurface cisternae at sites apposed to afferent nerve fibers. Our results support the validity of a previously proposed structural model for channel synapses and provide insights into the function of subsurface cisternae whose function in taste receptor cells is unknown. We also examined the localization of CALHM1 in hybrid synapses of type III cells, which are conventional chemical synapses accompanied by mitochondria similar to atypical mitochondria of channel synapses. CALHM1 was not detected in the six hybrid synapses examined using immunoelectron microscopy. We further performed double immunolabeling for CALHM1 and Bassoon, which is detected as puncta corresponding to conventional vesicular synapses in type III cells. Our observations suggest that at least some, and probably most, hybrid synapses are not accompanied by CALHM1.

The presynaptic active zone protein Bassoon as a marker for synapses between Type III cells and afferent nerve fibers in taste buds.

Taste buds are receptor organs for gustation. Two types of taste receptor cells have been identified in taste buds: Type II and Type III cells. Type III cells connect with afferent fibers through conventional chemical synapses. In the present study, we used immunocytochemistry to examine the distribution pattern of Bassoon, a scaffolding protein of the cytomatrix at the active zones of conventional synapses in mouse taste buds. Bassoon was predominantly detected as small puncta in Type III cells. Bassoon-immunoreactive puncta were observed in proximity to or partially overlapping with intragemmal nerve fibers. The distribution pattern of Bassoon in taste buds was similar among circumvallate, fungiform, and foliate taste buds. Immunoelectron microscopy showed Bassoon at the active zones of the conventional synapses of Type III cells in circumvallate taste buds. The present results demonstrate that Bassoon is a marker for synapses between Type III cells and afferent fibers in mouse taste buds.

ZSWIM8 is a myogenic protein that partly prevents C2C12 differentiation.

Cell adhesion molecule-related/downregulated by oncogenes (Cdon) is a cell-surface receptor that mediates cell-cell interactions and positively regulates myogenesis. The cytoplasmic region of Cdon interacts with other proteins to form a Cdon/JLP/Bnip-2/CDC42 complex that activates p38 mitogen-activated protein kinase (MAPK) and induces myogenesis. However, Cdon complex may include other proteins during myogenesis. In this study, we found that Cullin 2-interacting protein zinc finger SWIM type containing 8 (ZSWIM8) ubiquitin ligase is induced during C2C12 differentiation and is included in the Cdon complex. We knocked-down Zswim8 in C2C12 cells to determine the effect of ZSWIM8 on differentiation. However, we detected neither ZSWIM8-dependent ubiquitination nor the degradation of Bnip2, Cdon, or JLP. In contrast, ZSWIM8 knockdown accelerated C2C12 differentiation. These results suggest that ZSWIM8 is a Cdon complex-included myogenic protein that prevents C2C12 differentiation without affecting the stability of Bnip2, Cdon, and JLP.

N-cadherin localization in taste buds of mouse circumvallate papillae.

Taste buds, the receptor organs for taste, contain 50-100 taste bud cells. Although these cells undergo continuous turnover, the structural and functional integrity of taste buds is maintained. The molecular mechanisms by which synaptic connectivity between taste buds and afferent fibers is formed and maintained remain ambiguous. In the present study, we examined the localization of N-cadherin in the taste buds of the mouse circumvallate papillae because N-cadherin, one of the classical cadherins, is important for the formation and maintenance of synapses. At the light microscopic level, N-cadherin was predominantly detected in type II cells and nerve fibers in the connective tissues in and around the vallate papillae. At the ultrastructural level, N-cadherin immunoreactivity appears along the cell membrane and in the intracellular vesicles of type II cells. N-cadherin immunoreactivity also is evident in the membranes of afferent terminals at the contact sites to N-cadherin-positive type II cells. At channel type synapses between type II cells and nerve fibers, N-cadherin is present surrounding, but not within, the presumed neurotransmitter release zone, identified by large mitochondria apposed to the taste cells. The present results suggest that N-cadherin is important for the formation or maintenance of type II cell afferent synapses in taste buds.