Bert Ph. M. Menco

Small-volume extrusion apparatus for preparation of large, unilamellar vesicles

The design and performance of a filter holder which enables convenient preparation of volumes of up to a milliliter of large, unilamellar vesicles formed by extrusion (LUVETs) from multilamellar vesicles (MLVs) are described. The filter holder provides for back-and-forth passage of the sample between two syringes, a design that minimizes filter blockage, eliminates the need to change filters during LUVET preparation and reduces preparation time to a few minutes. Replicas of slam-frozen LUVETs in the electron microscope are unilamellar and reasonably homogeneous with an average diameter close to the pore size of the filters used to extrude them. Extrusion per se does not destabilize the vesicles, which trapped a fluorescent dye only when they were disrupted on freeze-thawing and during the first extrusion when most of the MLVs were apparently converted to LUVETs.

Ultrastructural localization of olfactory transduction components: the G protein subunit Golfα and type III adenylyl cyclase

Electron microscopy and postembedding immunocytochemistry on rapidly frozen, freeze-substituted specimens of rat olfactory epithelia were used to study the subcellular localization of the transduction proteins Golf alpha and type III adenylyl cyclase. Antibody binding sites for both of these proteins occur in the same receptor cell compartments, the distal segments of the olfactory cilia. These segments line the boundary between organism and external environment inside the olfactory part of the nasal cavity. Therefore, they are the receptor cell regions that most likely first encounter odorous compounds. The results presented here provide direct evidence to support the conclusion that the distal segments of the cilia contain the sites of the early events of olfactory transduction.

Developmental expression of G-proteins and adenylyl cyclase in peripheral olfactory systems. Light microscopic and freeze-substitution electron microscopic immunocytochemistry

SummaryLight microscopic immunohistochemistry coupled with freeze-substitution electron microscopic immunocytochemistry was used to localize α-subunits of G-proteins and type III adenylyl cyclase in developing rat olfactory epithelia. Some cilia immunoreacted with antibodies to Gsα and type III adenylyl cyclase as early as prenatal day 15 (E15; El=sperm-positive), but immunolabelling with antibodies to Golfα was not observed until E16. From then on numbers of receptor cells with immunolabelled cilia increased for all three probes. Immunoreactivity for antibodies to the olfactory signal-transduction proteins tended to parallel cilium development, though Golfα lags somewhat behind. Newly formed cilia labelled along their lengths, whereas mature cilia labelled predominantly along their long distal parts. Dendritic knobs and ciliary necklaces showed little or no labelling. While at E22 most multiciliate cells immunolabelled with antibodies to Gsα, Golfα, and type III adenylyl cyclase, not all of these cells labelled with antibodies to olfactory marker protein. Olfactory axons immunoreacted more intensely than epithelial surface structures with antibodies to Gsα at E15; the reverse occurred by about E18. Immunoreactivity with antibodies to a-subunits of the G-proteins Go, Gq/G11, and Gi was also found as early as E15. Antibodies to Goα labelled receptor cell dendritic knobs and cilia during development only. Antibodies to Giα labelled Bowmans glands, whereas those to Gqα/G11αbound to receptor cell cilia and axons (primarily vomeronasal), and supporting cell microvilli. We propose that Gs is the predominant G protein in cilia of immature olfactory receptor cells, while Goif is predominant in cilia of mature cells. Axonal immunoreactivity for some G-protein antibodies suggests G-protein participation in processing of olfactory axon and/or axon terminal-bound signals.

Ultrastructural localization of G-proteins and the channel protein TRP2 to microvilli of rat vomeronasal receptor cells

Microvilli of vomeronasal organ (VNO) sensory epithelium receptor cells project into the VNO lumen. This lumen is continuous with the outside environment. Therefore, the microvilli are believed to be the subcellular sites of VNO receptor cells that interact with incoming VNO‐targeted odors, including pheromones. Candidate molecules, which are implicated in VNO signaling cascades, are shown to be present in VNO receptor cells. However, ultrastructural evidence that such molecules are localized within the microvilli is sparse. The present study provides firm evidence that immunoreactivity for several candidate VNO signaling molecules, notably the G‐protein subunits Giα2 and Goα, and the transient receptor potential channel 2 (TRP2), is localized prominently and selectively in VNO receptor cell microvilli. Although Giα2 and Goα are localized separately in the microvilli of two cell types that are otherwise indistinguishable in their apical and microvillar morphology, the microvilli of both cell types are TRP2(+). VNO topographical distinctions were also apparent. Centrally within the VNO sensory epithelium, the numbers of receptor cells with Giα2(+) and Goα(+) microvilli were equal. However, near the sensory/non‐sensory border, cells with Giα2(+) microvilli predominated. Scattered ciliated cells in this transition zone resembled neither VNO nor main olfactory organ (MO) receptor cells and may represent the same ciliated cells as those found in the non‐sensory part of the VNO. Thus, this study shows that, analogous to the cilia of MO receptor cells, microvilli of VNO receptor cells are enriched selectively in proteins involved putatively in signal transduction. This provides important support for the role of these molecules in VNO signaling. J. Comp. Neurol. 438:468–489, 2001.

Putative odour receptors localize in cilia of olfactory receptor cells in rat and mouse: a freeze-substitution ultrastructural study

Two different polyclonal antibodies were raised to synthetic peptides corresponding to distinct putative odour receptors of rat and mouse. Both antibodies selectively labelled olfactory cilia as seen with cryofixation and immunogold ultrastructural procedures. Regions of the olfactory organ where label was detected were consistent with those found at LM levels. Immunopositive cells were rare; only up to about 0.4% of these receptor cells were labelled. Despite chemical, species, and topographic differences both antibodies behaved identically in their ultrastructural labelling patterns. For both antibodies, labelling was very specific for olfactory cilia; both bound amply to the thick proximal and the thinner and long distal parts of the cilia. Dendritic knobs showed little labelling if any. Dendritic receptor cell structures below the knobs, supporting cell structures, and respiratory cilia did not immunolabel. There were no obvious differences in morphology between labelled and unlabelled receptor cells and their cilia. Labelling could be followed up to a distance of about 15 μm from the knobs along the distal parts of the cilia. When labelled cells were observed, this signal was detectable in two, sometimes three, sections taken through these cells while being consistently absent in neighbouring cells. This pattern argues strongly for the specificity of the labelling. In conclusion, very few receptor cells labelled with the antibodies to putative odour receptors. Additionally the olfactory cilia, the cellular regions that first encounter odour molecules and that are thought to transduce the odorous signal, displayed the most intense labelling with both antibodies. Consequently, the results showed these cilia as having many copies of the putative receptors. Finally, similar patterns of subcellular labelling were displayed in two different species, despite the use of different antibodies. Thus, this study provides compelling evidence that the heptahelical putative odour receptors localize in the olfactory cilia.

Ciliated and microvillous structures of rat olfactory and nasal respiratory epithelia

SummaryThe olfactory epithelium of the Sprague-Dawley rat showed structures which indicate that freeze-substitution after ultra-rapid cryo-fixation is a better method for its preservation than conventional fixation techniques. A new feature is that matrices of the distal parts of olfactory cilia range in their staining intensity from very dense to electron-lucent. Outlines of structures are smooth and membrane features can be clearly seen.The textures of mucus from olfactory and respiratory epithelia are distinctly different after freeze-fracturing and deep-etching following cryo-fixation. Olfactory cilia show no microtubule-attached axonemal structures. Cross-sectional diameters are smaller after freeze-substitution than after freeze-fracturing.Intramembranous particle densities are lower in nine regions of three cell types in cryo-fixed olfactory and respiratory epithelia than in those chemically fixed and cryoprotected. The fracture faces of membranes from etched, cryo-fixed cells have holes, a result which probably accounts for differences in particle density between cryo-fixed and chemically-fixed, cryo-protected cells. Particle diameters are usually the same using both methods. Densities of intramembranous particles and particles plus holes are highest in supporting cell processes, followed by endings and cilia of olfactory receptor cells, and are lowest in respiratory cilia. Particle densities at outer and inner surfaces are higher than those in either fracture face. Outer surfaces show a good correlation from region to region with densities summated over both fracture faces.

Observations on axonemes and membranes of olfactory and respiratory cilia in frogs and rats using tannic acid-supplemented fixation and photographic rotation

With tannic acid-supplemented fixation and a photographic rotation technique, ultrastructural features of axonemes of frog olfactory cilia resemble those of respiratory cilia in virtually all respects. Different types of ciliary axonemes corresponding to motile and immotile olfactory cilia are not discernible. In rats, however, axonemes of olfactory cilia are quite different from those of respiratory cilia and always lack microtubule-attached structures in proximal parts. Their distal parts usually have only two microtubules which terminate in a cap-like structure. In either species, tannic acid-supplemented fixation reveals that outer leaflets of membranes of olfactory cilia are thicker than inner leaflets. This is not the case for the respiratory cilia, and the overall thickness of ciliary membranes is smaller in respiratory cilia. From our observations and literature data on vertebrates and invertebrates, it is inferred that the ultrastructure of axonemes of olfactory cilia is not evolutionarily stable. This implies that this structure does not play any specific role in the olfactory transduction process. However, the motility associated with microtubule-attached arms of those cilia which have complete axonemes may be involved in the efficacy of the olfactory process. The consistent differences between membranes of olfactory and respiratory cilia suggest that membranes of olfactory cilia may have specific properties important to the initial events of the olfactory transduction process.

Ultrastructural evidence for multiple mucous domains in frog olfactory epithelium.

SummaryThis study showed that the olfactory mucus is a highly structured extracellular matrix. Several olfactory epithelial glycoconjugates in the frog Rana pipiens were localized ultrastructurally using rapid-freeze, freeze-substitution and post-embedding (Lowicryl K11M) immunocytochemistry. Two of these conjugates were obtained from membrane preparations of olfactory cilia, the glycoproteins gp95 and olfactomedin. The other conjugates have a carbohydrate group which in the olfactory bulb appears to be mostly on neural cell-adhesion molecules (N-CAMs); in the olfactory epithelium this carbohydrate is present on more molecules. Localization of the latter conjugates was determined with monoclonal antibodies 9-OE and 5-OE. Ultrastructurally all antigens localized in secretory granules of apical regions of frog olfactory supporting cells and in the mucus overlying the epithelial surface, where they all had different, but partly overlapping, distributions. Monoclonal antibody 18.1, to gp95, labeled the mucus throughout, whereas poly- and monoclonal anti-olfactomedin labeled a deep mucous layer surrounding dendritic endings, proximal parts of cilia, and supporting cell microvilli. Labeling was absent in the superficial mucous layer, which contained the distal parts of the olfactory cilia. Monoclonal antibody 9-OE labeled rather distinct areas of mucus. These areas sometimes surrounded dendritic endings and olfactory cilia. Monoclonal antibody 5-OE labeled membranes of dendritic endings and cilia, and their glycocalyces, and also dendritic membranes.

Electron-microscopic demonstration of olfactory-marker protein with protein G-gold in freeze-substituted, Lowicryl K11M-embedded rat olfactory-receptor cells

SummaryIn this study electron-microscopic immunocytochemistry was used to localize olfactory marker protein in olfactory epithelia. Rat olfactory-epithelial samples were rapidly frozen, freeze-substituted with acetone, embedded at low temperatures with Lowicryl K11M and labelled on the sections with polyclonal antibodies raised against olfactory marker protein and with protein G conjugated to colloidal gold. Apart from the aforementioned use of acetone, substitution was carried out in the complete absence of chemical fixation, i.e., neither aldehydes nor OsO4 were used. This procedure resulted in localization concurrent with a good ultrastructural preservation. Olfactory-marker protein was present throughout the cytoplasmic compartments of dendrites and dendritic endings of olfactory-receptor cells, but it was not found in organelles such as mitochondria. Olfactory-marker protein was found only in dendriticendings of olfactory-receptor cells mature enough to have given rise to cilia, but these cilia displayed less labelling than dendrites and dendritic endings. Olfactory-marker protein was not found in apices and microvilli of neighboring olfactory-supporting cells.

The interaction of Bex and OMP reveals a dimer of OMP with a short half‐life

Olfactory marker protein (OMP) participates in the olfactory signal transduction pathway. This is evident from the behavioral and electrophysiological deficits of OMP‐null mice, which can be reversed by intranasal infection of olfactory sensory neurons with an OMP‐expressing adenovirus. Bex, brain expressed X‐linked protein, has been identified as a protein that interacts with OMP. We have now further characterized the interaction of OMP and Bex1/2 by in vitro binding assays and by immuno‐coprecipitation experiments. OMP is a 19 kDa protein but these immunoprecipitation studies have revealed the unexpected presence of a 38 kDa band in addition to the expected 19 kDa band. Furthermore, the 38 kDa form was preferentially co‐immunoprecipitated with Bex from cell extracts. In‐gel tryptic digestion, mass spectrometry, and two‐dimensional gel electrophoresis indicate that the 38 kDa protein behaves as a covalently cross‐linked OMP‐homodimer. The 38 kDa band was also identified in western blots of olfactory epithelium demonstrating its presence in vivo. The stabilities and subcellular localizations of the OMP‐monomer and ‐dimer were studied in transfected cells. These results demonstrated that the OMP‐dimer is much less stable than the monomer, and that while the monomer is present both in the nuclear and cytosolic compartments, the dimer is preferentially located in a Triton X‐100 insoluble cytoskeletal fraction. These novel observations led us to hypothesize that regulation of the level of the rapidly turning‐over OMP‐dimer and its interaction with Bex1/2 is critical for OMP function in sensory transduction.