Surface charges of the membrane and cell adhesion substances determine the structural integrity of hair bundles from the inner ear of fish

Summary The hairs (stereocilia = stereovilli) of sensory cells from the inner ear of vertebrates are interconnected by several types of connectors, whose role is unknown. They appear to stabilize the hair bundle mechanically, and may be directly involved in mechano-electric transduction. Our transmission electron-microscopical investigation of sensory epithelia from two species of fish (Rutilus rutilus, Scardinius erythrophthalmus, both Leuciscidae) has shown that not only the connectors but also the surface charges of the membrane are important factors for determining the shape of the hair bundle and the spatial interrelation of the stereovilli. A reduction of the ionic strength in the medium leads to an increase in distance between the stereovilli. This may be the result of an extension of the spread of the surface potential of the membrane at low ionic strength. The connectors are not broken by the increase in distance between the stereovilli. They are EDTA (ethylene-diamine-tetra-acetic-acid) resistant as are some cell adhesion molecules such as N-CAM (nerve-cell adhesion molecule) and protein A from Dictyostelium discoideum. The connectors do not prevent polycation-induced fusion of adjacent stereovillar membranes.     

N-glycans and glycosylphosphatidylinositol-anchor act on polarized sorting of mouse PrP(C) in Madin-Darby canine kidney cells

The cellular prion protein (PrP(C)) plays a fundamental role in prion disease. PrP(C) is a glycosylphosphatidylinositol (GPI)-anchored protein with two variably occupied N-glycosylation sites. In general, GPI-anchor and N-glycosylation direct proteins to apical membranes in polarized cells whereas the majority of mouse PrP(C) is found in basolateral membranes in polarized Madin-Darby canine kidney (MDCK) cells. In this study we have mutated the first, the second, and both N-glycosylation sites of PrP(C) and also replaced the GPI-anchor of PrP(C) by the Thy-1 GPI-anchor in order to investigate the role of these signals in sorting of PrP(C) in MDCK cells. Cell surface biotinylation experiments and confocal microscopy showed that lack of one N-linked oligosaccharide leads to loss of polarized sorting of PrP(C). Exchange of the PrP(C) GPI-anchor for the one of Thy-1 redirects PrP(C) to the apical membrane. In conclusion, both N-glycosylation and GPI-anchor act on polarized sorting of PrP(C), with the GPI-anchor being dominant over N-glycans.     

Mechanoelectric transduction in nematocytes of a hydropolyp (Corynidae)

In sensitivity and ultrastructure of their cnidocil apparatus (CA), the nematocytes (stinging cells) of hydrozoans are analogous to hair cells of vertebrates and epidermal mechanoreceptors of insects. Intracellular recordings using current and voltage clamp in the capitate tentacles of the marine hydropolyp Stauridiosarsia producta (Corynidae) now revealed that depolarizing receptor potentials and receptor currents are generated in nematocytes (stenotele type) in response to mechanical stimulation of the CA. The responsive cells were identified by injection of Lucifer Yellow. For recording, the tentacles were isolated from the polyp and held by a suction capillary. Stimuli were applied by a glass probe moved electromagnetically or piezoelectrically.The mechanosensitivity of the nematocytes was found to be strictly limited to the CA. The characteristics of the mechanoelectric transduction were those typical of mechanoreceptor cells: phasic-tonic time course of an increase in membrane conductance; latency between stimulus and receptor response < 50 s; sigmoid relationship between receptor-response amplitude and stimulus amplitude; maximal increase in conductance of 15 nS; reversal potential between + 35 mV and — 10 mV; unspecific cation dependence and reversible blocking by streptomycin. The results suggest a direct mechanical control of unspecific cation channels such as has been found for mechanoreceptor cells.Suprathreshold receptor potentials elicit two forms of regenerative depolarization: non-inactivating, steplike potentials and action potentials. The latter can trigger discharge of the nematocyst.The discharge of nematocysts in the intact animal (without recording) in response to adequate stimuli was blocked by streptomycin and Na⁺ depletion in the same way as the receptor potential.Mechanoreceptor potentials are thus the beginning of a stimulus-induced electrical reaction cascade that ends in nematocyst discharge.     

Cytoskeletal modifications of the sensorimotor interneurons ofHydra vulgaris (Cnidaria,Hydrozoa), indicating a sensory function similar to chordotonal receptors of insects

Summary The battery mother cell complexes in the tentacles ofHydra vulgaris contain a neuronal cell known as sensorimotor interneuron that is characterized by a modified cilium lying parallel to the mesoglea. The cilium is surrounded by up to three rings of microvilli. Microvilli and cilium arise in an unusual antiparallel orientation from the opposite poles of a central cellular cavity. The lumen of this cavity communicates with the extracellular environment by way of a straight channel-like opening that is encircled by the microvillar rings. The modified cilium extends into the channel and terminates outside in the intercellular space. The wall of the cavity and the channel are stabilized by bundles of microtubules. A prominent glycocalyx interconnects all microvilli and links the innermost microvillar ring to the cilium. Within this contact region approximately 0.7 m in length the ciliary axoneme is specifically modified: all nine microtubule doublets and up to six additional microtubules are embedded in electron-dense material. The microtubule doublets are connected to the ciliary membrane by ledges of Y-shaped cross-bridging elements. These axonemal modifications resemble those known from the hydrozoan cnidocil complex or from the outer segments of insect mechanoreceptor cells. Distribution and orientation of the sensorimotor interneuron within the tentacles indicate a mechanosensory function of the cell similar to that of chordotonal receptors of insects.     

Structures transmitting stimulatory force to the sensory hairs of vestibular ampullae of fishes and frog

Summary Serial sections of the vestibular ampullae of two species of fish and one species of frog were investigated by electron microscopy. The kinocilium is the only connection between the sensory cells and the auxiliary structure (cupula). The cupula possesses canals that traverse its entire height. Each canal contains a single kinocilium in its proximal part; distally, it is filled with material that stains with colloidal silver. The matrix of the cupula consists of filaments running perpendicular to the canals. These filaments do not stain with colloidal silver. The kinocilium is connected to the wall of the canal via structures that differ in the studied species of fish and frog. The filamentous links between the kinocilium and the longest stereovilli of the sensory hair bundle are similar in all the investigated species. The stereovilli are interconnected by basal and shaft links, and by horizontal and oblique tip connectors, similar to those described by other authors for macula organs and the organ of Corti, although differences in structural details, especially of the horizontal tip and the shaft connectors, are present. Some of these are species specific and some are related to the position of the sensory cell in the epithelium and/or specific to the organ (ampulla or macula organ). Some attachment sites of the links are associated with osmiophilic submembranous material. These differences in the structure, distribution and attachment sites of the links are possibly of functional importance.     

Protein kinase CK2 is coassembled with small conductance Ca(2+)-activated K+ channels and regulates channel gating

Small conductance Ca(2+)-activated K+ channels (SK channels) couple the membrane potential to fluctuations in intracellular Ca2+ concentration in many types of cells. SK channels are gated by Ca2+ ions via calmodulin that is constitutively bound to the intracellular C terminus of the channels and serves as the Ca2+ sensor. Here we show that, in addition, the cytoplasmic N and C termini of the channel protein form a polyprotein complex with the catalytic and regulatory subunits of protein kinase CK2 and protein phosphatase 2A. Within this complex, CK2 phosphorylates calmodulin at threonine 80, reducing by 5-fold the apparent Ca2+ sensitivity and accelerating channel deactivation. The results show that native SK channels are polyprotein complexes and demonstrate that the balance between kinase and phosphatase activities within the protein complex shapes the hyperpolarizing response mediated by SK channels.     

The ciliated sensory cell of Stauridiosarsia producta (Cnidaria,Hydrozoa) — a nematocyst-free nematocyte?

Summary A mechanosensitive ciliated cell type of the polyp Stauridiosarsia producta (Hydrozoa) was investigated by means of electron microscopy. These cells bear at their apical cell surface a modified cilium, a set of seven stereovilli, a so-called pseudovillar system and a large vacuole. Cilium and stereovilli are interconnected like the cnidocil apparatus of hydrozoan nematocytes which is responsible for mechanoelectric transduction. The vacuole is enclosed by and linked to the pseudovillar system by a microtubular basket. Considering its structural organization and physiological activities the ciliated sensory cell closely resembles a nematocyte that has lost its ability ot produce a nematocyst.     

Identification of infant food as a vehicle in a nosocomial outbreak of Citrobacter freundii: epidemiological subtyping by allozyme, whole-cell protein and antibiotic resistance

A total of 38 Citrobacter freundii strains was isolated from patients and their environment at a neonatal intensive care unit of a large hospital where colonization and clinical diseases due to the agent had been observed. Epidemiological investigations were carried out by subtyping, for which a combination of allozyme, whole-cell protein and resistance pattern analysis was used. Infant formula was identified as a vehicle of nosocomial spread. This shows that the role of foods in the transmission of hospital infections should not be underestimated.
The combination of methods applied, in particular a limited enzyme set, is recommended also for epidemiological investigations of food-borne infections and establishment of their causes.     

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