Ellen R. Dirksen

Intercellular signaling in glial cells: Calcium waves and oscillations in response to mechanical stimulation and glutamate

Intercellular Ca2+ signaling in primary cultures of glial cells was investigated with digital fluorescence video imaging. Mechanical stimulation of a single cell induced a wave of increased [Ca2+]i that was communicated to surrounding cells. This was followed by asynchronous Ca2+ oscillations in some cells. Similar communicated Ca2+ responses occurred in the absence of extracellular Ca2+, despite an initial decrease in [Ca2+]i in the stimulated cell. Mechanical stimulation in the presence of glutamate induced a typical communicated Ca2+ wave through cells undergoing asynchronous Ca2+ oscillations in response to glutamate. The coexistence of communicated Ca2+ waves and asynchronous Ca2+ oscillations suggests distinct mechanisms for intra- and intercellular Ca2+ signaling. This intercellular signaling may coordinate cooperative glial function.

Mechanisms and function of intercellular calcium signaling

Intercellular Ca2+ waves initiated by mechanical or chemical stimuli propagate between cells via gap junctions. The ability of a wide diversity of cells to display intercellular Ca2+ waves suggests that these Ca2+ waves may represent a general mechanism by which cells communicate. Although Ca2+ may permeate gap junctions, the intercellular movement of Ca2+ is not essential for the propagation of Ca2+ waves. The messenger that moves from one cell to the next through gap junctions appears to be IP3 and a regenerative mechanism for IP3 may be required to effect multicellular communication. Extracellularly mediated Ca2+ signaling also exists and this could be employed to supplement or replace gap junctional communication. The function of intercellular Ca2+ waves may be the coordination of cooperative cellular responses to local stimuli.

Centriole and basal body formation during ciliogenesis revisited

Summary— This review is concerned with the formation during ciliogenesis of centrioles and basal bodies, primarily in epithelial multiciliated cells from the developing vertebrate respiratory and reproductive tracts. During ciliated cell differentiation, in these as well as in other cell types, cilium formation is preceded by the formation of centrioles assembled from precursor structures having little resemblance to the mature organelle. The origin, composition and function of the centriole precursor structures in generating large numbers of centrioles in a short period of time during ciliogenesis is discussed. This review also focuses on the biochemistry of centrioles and basal bodies and on recent experimental evidence that DNA might be associated with these structures.

Sequence-specific antibodies to connexins block intercellular calcium signaling through gap junctions

Mechanical stimulation of a single cell in primary airway epithelial cell cultures induces an intercellular Ca2+ wave that has been proposed to be mediated via gap junctions. To investigate directly the role of gap junctions in this multicellular response, the effects of intracellularly-loaded sequence-specific connexin (gap junction) antibodies on the propagation of intercellular Ca2+ waves were evaluated. Electroporation of antibodies to the cytosolic loop (Des 1, generated to amino acids 102-112 + 116-124; and Des 5, amino acids 108-119), or to the carboxyl tail (Gap 9, amino acids 264-283) of connexin 32 inhibited the propagation of intercellular Ca2+ waves. The inhibitory effect of Des 1 antibody was competitively reversed by the co-loading of a peptide derived from a similar cytosolic loop sequence (Des 5 peptide). Conversely, the inhibitory effects on intercellular Ca2+ wave propagation of Gap 9 antibody was not altered by co-loading with the Des 5 peptide. Antibodies raised to peptide sequences within the extracellular loop (Gap 11, amino acids 151-187), or the cytoplasmically located amino terminus (Gap 10, amino acids 1-21) of connexin 32 did not inhibit mechanically-induced intercellular communication. Also ineffective in perturbing intercellular communication were antibodies raised to peptide sequences of the cytosolic loops of connexin 43 (Gap 15, amino acids 131-142) or connexin 26 (Des 3, amino acids 106-119). These data suggest that mechanically-induced Ca2+ waves in airway cell cultures are propagated through gap junctions made up of connexin 32 proteins.

Possible communication between murine macrophages oriented in linear chains in tissue culture

Abstract Macrophages from mouse tissues were observed to assume a linear orientation when cultivated on plastic or glass Petri dishes in tissue culture. The cells demonstrated cytoplasmic bridging and electrotonic coupling. These results are discussed as evidence for possible communications among macrophages and the requirement for this cell contact for certain normal functions.

PREPARATION OF EXPLANT AND ORGAN CULTURES AND SINGLE CELLS FROM AIRWAY EPITHELIUM

Publisher Summary In this chapter, the methods for preparing explant and organ cultures as well as the enzymatic isolation of cells are described. The inaccessibility of the ciliated epithelium of the mammalian respiratory tract has hampered in vivo studies of its structure and function, including those regarding the regulatory mechanisms of mucociliary transport. Consequently, several in vitro approaches have been developed to maintain and study airway tissues in culture. These include explant cultures, where pieces of tissue are seeded on a substrate to encourage the spreading and outgrowth of the epithelial cells; organ cultures, in which the tissue is maintained to preserve its original structural and functional form; monolayer cell cultures, where enzymatically dissociated cells are plated onto a noncellular substrate; and feeder layer cultures, where isolated epithelial cells are grown on a cellular substrate such as treated fibroblasts. Explant cultures have been utilized for the measurement of ciliary beat frequency, including the timings of the phases of the beat cycle, of single and multiple cells, as well as for the analysis of intercellular Ca2+ signaling. Isolated epithelial cells have been used in patch–clamp studies and in assays to measure inositol trisphosphate levels. There is description of preparation of rat tail collagen, of collagen-coated coverslips, and tracheal epithelial explant culture—the materials to be used and the procedure. The technique is sterile. There is description of dissection of rabbit trachea, technique is aseptic. Details of preparation of organ culture and preparation of enzymatically isolated cells have been covered. In organ culture, the tracheal mucosa is supported at a liquid-air interface to mimic in vivo conditions of nutrient and gas exchange. Isolated cell preparations are obtained either from freshly dissected tissues or from tissues that had previously been placed in organ culture.

Regulation of ciliary activity in the mammalian respiratory tract

A computer-assisted transillumination, photoelectronic technique has been used to measure the beat frequency of cilia of rabbit tracheal cells grown in culture. When ciliated cells are mechanically stimulated with a microprobe the cells respond rapidly by increasing the beat frequency of their cilia. This mechanosensitive response is not limited to the stimulated cell, but is communicated in all directions to neighboring cells. To characterize the progression of this communicated response we used an automated computer-assisted imaging system to examine high-speed films of responding cells. The time it takes for the response to be transmitted between cells is slow (1-3 sec) with each cell responding after a lag-time that is proportional to the distance of the cell from the stimulated cell. We have confirmed that gap junctions are present between cells and that adjacent or non-adjacent ciliated, as well as non-ciliated, cells are electrically coupled. To correlate the mechanosensitive response with intracellular calcium fluxes we have used fura-2, a calcium-specific fluorescent dye, and digital video microscopy. Mechanical stimulation of the cultured ciliated cells, in the presence of extracellular calcium, resulted in an initial increase in intracellular calcium, which was communicated to neighboring cells. Without extracellular calcium, mechanosensitivity of cultured cells was lost and a small decrease in intracellular calcium was observed in the stimulated cell. However, neighboring cells still displayed an increase in intracellular calcium. The time course and general pattern of calcium increase in adjacent cells was similar to the responses in ciliary activity produced by mechanical stimulation. Ciliary beat frequency is also elevated by beta-adrenergic drugs independently of mechanosensitivity. These responses are important because they could provide a dual regulatory mechanism for the control of mucus transport. Adrenergic agonists could provide non-specific control by increasing ciliary activity throughout the airways while mechanosensitivity could provide local control by increasing activity in those regions of heavy mucus load.

Quantification of ciliary beat frequency and metachrony by high-speed digital video.

Publisher Summary The quantification of ciliary activity depends on the detection and analysis of the rhythmic variation in light intensity of a microscope image induced by ciliary movement. This approach requires the simplification and optimization of signal contrast and waveform and its success depends on the photo-detection method, the microscope optics, and the type of cell preparation used. For beat frequency measurements, a single detector system consisting of a photomultiplier, phototransistor, or fiberoptic device is sufficient. For the analysis of ciliary metachrony, spatial information is essential and a system requires a multipoint detector such as a digital video camera. This chapter highlights the advantages and limitations of earlier techniques for measuring ciliary activity and describes the principles and methodology of an advanced high-speed digital video technique. Large light signal amplitude can be obtained from coordinated groups of active cilia of isolated or cultured cells with phase-contrast optics. The signal waveform needs to be simple and easily interpretable; however, cilia of isolated cells or epithelial sheets generally beat in a metachronal manner and this can result in a complicated signal waveform because of the orientation of the ciliary beat cycle with respect to the detector and the number of cilia simultaneously contributing to the signal. An improvement in signal waveform can be achieved by monitoring only the effective stroke by recording close to the ciliary tips. The chapter discusses quantification of ciliary metachronism and video techniques for measuring ciliary activity. The high-speed digital video photodetection technique discussed has a high temporal and spatial resolution that can be used for the measurement of ciliary beat frequency and metachrony. The combination of this technique with a fluorescence imaging system that can simultaneously measure intracellular ions or messengers provides a unique opportunity for investigation of the physiological control of ciliary activity.

Rapid tubulin synthesis during ciliated cell differentiation

Using pulse-chase conditions in culture we have investigated the incorporation of 3H-leucine into tubulin of isolated oviducts from 5 day-old mice. Label appears in soluble, particulate and axonemal fractions minutes after incubation. In the latter two fractions, but not in the soluble fraction, this label is rapidly diluted under chase conditions. The data do not fit a simple model of sequential transfer of radioactively labeled, newly synthesized tubulin from a soluble fraction through centriole precursors to assembled ciliary axonemes.