Centriole morphogenesis in developing ciliated epithelium of the mouse oviduct.

The differentiating mouse oviduct has been used for the study of centriole morphogenesis because its epithelium is extensively ciliated and centriole formation occurs in a brief period after birth. Proliferative elements, consisting of an extensive fibrillar meshwork encrusted with 75 mmicro granules, were encountered at all ages, but were the only centriole precursors present in younger animals (2-3 days). These large aggregates were found either physically associated with a mature centriole or alone, but never associated with procentrioles. It is likely, therefore, that although proliferative elements may be derived from preexisting centrioles, they do not directly produce new centrioles. An intermediate structure, the condensation form, found primarily in older animals (4-6 days), and produced by the packing of the proliferative element material, gives rise to daughter procentrioles. This association of procentriole and condensation form has been called a generative complex. Condensation forms undergo various stages of depletion, producing hollow spheres with thin walls or small osmiophilic aggregates as procentrioles grow in length and assemble their microtubules. From these observations it is concluded that synthesis of microtubular precursor protein is mediated by the mature centriole and that this protein is packaged into many condensation forms in order to allow the rapid assembly of a large number of centrioles in a brief period of time.

Microtubule protein during ciliogenesis in the mouse oviduct.

A colchicine-binding assay and quantitative sodium dodecyl sulfate gel electrophoresis have been used to determine the changes which occur in microtubule protein (tubulin) concentrations in the particulate and soluble fractions of mouse oviduct homogenates during that period of development when centriole formation and cilium formation are at a maximum. When mouse oviducts, at various ages after birth, are homogenized in Tris-sucrose buffer, tubulin concentration is partitioned between the soluble (70%) and particulate (30%) fractions. During the period of most active organelle formation (3-12 days), there is a marked increase in colchicine-binding specific activity, in both the soluble and particulate fractions. Microtubule protein concentration increases from 16 to 24% in the soluble fraction, declining to 14% in the adult. In the particulate fractions, microtubule protein concentration increases from 16 to 27%, leveling off at 16% in the adult. We have concluded from these observations and from electron microscopy that colchicine-binding activity in the particulate fractions is related to the presence of centriole precursors in the pellets of homogenized oviducts from newborn mice. These data further suggest that centriole precursor structures are conveniently packaged aggregates of microtubule protein actively synthesized between 3 and 5 days, and maintained at a maximum during the most active period of organelle assembly.

Nucleolar aging in Tetrahymena during the cultural growth cycle.

Nucelolar morphology was studied by electron microscopy in control and actinomycin D-treated populations of Tetrahymena pyriformis (W) during the cultural growth cycle. Nucleoli exhibit an "aging" cycle concomitant with the cultural growth cycle, but independent of the individual cell cycle. Four different stages in the course of this aging process have been defined. Stage 1 occurs upon inoculation (low number of cells per milliliter) and lasts through lag and accelerating growth phases. In this stage, many small nucleoli are found at the nuclear periphery. In stages 2 and 3, nucleolar fusion begins. Stage 2 dominates the first half of logarithmic growth, and stage 3 dominates the second half. In late decelerating growth phase, the nucleoli enter stage 4. In this stage, only a few large nucleoli are present and these are apparently inactive in ribosome production. In stationary phase, where total RNA remains constant, only stage 4 nucleoli are present. The relative preponderance of granular vs. fibrous components in the nucleoli changes during this cycle, the granular component dominating stage 1 nucleoli and the fibrillar, stage 4 nucleoli. There is a shortening of the intermediate nucleolar stages in the treated cultures; fusion occurs early and is now pronounced. Not enough ribosomes accumulate to carry the treated cultures through the number of generations equivalent to those of the control, which produces a premature stationary phase.

Intercellular calcium signaling via gap junctions in glioma cells.

Calcium signaling in C6 glioma cells in culture was examined with digital fluorescence video microscopy. C6 cells express low levels of the gap junction protein connexin43 and have correspondingly weak gap junctional communication as evidenced by dye coupling (Naus, C. C. G., J. F. Bechberger, S. Caveney, and J. X. Wilson. 1991. Neurosci. Lett. 126:33-36). Transfection of C6 cells with the cDNA encoding connexin43 resulted in clones with increased expression of connexin43 mRNA and protein and increased dye coupling, as well as markedly reduced rates of proliferation (Zhu, D., S. Caveney, G. M. Kidder, and C. C. Naus. 1991. Proc. Natl. Acad. Sci. USA. 88:1883-1887; Naus, C. C. G., D. Zhu, S. Todd, and G. M. Kidder. 1992. Cell Mol. Neurobiol. 12:163-175). Mechanical stimulation of a single cell in a culture of non-transfected C6 cells induced a wave of increased intracellular calcium concentration ([Ca2+]i) that showed little or no communication to adjacent cells. By contrast, mechanical stimulation of a single cell in cultures of C6 clones expressing transfected connexin43 cDNA induced a Ca2+ wave that was communicated to multiple surrounding cells, and the extent of communication was proportional to the level of expression of the connexin43 cDNA. These results provide direct evidence that intercellular Ca2+ signaling occurs via gap junctions. Ca2+ signaling through gap junctions may provide a means for the coordinated regulation of cellular function, including cell growth and differentiation.

Intercellular propagation of calcium waves mediated by inositol trisphosphate.

Two types of calcium (Ca2+) signaling-propagating intercellular Ca2+ waves of increasing intracellular Ca2+ concentration ([Ca2+]i) and nonpropagating oscillations in [Ca2+]i-co-exist in a variety of cell types. To investigate this difference in Ca2+ signaling, airway epithelial cells were loaded with heparin, an inositol 1,4,5-triphosphate (IP3) receptor antagonist, by pulsed, high-frequency electroporation. Heparin inhibited propagation of intercellular Ca2+ waves but not oscillations of [Ca2+]i. In heparin-free cells, Ca2+ waves propagated through cells displaying [Ca2+]i oscillations. Depletion of intracellular Ca2+ pools with the Ca2+-pump inhibitor thapsigargin also inhibited the propagation of Ca2+ waves. These studies demonstrate that the release of Ca2+ by IP3 is necessary for the propagation of intercellular Ca2+ waves and suggest that IP3 moves through gap junctions to communicate intercellular Ca2+ waves.

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.

Virus-like particles in placentas from normal individuals and patients with systemic lupus erythematosus.

Nineteen human placentas, obtained from 9 normal women and 10 women with diagnosed systemic lupus erythematosus (SLE), were extensively examined by electron microscopy for the presence of virus-like particles. Free and budding viral forms were found in the placentas of 4 of the normal women and 3 of the women with SLE. In every case, these virus-like particles were found in the basal infoldings of the syncytiotrophoblast cells of the chorionic villi. These particles could be distinguished from typical mammalian type C viruses by the absence of a space between the envelope and the nucleocapsid.

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.

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.

Selected contribution: PKC activation inhibits Ca(2+) signaling in tracheal epithelial cells kept in simulated microgravity.

Microgravity has been shown to alter protein kinase C (PKC) activity; therefore, we investigated whether microgravity influences mechanically stimulated Ca(2+) signaling and ATP-induced Ca(2+) oscillations, both of which are modulated by PKC. Rabbit tracheal epithelial outgrowth cultures or suspended epithelial sheets were rotated in bioreactors to simulate microgravity. Mechanical stimulation of a single cell increased the cytosolic Ca(2+) concentration in 35-55 cells of both outgrowth cultures and epithelial sheets kept at unit gravity (G) or in simulated microgravity (smicroG). In outgrowth cultures, 12-O-tetradecanoylphorbol-13-acetate (TPA; 80 nM), a PKC activator, restricted Ca(2+) "waves" to about 10 cells in unit G and to significantly fewer cells in smicroG. TPA only slightly reduced the spread of Ca(2+) waves in epithelial sheets kept in smicroG but did not inhibit Ca(2+) waves of sheets kept in unit G. In both cell preparations from both conditions, TPA inhibited ATP-induced Ca(2+) oscillations; however, the effect was more pronounced in cells kept in smicroG. These results suggest that PKC activation is more robust in cells subjected to smicroG.

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.

Centriole and basal body formation during ciliogenesis revisited.

This review is concerned with the formation during ciliogenesis of centrioles and basal bodies, primarily in epithelial multi-ciliated 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.

Mechanosensitivity of cultured ciliated cells from the mammalian respiratory tract: implications for the regulation of mucociliary transport.

Mechanical stimulation of the cell surface or cilia of cultured ciliated epithelial cells derived from the rabbit tracheal mucosa resulted in a transitory ciliary beat frequency increase of 20% or more. This response was composed of a lag, rise, and recovery phase. The duration of the response, but not the maximal frequency, was increased by stronger stimulation. The ability of these ciliated cells to respond to stimulation depended on culture age; generally, cultures younger than 4 days were insensitive. The frequency response was transmitted to adjacent and more distal cells in all directions. The lag phase but not the rise time of the adjacent cell response was extended. These ciliary responses were lost when extracellular Ca2+ was removed. Replacement of Ca2+ resulted in a restoration of mechanosensitivity. In 1 mM verapamil and Ca2+ the frequency response was also lost. These results suggest that the frequency response is dependent on Ca2+ influx (although its intracellular action is unknown) and imply that mucociliary clearance is a localized self-regulating process.

Mechanosensitive and beta-adrenergic control of the ciliary beat frequency of mammalian respiratory tract cells in culture.

Respiratory tract ciliated cells, obtained from the rabbit trachea and maintained in culture, were sensitive to mechanical stimulation. The mechanical deformation of the cell surface induced a rapid, but transient, increase in ciliary beat frequency. In addition, the beat frequency of these ciliated cells was also increased in a dose-dependent manner by the beta-adrenergic drug isoproterenol (10(-7) to 10(-4) M) and by the calcium ionophore A23187 (10(-6) and 10(-5) M). To determine if drug and mechanosensitive activation of ciliary beat frequency arise from a common or different cellular mechanism, we investigated the effect of mechanical stimulation on beat frequency in the presence of isoproterenol or A23187. In isoproterenol (10(-8) to 10(-4) M), none of the parameters used to describe the ciliary beat frequency response to mechanical stimulation was altered. In A23187 (10(-6) M or above), the magnitude of the beat frequency response was significantly reduced or almost abolished, suggesting that mechanical stimulation acts, like A23187, to increase ciliary beat frequency by increasing intracellular calcium. Lower concentrations of A23187 had no effect. These results suggest that respiratory tract ciliated cells have at least two independent mechanisms for the control of ciliary beat frequency: one probably utilizing calcium, the other probably cAMP.

Microtubule sliding in cilia of the rabbit trachea and oviduct.

Evidence for active sliding of microtubules during ciliary activity has been demonstrated in a number of organisms: sea urchin sperm flagella, protozoan cilia, and mollusc gill cilia. Although there is evidence that active sliding also occurs in mammalian sperm flagella, there is little or no information on whether active sliding of microtubules also occurs in the short (5-micron) cilia of the mammalian trachea or oviduct. Since these cilia are important in tracheobronchial clearance and ovum transport, respectively, it has been important to demonstrate that microtubule sliding is also involved in the activity of somatic cilia. Ciliated apical portions (cortices) and cilia were isolated from rabbit trachea and oviduct, using Triton X-100 to demembranate the cilia. Most of the ciliated cortices reactivated upon addition of ATP, whereas isolated cilia reactivated to a lesser extent. When preparations of cilia were digested with trypsin before or after ATP addition, disintegration of axonemal doublets occurred with about the same frequency as reactivation. These events were recorded using Nomarski optics and dark-field microscopy. When isolated cilia which had been digested by trypsin and exposed to ATP were also prepared for electron microscopy by negative staining, telescoping of doublet microtubules from axonemes could be shown. These results demonstrate that mammalian somatic ciliary doublet microtubules actively slide in a manner similar to that described for invertebrate cilia.