Factors controlling axonal and dendritic arbors.

The sculpting and maintenance of axonal and dendritic arbors is largely under the control of molecules external to the cell. These factors include both substratum-associated and soluble factors that can enhance or inhibit the outgrowth of axons and dendrites. A large number of factors that modulate axonal outgrowth have been identified, and the first stages of the intracellular signaling pathways by which they modify process outgrowth have been characterized. Relatively fewer factors and pathways that affect dendritic outgrowth have been described. The factors that affect axonal arbors form an incompletely overlapping set with those that affect dendritic arbors, allowing selective control of the development and maintenance of these critical aspects of neuronal morphology.

The Neurospora crassa genome: cosmid libraries sorted by chromosome.

A Neurospora crassa cosmid library of 12,000 clones (at least nine genome equivalents) has been created using an improved cosmid vector pLorist6Xh, which contains a bacteriophage lambda origin of replication for low-copy-number replication in bacteria and the hygromycin phosphotransferase marker for direct selection in fungi. The electrophoretic karyotype of the seven chromosomes comprising the 42.9-Mb N. crassa genome was resolved using two translocation strains. Using gel-purified chromosomal DNAs as probes against the new cosmid library and the commonly used medium-copy-number pMOcosX N. crassa cosmid library in two independent screenings, the cosmids were assigned to chromosomes. Assignments of cosmids to linkage groups on the basis of the genetic map vs. the electrophoretic karyotype are 93 +/- 3% concordant. The size of each chromosome-specific subcollection of cosmids was found to be linearly proportional to the size of the particular chromosome. Sequencing of an entire cosmid containing the qa gene cluster indicated a gene density of 1 gene per 4 kbp; by extrapolation, 11,000 genes would be expected to be present in the N. crassa genome. By hybridizing 79 nonoverlapping cosmids with an average insert size of 34 kbp against cDNA arrays, the density of previously characterized expressed sequence tags (ESTs) was found to be slightly <1 per cosmid (i.e., 1 per 40 kbp), and most cosmids, on average, contained an identified N. crassa gene sequence as a starting point for gene identification.

Glutamate-induced changes in the pattern of hippocampal dendrite outgrowth: a role for calcium-dependent pathways and the microtubule cytoskeleton.

Glutamate regulation of a variety of aspects of dendrite development may be involved in neuronal plasticity and neuropathology. In this study, we examine the calcium-dependent pathways and alterations in the microtubule (MT) cytoskeleton that may mediate glutamate-induced changes in the pattern of dendrite outgrowth. We used Fura-2 AM and inhibitors of the calcium-dependent proteins, calmodulin and calpain, to identify the role of specific calcium-dependent pathways in glutamate-regulated dendrite outgrowth. Additionally, we used a quantitative fluorescence technique to correlate changes in MT levels with glutamate-induced changes in dendrite outgrowth. We show that the intracellular calcium concentration ([Ca(2+)](i)) changes in a biphasic manner over a 12-h period in the presence of glutamate. A transient increase in [Ca(2+)](i) over the first hour of glutamate exposure correlated with a calmodulin-associated increase in the rate of dendrite outgrowth, whereas a sustained increase in [Ca(2+)](i) was correlated with calpain-associated dendrite retraction. Quantitative fluorescence measurements showed no net change in the level of MTs during calmodulin-associated increases in dendrite outgrowth, but showed a significant decline in the level of MTs during calpain-associated dendrite retraction. These findings provide insights into the intracellular mechanisms involved in activity-dependent regulation of dendrite morphology during development and after pathology.

Micro-perfusion flow cell for imaging cultured cells.

We present a unique design for a flow cell with a small working volume that allows rapid displacement of media viewed under high power and short working distance objectives. The flow cell has a small internal depth (ca. 0.033 cm) and volume (ca. 0.05 mL) and is easy to handle. Made of Delrin, the flow cell is biologically inert. We have used the flow cell for fluorescence imaging of PC12 cells loaded with tetramethylrhodamine dextran (TMRD) and other dyes.

Coherent backscatter enhances reflection confocal microscopy.

When thin optically transparent specimens are grown on reflective substrates, contrast in reflection confocal microscopy is markedly enhanced. This enhanced contrast allows for the visualization of the thin filopodia and organelles contained within the neuritic processes of PC12 cells in culture. The characteristics of this contrast enhancement suggest that it arises because of interference between light scattered from the specimen and coherently backscattered illumination reflected off the substrate. This technique provides a method for visualizing living cells or other similarly transparent objects on opaque substrates in a nondestructive manner.

Glutamate modulation of dendrite outgrowth: alterations in the distribution of dendritic microtubules.

Glutamate can both facilitate and inhibit dendrite outgrowth in vitro. The major effects of low levels of glutamate occur only on the dendrites (not the axon) of pyramidal neurons and may be important for modulating dendrite outgrowth during neuronal development in vivo. Cytoskeletal changes resulting from glutamate exposure must underlie these changes in dendrite outgrowth. In the present study, hippocampal neuron cultures were used to measure the outgrowth of both axons and immature dendrites in the presence or absence of 50 microM glutamate. Subsequently, neurons were extracted and fixed for immunofluorescent labeling of microtubules and rhodamine phalloidin labeling of microfilaments. Additionally, neurons were prepared for electron microscopy to examine dendritic microtubules at the ultrastructural level. Glutamate led to increased dendrite outgrowth in the short term (4 hr) and dendrite retraction in the long term (8 hr). After short-term glutamate exposures, no obvious morphological changes occur in either the microtubules or microfilaments. However, longer glutamate exposure causes a decrease in the number of microtubules in the distal region of retracting dendrites, and causes an increase in microtubule number in the dendritic shaft of both retracting and growing dendrites. Thus, the microtubule cytoskeleton may be involved in producing the changes in dendrite outgrowth caused by glutamate exposure.

Free cyclic AMP increases in PC12 cells on depolarization.

The temporal dynamics of the intracellular second messenger cyclic AMP (cAMP) were monitored in living PC12 cells by digital fluorescence ratio imaging using FlCRh, a single-excitation dual-emission cAMP indicator. When the cells were depolarized by exposure to high K+, the free cAMP concentration was elevated, and then slowly decreased back to resting levels when the depolarizing stimulus was removed. Furthermore, the cAMP elevation due to depolarization decreased with successive depolarizations.

Microtubule behavior in PC12 neurites: variable results obtained with photobleach technology.

We have examined the effects of various means of photobleaching on the recovery of fluorescence, movement, and morphology of the microtubules in the neurites of rhodamine-tubulin-injected PC12 cells. We find that, depending on power of and time of exposure to the bleaching beam, we can generate at least three different patterns of fluorescence recovery in regenerating PC12 neurites. If bleaching is performed with a relatively low-power beam for an extended period, fluorescence in polymer recovers very little after 1 hour. Under these conditions, however, tubulin immunostaining is seen extending through the bleach zone, and microtubules are present through the bleached zone in thin section electron micrographs. If bleaching is performed with a high-power laser, for 0.5-5 seconds, fluorescence recovery also is quite slow, but electron microscopic observations reveal that no microtubules extend through the bleached region of the neurite, and the uranyl acetate-stained cytoplasm appears more electron lucent than in the unbleached neurite. Finally, if bleaching is performed by very brief exposure to a high-intensity laser beam, resulting in an incomplete reduction of fluorescence intensity through the bleach zone, fluorescence recovery occurs within 20-30 minutes, and immunostained microtubules appear intact through the bleach zone; electron microscopy confirms that microtubules extend through the bleached zone of such neurites. In all three cases, movement of the bleach zone is observed in approximately half of the experimental neurites. These results indicate that highly variable microtubule behaviors can be obtained with photobleach technology, presumably due to different levels and pathways of photodamage generated by different bleach protocols. Nevertheless, it is clear that both turnover and movement of microtubules occur in PC12 neurites, and both are likely to be involved in neurite maintenance and growth.

Trypanosoma cruzi infection of BSC-1 fibroblast cells causes cytoskeletal disruption and changes in intracellular calcium levels.

The disruption of vimentin and actin filaments of host BSC-1 fibroblast cells by Trypanosoma cruzi was investigated using a mouse monoclonal anti-vimentin antibody and rhodamine phalloidin, respectively. Indirect immunofluorescence microscopy demonstrated that infection of BSC-1 cells by T. cruzi caused disruption of both cytoskeletal components. The disruption was greater as infection progressed. Mechanisms other than mechanical ones may play a role in the disruption since disrupted cytoskeletal elements were well removed from the parasites. In the determination of intracellular calcium concentrations using Fura-2 AM, infected and uninfected cells both showed an initial increase in intracellular calcium levels. At later times of infection (3 to 5 days), intracellular calcium levels of infected cells were significantly lower than those of control cells. There was no specific localization of intracellular calcium in the infected host cells as determined by image analysis.

Intracellular pH of tissue-cultured bovine corneal endothelial cells.

Intracellular pH (pHi) of bovine tissue-cultured corneal endothelial cells has been measured under several experimental conditions. Determinations were made on individual cells using video-imaging techniques that allowed assessment of 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein fluorescence at 440 and 490 nm. Each experiment had a calibration performed on a cell monolayer: this was performed using a high K(+)-nigericin solution. Resting pHi was 7.25 +/- 0.03 (n = 18) in bicarbonate solution at pH 7.4. Amiloride (1 mM) caused an acidification of approximately 0.2 U within 2 min: replacement with normal Ringer allowed a return to normal pHi after an alkali overshoot. Exposure to 20 mM NH4Cl caused alkalinization that became acidic upon washout of NH4Cl. In Na(+)-rich solution pHi returned to normal after acidification but pHi remained low in Na(+)-free solution until substituted by Na(+)-rich solution. Removal of HCO3- from the bathing solution caused a nonsignificant acidification of pHi by 0.1 U at 2 and 4 min, and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS; 1 mM) acidified pHi by 0.14 U at 2 min and 0.24 U at 4 min. Addition of DIDS (1 mM) in a HCO3(-)-free solution had no effect on pHi. Hydrogen peroxide acidified pHi by 0.3 U at 50 microM and 1 mM. These results indicate that a Na+:H+ antiport exists that regulates pHi even at normal ambient pH in the presence of bicarbonate: this process becomes highly activated after an acid load. There is a DIDS-sensitive HCO3- movement that is probably coupled to Na+ or Cl-.

Quantitative fluorescence techniques for the determination of local microtubule polymerization equilibria in cultured neurons.

The local control of intracellular microtubule polymerization equilibria has been hypothesized to be an important factor in the determination of neurite extension and other examples of cellular asymmetry. Provided that the quantum yield of the fluorophore remains constant, the combination of fluorescent analogue cytochemistry, differential extraction protocols, and quantitative video microscopy makes it possible to measure local fractions of cytoskeletal protein in polymer, even when it is impossible to resolve individual fibrils of the polymer. We have developed appropriate quantitative video microscopic techniques for measuring the fluorescence of a fluorescent analogue-injected neurite before and after extraction under microtubule-stabilizing conditions. We have used these methods to demonstrate that tetramethylrhodamine-n-hydroxysuccinimide tubulin is an appropriate fluorescent analogue, allowing us to measure fractions of tubulin in polymer locally within PC12 neurites. As would be expected, the fraction of tubulin fluorescent analogue in polymer approaches 1.0 in neurites exposed to the microtubule-stabilizing drug taxol and is close to 0 in neurites injected and extracted in the cold, or extracted under microtubule-destabilizing conditions. We have, therefore, developed a tool that allows us to measure microtubule polymerization equilibria out the neurites of cells in culture, which will allow us to test hypotheses that factors which affect neurite outgrowth do so by means of effects on microtubule polymerization equilibria.

Sites of tubulin polymerization in PC12 cells.

The site at which tubulin enters into polymer in the neuritic process is a very important datum in terms of our understanding of the mechanism of transport of the microtubular cytoskeleton out the axon. If the form of tubulin being transported out the axon is the microtubule, then assembly of tubulin into microtubules should occur at or near the cell body; if, however, the form of tubulin transported is free tubulin dimer, then assembly can occur at any free microtubule end out the neurite. We have injected a fluorescent analog of tubulin into differentiated PC 12 cells and used differential extraction protocols to extract free dimer but not microtubules. We have imaged these cells before and after extraction by low-light-level video fluorescence microscopy and have used image analysis to examine the sites of tubulin incorporation into polymer or other unextracted components as a function of time. We find that tubulin in the distal reaches of the neurite is found initially as monomer and that its appearance in the unextracted component occurs later. This pattern of appearance of fluorescent tubulin initially in the soluble fraction and later in the unextractable component is qualitatively similar to that reported by other workers for biotinylated tubulin, but we see a larger gap between the rates of appearance in soluble fraction and in polymer. Quantitative analysis of fluorescence intensities in the two compartments with distance out the neurite reveals substantial variation between different neurites: In some neurites, the pattern of variation of unextracted/total tubulin suggests that tubulin enters into the unextracted component primarily near the cell body and that this unextracted component moves out the neurite with time, and in other neurites it suggest that monomer adds into microtubule ends staggered out the neurite. In no case do we see a pattern suggesting that distal addition predominates. These analyses of fluorescence intensities in extracted and unextracted neurites suggest that both transport of polymerized microtubules and monomer addition onto staggered microtubule ends occur in PC12 neurites and that in individual neurites one or the other of these two behaviors may predominate.

Neurite elongation is blocked if microtubule polymerization is inhibited in PC12 cells.

We have injected process-bearing PC12 cells with colchicine-tubulin mixed with either fluorescein-dextran or a rhodamine-labelled tubulin analogue to determine the role of microtubule polymerization in neurite elongation. Colchicine-tubulin is a specific, substoichiometric poison of microtubule assembly. We have shown that colchicine-tubulin does not cause existing PC12 microtubules to disassemble, and yet can inhibit the assembly of rhodamine-tubulin injected along with it. In population studies of neurite outgrowth in injected and uninjected cells, we find that colchicine-tubulin substantially inhibits neurite extension from injected cells over a wide variety of concentrations. In acute time-course studies of injected cells, we find that colchicine-tubulin does not block neurite outgrowth until the injectate reaches the neurite tip. Thereafter, however, it blocks process elongation completely. Thus we can conclude that microtubule polymerization in the region of the growth cone is an important element in neurite elongation. While polymerization at the cell body may be important in supplying subunits to the distal neurite, it does not play a direct role in process extension.

Inhibition of mitosis in PtK2 cells by CAPP1-calmodulin.

Various indirect evidence has indicated that calcium ions and the calcium-binding regulator protein, calmodulin, may regulate mitosis in higher eukaryotes. We have used the competitive antagonist, CAPP1-calmodulin, to antagonize intracellular calmodulin and test the hypothesis that calmodulin serves as a regulator of mitosis. We find that CAPP1-calmodulin inhibits the transit of cells through metaphase at estimated intracellular concentrations up to that of native calmodulin; beyond that level, the inhibition of mitosis vanishes. The membrane-permeant anticalmodulin agents, W7 and calmidazolium, also inhibit the progress of cells through metaphase. The similarity of the inhibitory curves for CAPP1-calmodulin, W7, and calmidazolium suggests that all these agents inhibit mitosis by antagonizing intracellular calmodulin. In order to test whether this inhibition of metaphase transit is due to an effect of the agents on intracellular free calcium, we used the calcium indicator Fura-2 to measure intracellular calcium levels after CAPP1-calmodulin injection or during calmidazolium treatment. We found that, while intracellular calcium levels are modestly elevated during calmidazolium treatment, they were unaffected by CAPP1-calmodulin, a result suggesting that mitosis inhibition was not due to an effect on intracellular free calcium. The reasons for the anomalous dose-response behavior of these drugs are not known; however, the behavior of cells at drug levels below the point of anomaly supports the hypothesis that calmodulin acts as a regulator of mitosis in these cells.

A monoclonal antibody to alpha tubulin recognizes host cell and Trypanosoma cruzi tubulins.

A mouse monoclonal anti-alpha-tubulin antibody was used to investigate the disposition of the cytoskeletal microtubules of three tissue culture cell lines--J774 macrophages, BSC-1, and Vero cells--infected with the Brazil strain of Trypanosoma cruzi. Indirect immunofluorescence light microscopy was used to demonstrate the antigenic response in host cells and parasites, simultaneously. In all morphotypes of T. cruzi, the monoclonal antibody reacted with all subpopulations of microtubules, inclusively, the subpellicular, flagellar, cytopharyngeal, and mitotic. The host cell cytoskeletal microtubule framework was revealed and the redistribution and destruction of the microtubular lattice in response to parasite infection over a 120 h period recorded. Our results show that after the initial inoculation of tissue cultures with trypomastigotes, the parasites penetrate the cells and locate in the perinuclear region of the cell where they multiply. The number and distribution of host cell microtubules were altered during the infection. The normal radial distribution of microtubules extending from the center of the cell to the periphery was destroyed. The remaining microtubules were observed at the periphery encircling, but well removed from the proliferating parasites. The complete transformation of the parasites was monitored throughout the infection with the end result being the liberation of parasites and the near complete destruction of the microtubular framework of the host cell. A residual population of dividing spheromastigotes was observed in cells liberating trypomastigotes. Colloidal gold labeling of thin sections as seen in the electron microscope affirmed the specificity of our monoclonal antibody to all subpopulations of microtubules in T. cruzi.

Effect of microinjected calcium-calmodulin on mitosis in PtK2 cells.

Calcium and calmodulin are believed to play a significant role in the regulation of mitosis, because they are both localized in the mitotic spindle and because they can potentiate microtubule depolymerization in the test tube and in the living cell. It has been hypothesized, specifically, that calcium-saturated calmodulin drives the shortening of the kinetochore microtubules that must occur during prometaphase, when the chromosomes congress to the metaphase plate, and during anaphase A, when the half-spindles shorten. We have examined the role of calmodulin in mitosis by observing the consequences of calmodulin microinjection on the progress of mitosis and morphology of the mitotic spindle in PtK2 cells. We have found that the injection of excess calcium-saturated calmodulin during early prometaphase significantly prolongs the time required for the cell to go into anaphase, and that neither calcium-depleted calmodulin nor buffer alone produce a similar perturbation. Calcium ion alone produces a similar but much smaller retardation of mitosis. Immunofluorescence and fluorescent analogue cytochemical studies of spindle morphology reveal that the immediate (less than 5-min) effect of calcium-saturated calmodulin on prometaphase spindles is a significant shortening of the kinetochore fibers and "interpolar" microtubules but not the astral microtubules. After this perturbation, however, the spindle quickly recovers its normal form. An equivalent transient shortening of the spindle fibers is seen following the injection of calcium chloride solutions but not after the injection of calcium-depleted calmodulin or buffer alone. Taken together, these observations suggest that calcium-saturated calmodulin plays a significant role in the regulation of mitosis, and that this regulatory pathway involves more than spindle fiber shortening.

Slow transport of tubulin in the neurites of differentiated PC12 cells.

In order to study the rate and form of tubulin transport in cultured neuronal cells, the fluorescence recovery after the photobleaching of a fluorescent tubulin analog has been followed within the neuritic processes of differentiated PC12 cells. In these cells, as in peripheral axons, tubulin is transported in coherent, nondiffusing waves at two different slow rates that are within the range of the slow components a and b of axonal transport measured in vivo. Finally, it appears that most, if not all, of the tubulin analog is moving out these processes. Thus, slow neuroplasmic transport in cultured neuron-like cells is a good model of axonal transport, in which experimental manipulations of the system can be performed that would be difficult in the whole animal.

Local cytoplasmic calcium gradients in living mitotic cells.

Cytoplasmic free calcium has been proposed as a regulator of many microtubule-mediated processes, including mitosis. It has been difficult to test this hypothesis because methods for local measurement of free Ca2+ in the living cell have not been available. We have used the fluorescent calcium indicator dye Quin-2 (methoxyquinoline-1bis(o-aminophenoxy)ethane-N,N,N',N' -tetra acetic acid), which allows such observations to be made by digital processing of fluorescent images from the light microscope. Here we report the application of this technique to the study of local Ca2+ concentrations in mitotic endosperm cells of Haemanthus sp., and show that there is transient increase in free Ca2+ at the mitotic spindle poles during anaphase. This locally high Ca2+ may provide a mechanism for the regional control of microtubules and other cytoskeletal elements during anaphase.

Intracellular free calcium levels are reduced in mitotic Pt K2 epithelial cells.

Using a fluorescence ratio method, we have studied the intracellular free calcium levels in individual quin-2-loaded mitotic cells under the microscope. We have found that intracellular free calcium concentrations in Pt K2 epithelial cells drop by approximately 50% as they pass through mitosis. Calcium levels in interphase cells were 53 +/- 7 nM. During prophase, free cytoplasmic calcium begins to decrease, reaching 28 +/- 3 nM in prometaphase. Calcium levels remain low until the nuclear envelope is re-formed in late telophase, when they increase again to interphase levels. This decrease in overall free calcium in mitosis suggests that the mitotic cell has mechanisms for the general sequestration, and perhaps local release, of calcium ions.