Proliferation Of Microtubules Research Articles

Acute microtubule changes linked to DMD pathology are insufficient to impair contractile function or enhance contraction-induced injury in healthy muscle.

Duchenne muscular dystrophy (DMD) is marked by the genetic deficiency of the dystrophin protein in striated muscle whose consequence is a cascade of cellular changes that predispose the susceptibility to contraction injury central to DMD pathology. Recent evidence identified the proliferation of microtubules enriched in post-translationally modified tubulin as a consequence of dystrophins absence that increases the passive mechanics of the muscle fiber and the excess mechanotransduction elicited reactive oxygen species and calcium signals that promote contraction injury. Motivated by evidence that acutely normalizing the disease microtubule alterations reduced contraction injury in murine DMD muscle ( mdx ), here we sought the direct impact of these microtubule alterations independent of dystrophins absence and the multitude of other changes consequent to dystrophic disease. To this end we used acute pharmacologic (epithiolone-D, EpoD; 4 hours) or genetic (vashohibin-2 and small vasohibin binding protein overexpression via AAV9; 2 weeks) strategies to effectively model the proliferation of detyrosination enriched microtubules in the mdx muscle. Quantifying in vivo nerve evoked plantarflexor function we find no alteration in peak torque nor contraction kinetics in WT mice modeling these DMD relevant MT alterations. Quantifying the susceptibility to eccentric contraction injury we show EpoD treatment proffered a small but significant protection from contraction injury while VASH/SVBP had no discernable impact. We conclude that the disease dependent MT alterations act in concert with additional cellular changes to predispose contraction injury in DMD.

Abstract P3115: Disruption Of Assembly By Estrogen Limits Microtubule Proliferation In Failing Heart Cells

Right heart failure, resulting from high pressure in the pulmonary artery, is the leading cause of mortality in pulmonary arterial hypertension (PAH). Interestingly, females with PAH have superior right ventricular (RV) function, which can mostly explain improved survival in female patients. Microtubules are long filaments that regulate cellular contractility and transport within cardiac cells. The excess proliferation of microtubules is associated with pathological t-tubule remodeling in failing RV cardiomyocytes. Previous work suggests that the female hormone estrogen disrupts microtubule assembly. Here, we explored whether estrogen treatment could ameliorate the pathological proliferation of microtubules. By utilizing iPSC-derived cardiomyocytes with live-cell confocal imaging, we show that estrogen treatment leads to long pausing during microtubule growth, like the microtubule inhibitor colchicine. Additionally, estrogen treatment rescues microtubule overgrowth in endothelin-stressed primary cardiomyocytes (Fig. 1). We examined the molecular mechanism using purified tubulin, the building block of microtubules, and estrogen. In cell-free experiments, estrogen induced long pauses in microtubule growth. Our preliminary data suggest that estrogen, like colchicine, binds to free tubulin and co-polymerizes with it, disrupting microtubule assembly independent of estrogen receptors. We conclude that estrogen’s ability to limit microtubule proliferation could contribute to sex differences in RV function in PAH. Figure 1: Primary cardiomyocytes. Microtubule proliferation in endothelin-treated cells was rescued with estradiol.

Microtubule-Dependent Alterations to Mechanical Properties and Mechanotransduction in Skeletal Muscle

In striated muscle, we discovered that force transduced through the microtubule (MT) network activates NADPH Oxidase 2 (NoX2) ROS signals (i.e., X-ROS) that target calcium (Ca2+) channels. The significance of our discovery was revealed in dystrophin-deficient heart and skeletal muscle, where MT proliferation leads to an increase in both the cytoskeletal stiffness and the mechano-activated X-ROS and Ca2+ signaling that underscores disease progression and enhanced contraction-induced injury. Our most recent work (Kerr, J.P., et. al., Nat. Comm. http://dx.doi.org/10.1038/ncomms9526) implicated the abundance of detyrosinated MT filaments, rather than MT network proliferation per se, in the amplification of X-ROS mechanotransduction and depressed contractile mechanics through the alteration of cytoskeletal stiffness. Further, reducing MT detyrosination in vivo protected against contraction-induced injury in the mdx mouse, highlighting the significance of X-ROS and detyrosinated MT in the disease process. Here we extend our discovery by examining the passive mechanical properties of single, intact skeletal myofibers using novel technology and techniques. We show that the longitudinal stress-strain relationship and shear stiffness of the muscle fiber is regulated by the abundance of detyrosinated MTs. These results are consistent with the MT network acting as a compression-resistant element both within the myofilament lattice and beneath the sarcolemmal membrane, and that detyrosination of the MT network ‘tunes’ this behavior. Our ongoing studies will inform an integrated understanding and computational modeling of the cytoskeleton's role in cell mechanics and mechanotransduction, forwarding our goal of defining novel therapeutic targets for disease.

Microtubule proliferation in right ventricular myocytes of rats with monocrotaline-induced pulmonary hypertension

Microtubules are components of the cardiac cytoskeleton that can proliferate in response to pressure-overload in animal and human heart failure. We wished to test whether there was a proliferation of the microtubule cytoskeleton in the right ventricle of rats with pulmonary hypertension induced by monocrotaline (MCT) and whether this contributed to contractile dysfunction. Male Wistar rats were injected with 60mg/kg of MCT in saline or an equivalent volume of saline (CON). MCT produced clinical signs of heart failure within 4weeks of injection. Expression of right ventricular mRNA for α-tubulin was measured by real-time reverse transcription polymerase chain reaction. Free and polymerised fractions of β-tubulin protein were assessed using Western blot analysis and immunofluorescence microscopy was used to assess tyrosinated and acetylated (stabilized) microtubules. Right ventricular myocyte contraction was measured in response to the microtubule de-polymeriser colchicine (10μmol/l for at least 1h). Compared to CON, in MCT right ventricles there was a small but statistically significant increase in the expression of mRNA for α-tubulin (P<0.001); total (P<0.05) and polymerised fraction (P<0.01) of β-tubulin protein and level of acetylated tubulin (P<0.01). However colchicine treatment did not increase the contraction of MCT myocytes (P>0.05) or affect their response to increased stimulation frequency. Our observations support the hypothesis that microtubule proliferation is a common response to pulmonary hypertension in failing right ventricles but suggest that the effect this has on contraction depends upon the specific experimental or clinical conditions that prevail and the subsequent level of microtubule proliferation.

Opposing Actions of Extracellular Signal-regulated Kinase (ERK) and Signal Transducer and Activator of Transcription 3 (STAT3) in Regulating Microtubule Stabilization during Cardiac Hypertrophy

Excessive proliferation and stabilization of the microtubule (MT) array in cardiac myocytes can accompany pathological cardiac hypertrophy, but the molecular control of these changes remains poorly characterized. In this study, we examined MT stabilization in two independent murine models of heart failure and revealed increases in the levels of post-translationally modified stable MTs, which were closely associated with STAT3 activation. To explore the molecular signaling events contributing to control of the cardiac MT network, we stimulated cardiac myocytes with an α-adrenergic agonist phenylephrine (PE), and observed increased tubulin content without changes in detyrosinated (glu-tubulin) stable MTs. In contrast, the hypertrophic interleukin-6 (IL6) family cytokines increased both the glu-tubulin content and glu-MT density. When we examined a role for ERK in regulating cardiac MTs, we showed that the MEK/ERK-inhibitor U0126 increased glu-MT density in either control cardiac myocytes or following exposure to hypertrophic agents. Conversely, expression of an activated MEK1 mutant reduced glu-tubulin levels. Thus, ERK signaling antagonizes stabilization of the cardiac MT array. In contrast, inhibiting either JAK2 with AG490, or STAT3 signaling with Stattic or siRNA knockdown, blocked cytokine-stimulated increases in glu-MT density. Furthermore, the expression of a constitutively active STAT3 mutant triggered increased glu-MT density in the absence of hypertrophic stimulation. Thus, STAT3 activation contributes substantially to cytokine-stimulated glu-MT changes. Taken together, our results highlight the opposing actions of STAT3 and ERK pathways in the regulation of MT changes associated with cardiac myocyte hypertrophy.

The effects of tubulin-binding agents on stretch-induced ventricular arrhythmias

Stretch-activated ion channels have been identified as transducers of mechanoelectric coupling in the heart, where they may play a role in arrhythmogenesis. The role of the cytoskeleton in ion channel control has been a topic of recent study and the transmission of mechanical stresses to stretch-activated channels by cytoskeletal attachment has been hypothesized. We studied the arrhythmogenic effects of stretch in 16 Langendorff-perfused rabbit hearts in which we pharmacologically manipulated the microtubular network of the cardiac myocytes. Group 1 ( n=5) was treated with colchicine, which depolymerizes microtubules, and Group 2 ( n=6) was treated with taxol, which polymerizes microtubules. Stretch-induced arrhythmias were produced by transiently increasing the volume of a fluid-filled left ventricular balloon with a volume pump driven by a computer-controlled stepper motor. Electrical events were recorded by a contact electrode which provided high-fidelity recordings of monophasic action potentials and stretch-induced depolarizations. The probability of eliciting a stretch-induced arrhythmia increased (0.22±0.11 to 0.62±0.19, p=0.001) in hearts treated with taxol (5 μM), whereas hearts treated with colchicine (100 μM) showed no statistically significant change. We conclude that proliferation of microtubules increased the arrhythmogenic effect of transient left ventricle diastolic stretch. This result indicates a possible mode of arrhythmogenesis in chemotherapeutic patients and patients exhibiting uncompensated ventricular hypertrophy. The data would indicate that the cytoskeleton represents a possible target for antiarrhythmic therapies.

Modulation of Ca 2+ Signaling by Microtubule Disruption in Rat Ventricular Myocytes and Its Dependence on the Ruptured Patch-Clamp Configuration

In the absence of hypertrophic proliferation of microtubules, microtubule disruption by colchicine does not modulate contraction of adult cardiac myocytes. However, Gomez et al (Circ Res. 2000;86:30-36) recently reported that disruption of microtubules by colchicine in ruptured patch-clamped myocytes increased I(Ca,L) density and [Ca(2+)](i) transient amplitude and depressed the response of these parameters to the beta-adrenoceptor agonist isoproterenol. These effects were ascribed to stimulation of adenylyl cyclase by increased intracellular free tubulin. In the present study, we show that in intact rat ventricular myocytes, 2 to 4 hours of exposure to 10 micromol/L colchicine had no effect on shortening or [Ca(2+)](i) transient amplitude or on the amplitude of I(Ca,L) in perforated patch-clamped cells, under basal conditions and after stimulation with 1 micromol/L isoproterenol. However, in ruptured patch-clamped myocytes, basal I(Ca,L) was 2-fold higher after treatment with colchicine compared with vehicle and, in contrast to vehicle-treated cells, I(Ca,L) did not increase in response to isoproterenol. Cell width decreased during ruptured patch-clamp experiments in colchicine-treated but not vehicle-treated myocytes. We conclude that in cells with intact sarcolemma, colchicine does not modulate Ca(2+) signaling or the response to beta stimulation. However, the combination of microtubule disruption by colchicine and the ruptured patch configuration activates I(Ca,L) and attenuates the response to beta stimulation. We propose that these effects may be due to loss of free tubulin by intracellular dialysis or to increased sensitivity to mechanical stimulation as a result of microtubule disruption. These findings have important implications for cardiomyopathies associated with decreased free tubulin or a diminished microtubular network. The full text of this article is available at http://www.circresaha.org.

Mechanisms Associated with the Negative Inotropic Effect of Deuterium Oxide in Single Rat Ventricular Myocytes

Deuterium oxide (D2O) is known to cause a negative inotropic effect in muscle although the mechanisms associated with this response in cardiac muscle are not well understood. We studied the effects of D2O in single rat ventricular myocytes in order to characterise the mechanisms associated with its negative inotropic effect and to assess its possible use as an acute modulator of microtubules. D2O rapidly reduced the magnitude of contraction in rat ventricular myocytes, and there was some recovery of contraction in the presence of D2O. Colchicine, an agent known to depolymerise microtubules, did not modify the effect of D2O. D2O decreased the L-type Ca2+ current (ICa), measured under whole cell and perforated patch clamp conditions. Slowing of the time to peak and a delay in inactivation of ICa were observed. Intracellular calcium ([Ca2+]i) and sodium ([Na+]i) were measured using the fluorescent indicators fura-2 and SBFI, respectively. The fall in contraction upon exposure to D2O was not associated with a fall in the [Ca2+]i transient; this response is indicative of a reduction in myofilament Ca2+ sensitivity. Both the [Ca2+]i transient and [Na+]i increased during the partial recovery of contraction in the presence of D2O. We conclude that a decrease in the myofilament sensitivity for Ca2+ and a reduction in Ca2+ influx via ICa are principally responsible for the negative inotropic effect of D2O in cardiac muscle. We found no evidence to explain the negative inotropic effect of D2O in terms of microtubule proliferation. In addition we suggest that acute application of D2O is not a useful procedure for the investigation of the role of microtubules in excitation-contraction coupling in cardiac muscle.

Mechanisms associated with the negative inotropic effect of deuterium oxide in single rat ventricular myocytes

Deuterium oxide (D2O) is known to cause a negative inotropic effect in muscle although the mechanisms associated with this response in cardiac muscle are not well understood. We studied the effects of D2O in single rat ventricular myocytes in order to characterise the mechanisms associated with its negative inotropic effect and to assess its possible use as an acute modulator of microtubules. D2O rapidly reduced the magnitude of contraction in rat ventricular myocytes, and there was some recovery of contraction in the presence of D2O. Colchicine, an agent known to depolymerise microtubules, did not modify the effect of D2O. D2O decreased the L-type Ca2+ current (ICa), measured under whole cell and perforated patch clamp conditions. Slowing of the time to peak and a delay in inactivation of ICa were observed. Intracellular calcium ([Ca2+]i) and sodium ([Na+]i) were measured using the fluorescent indicators fura-2 and SBFI, respectively. The fall in contraction upon exposure to D2O was not associated with a fall in the [Ca2+]i transient; this response is indicative of a reduction in myofilament Ca2+ sensitivity. Both the [Ca2+]i transient and [Na+]i increased during the partial recovery of contraction in the presence of D2O. We conclude that a decrease in the myofilament sensitivity for Ca2+ and a reduction in Ca2+ influx via ICa are principally responsible for the negative inotropic effect of D2O in cardiac muscle. We found no evidence to explain the negative inotropic effect of D2O in terms of microtubule proliferation. In addition we suggest that acute application of D2O is not a useful procedure for the investigation of the role of microtubules in excitation-contraction coupling in cardiac muscle.

Effect of the microtubule polymerizing agent taxol on contraction, Ca2+ transient and L-type Ca2+ current in rat ventricular myocytes.

1. Microtubules form part of the cytoskeleton. Their role in adult ventricular myocytes is not well understood although microtubule proliferation has previously been linked with reduced contractile function. 2. We investigated the effect of the anti-tumour drug taxol, a known microtubule polymerizing agent, on Ca2+ handling in adult rat ventricular myocytes. 3. Treatment of cells with taxol caused proliferation of microtubules. 4. In taxol-treated cells there was a reduction in the amplitude of contraction, no significant effect on the amplitude of L-type Ca2+ current, but a significant reduction in the amplitude of the Ca2+ transient. 5. Caffeine was used to release Ca2+ from the sarcoplasmic reticulum (SR). There was a significant reduction in the ratio of electrically stimulated : caffeine-induced Ca2+ transients in taxol-treated cells. This observation is consistent with the hypothesis that taxol reduces fractional SR Ca2+ release. 6. We suggest that the negative inotropic effect of taxol may, at least in part, be the result of reduced release of Ca2+ from the SR. Microtubules may be important regulators of Ca2+ handling in the heart.

Transition from mitotic apparatus to cytokinetic apparatus in pollen mitosis of the slipper orchid

Cytokinesis following asymmetrical pollen mitosis was studied in the slipper orchidCypripedium fasciculatum using techniques of immunofluorescence, confocal laser scanning, and transmission electron microscopy. Data from stereo reconstructions of double labelled preparations (microtubules/nuclei) show that the contribution of residual spindle fibers to development of the interzonal array is minor; rather, new populations of microtubules are nucleated in association with the two groups of anaphase chromosomes. As kinetochores reach the poles, trailing arms of the chromosomes and nonkinetochore microtubules are displaced outward in the equatorial zone and by early telophase the interzone is left virtually free of microtubules. The interzonal apparatus has its origin in a massive proliferation of microtubules from the polar regions and surfaces of contracting chromosomes. Each polar region appears as a hub from which microtubules radiate in a spoke-like configuration and numerous tufts of microtubules appear to emanate from margins of the chromosomes themselves. These newly organized arrays of microtubules extend to the equatorial region where they interact to form the interzonal apparatus. Increasing organization of microtubules in the interzone results in development of a typical phragmoplast configuration consisting of opposing cone-like bundles of microtubules bisected by an unstained equatorial line.

The distribution of alpha- and gamma-tubulin in fresh and aged human and mouse oocytes exposed to cryoprotectant.

The distribution of alpha- and gamma-tubulin in human and mouse oocytes has been investigated immunocytochemically. Comparisons have been made between freshly recovered and aged oocytes (both human and mouse), and also between human oocytes before and after exposure to cryoprotectant. Control fresh human oocytes had compact anastral spindles oriented orthogonal to the oolemma, with the pole adjacent to the oolemma being smaller than that directed towards the centre of the oocyte. Each pole was associated with a ring of particulate gamma-tubulin staining that extended a short distance into the body of the spindle. No alpha- and gamma-tubulin staining was found elsewhere in the ooplasm. Human oocytes which had failed to fertilize after an 18 h incubation with spermatozoa and had spent a further 6-8 h in culture showed an increased incidence of spindle abnormalities and of the proliferation of ooplasmic microtubules, which became more pronounced with age post-ovulation. The gamma-tubulin staining pattern of these aged human oocytes revealed greater staining over the whole of the spindle than in fresh oocytes. Examination of mouse oocytes aged in vitro or in vivo showed similar evidence of microtubule proliferation and disorganization, and the gamma-tubulin staining pattern was a sensitive indicator of ageing. The spindles of most fresh human oocytes exposed to 1.5 M dimethyl sulphoxide (DMSO) at 4 degrees C differed from controls in being slightly reduced in size or in having more pointed spindle poles with smaller diameters, both indications that some dismantling of the microtubules had occurred. The distribution of gamma-tubulin in these oocytes extended over more of the spindle. Restoration of DMSO-exposed oocytes to control medium at 37 degrees C for an extended period restored spindle structure to a state closely resembling that in controls. However, recovery of an exclusively polar gamma-tubulin staining did not occur. In both controls and DMSO-exposed human oocytes, chromosomes were arranged on the metaphase equatorial plate. In contrast, exposure of oocytes to 4 degrees C in the absence of DMSO caused dismantling of the spindle. It is concluded that (i) changes in microtubule organization with ageing of oocytes makes them unsuitable for use therapeutically after re-insemination or intracytoplasmic sperm injection, (ii) conditions of cryoprotectant addition previously found optimal for the stabilization of the spindle in the mouse oocyte also appear to be effective in stabilizing the spindle of the human oocyte, and (iii) the distribution of gamma-tubulin in relation to the spindle of the human oocyte appears to be sensitive to age and conditions.

Calcitonin gene-related peptide- and substance P-immunoreactive axons in the nucleus gracilis of the rat with special reference to axonal dystrophy: light and electron microscopic observations.

Calcitonin gene-related peptide (CGRP) and substance P (SP)-immunoreactive (IR) axons in the nucleus gracilis of normal rats (1-15 months of age) were studied by light and electron microscopy. Besides many CGRP-IR and SP-IR varicosities with normal appearance, we found a few swollen (nearly round or oval) varicosities with either CGRP or SP immunoreactivity. Swollen CGRP-IR varicosities were more frequently seen than SP-IR ones, appearing from 3 months of age and increasing in number and size (up to approximately 25 microns in diameter) with advancing age. At the electron microscopic (EM) level, CGRP-IR and SP-IR swollen varicosities showed dystrophic changes, i.e., many membranous dense bodies, and proliferation of microtubules and neurofilaments. CGRP-IR or SP-IR dystrophic axons also contained many mitochondria and sometimes made synaptic contacts with nonreactive dendrites (occasionally with non-IR axons). These findings suggest that the dystrophic CGRP and SP axonal profiles represent a functionally distinct subpopulation of axonal dystrophy in the nucleus gracilis and use CGRP or SP as a neuroactive substance. Using a double-immunostaining method, many of normal CGRP-IR axons were identified to be SP-IR. However, no single dystrophic varicosity was found to contain both CGRP and SP immunoreactivities. These findings suggest that CGRP and SP afferents are independently affected and progress to dystrophic changes.

Recent studies on cytotoxic, anti-HIV and antimalarial agents from plants

Abstract

Proliferation of microtubules from discrete electron-dense bodies in the ciliary dendritic portion of mechanosensory setae of copepod antennae

Sensory setae on the appendages of most arthropods contain microtubules in their innervating dendrites. Both mechano- and chemoreceptors are of ciliary origin. Mechanoreceptive dendrites typically contain more microtubules than do chemoreceptive dendrites, perhaps reflecting a role for microtubules in stiffening the setae. The presence of microtubules may also increase the sensitivity of mechanotransduction.The overall arrangement of calanoid copepod mechanoreceptors has been described elsewhere. In calanoid copepods the arrangement of microtubules in the distal ciliary segment of the dendrites is dramatic: as many as 3000 microtubules per dendrite are found in parallel rows (fig. 1,3a). These bundles of microtubules extend some distance into the setae themselves. The basal bodies within these dendrites are especially electron-dense and give rise to a circlet of 9 + 0 microtubules (fig. 2,3f)- Just distal to the circlet electron-dense material begins to form adjacent to the microtubules (fig. 3e), eventually forming elongate electron-dense bodies radiating outward (fig. 3d).

THE QUADRIPOLAR MICROTUBULE SYSTEM AND MEIOTIC SPINDLE ONTOGENY IN HORNWORTS (BRYOPHYTA: ANTHOCEROTAE)

Ontogeny of the meiotic spindle in hornworts was studied by light microscopy of live materials, transmission electron microscopy, and indirect immunofluorescence microscopy. As in monoplastidic meiosis of mosses and Isoetes, the single plastid divides twice, and the four resultant plastids migrate into the future spore domains where they organize a quadripolar microtubule system (QMS). Additionally, a unique axial microtubule system (AMS) was found to parallel the plastid isthmus at each division in meiosis, much as in the single plastid division of mitosis. This finding is used to make a novel comparison of mitotic and meiotic spindle development. The AMS contributes directly to development of the mitotic spindle, whereas ontogeny of the meiotic spindle is more complex. Nuclear division in meiosis is delayed until after the second plastid division; the first AMS disappears without spindle formation, and the two AMSs of the second plastid division contribute to development of the QMS. Proliferation of microtubules at each plastid results in the QMS consisting of four cones of microtubules interconnecting the plastids and surrounding the nucleus. The QMS contributes to the development of a functionally bipolar spindle. The meiotic spindle is comparable to a merger of two mitotic spindles. However, the first division spindle does not terminate in what would be the poles of mitosis; instead the poles converge to orient the spindle axis midway between pairs of non‐sister plastids.

The Quadripolar Microtubule System and Meiotic Spindle Ontogeny in Hornworts (Bryophyta: Anthocerotae)

Ontogeny of the meiotic spindle in hornworts was studied by light microscopy of live materials, transmission electron microscopy, and indirect immunofluorescence microscopy. As in monoplastidic meiosis of mosses and Isoetes, the single plastid divides twice, and the four resultant plastids migrate into the future spore domains where they organize a quadripolar microtubule system (QMS). Additionally, a unique axial microtubule system (AMS) was found to parallel the plastid isthmus at each division in meiosis, much as in the single plastid division of mitosis. This finding is used to make a novel comparison of mitotic and meiotic spindle development. The AMS contributes directly to development of the mitotic spindle, whereas ontogeny of the meiotic spindle is more complex. Nuclear division in meiosis is delayed until after the second plastid division; the first AMS disappears without spindle formation, and the two AMSs of the second plastid division contribute to development of the QMS. Proliferation of microtubules at each plastid results in the QMS consisting of four cones of microtubules interconnecting the plastids and surrounding the nucleus. The QMS contributes to the development of a functionally bipolar spindle. The meiotic spindle is comparable to a merger of two mitotic spindles. However, the first division spindle does not terminate in what would be the poles of mitosis; instead the poles converge to orient the spindle axis midway between pairs of non-sister plastids.

Tumor promoters stimulate hyperplasia of microtubule organizing center and inhibit DNA synthesis in cultured cells.

Chemical tumor promoters induce significant morphologic changes in several cultured cell models. In this article we describe a new effect of two potent, chemically different tumor promoters, 12-O-tetradecanoylphorbol-13-acetate (TPA) and dihydroteleocidin B (DHTB) on cultured human HeLa and melanoma cells. Using immunofluorescence microscopy, we observed that TPA and DHTB induced a dramatic increase in the size (greater than or equal to 3X normal diameter) of the centrosome, a microtubule-organizing center, within 24 h of incubation. In HeLa cells the effect was serum- and dose-dependent, was observed in 76-92% of cells within 72 h of incubation, and was associated with an increase in cytoplasm-nucleus ratio and proliferation of microtubules from the centrosome. The tumor promoters inhibited serum-induced DNA synthesis in both cell lines. Electron microscopy revealed the presence of clumps of microcentriole bodies or fragments adjacent to the intact centriole.

Human blood monocytes: characterization of negatively selected human monocytes and their suspension cell culture derivatives.

Normal human monocytes were negatively selected from leucapheresis cell suspensions by countercurrent centrifugation-elutriation in high yield with a mean purity of 93.5%. The combination of the novel methods of negative cell selection and suspension cell culture has provided the opportunity to study serially over several days the morphologic and functional changes of monocytes from a single donor as they matured in culture to typical macrophages. Human monocytes nearly double in size during the first week of culture, experiencing near daily increases in cell volume. This was associated with changes in the ultrastructure of these cells, including the development of numerous small knob-like projections on the cell membrane and the proliferation of microtubules and filamentous structures within the cell cytoplasm during the first 6 days of culture. Peroxidase activity declined during the first 4 days of culture, whereas 5'-nucleotidase activity was acquired during the first 48 h of culture. Lysozyme activity in the cultures increased form day 2 to day 6 of culture. The phagocytic capacity of monocytes for igG-coated erythrocytes increased dramatically during the first week of culture, but the cytotoxic capability of monocytes against similar targets in an antibody-dependent cytotoxicity assay declined to nearly half of base-line levels by day 2 of culture and remained at this diminished level during subsequent days of culture.

Synaptic organization of the nucleus gracilis of the cat. Experimental identification of dorsal root fibers and cortical afferents.

AbstractThe synaptic organization throughout the nucleus gracilis has been investigated in unoperated cats. Axon terminals of variable size can establish synaptic contacts with neuronal somata, dendritic processes, initial segment of axons or with other axon terminals at “complex synaptic arrangements.” Large boutons with rounded vesicles are regularly associated with smaller boutons containing flattened vesicles; the latter type of bouton forms frequently a double synapse being presynaptic to the large bouton and to the element postsynaptic to this (“complex synaptic arrangements”). Medium‐sized to small axon terminals of the “isolated” type contain primarily either rounded or flattened vesicles. These boutons are surrounded by a thin glial process which also wraps the postsynaptic element, mostly represented by a small dendritic profile. The “isolated” type of bouton seems to be more abundant in the rostral than in the caudal part of the nucleus. In all unoperated control animals altered axons and axon terminals are present. They are enlarged and display hyperplasia and dilatation of tubular profiles of smooth endoplasmic reticulum as well as proliferation of microtubules and various aspects of mitochondrial degeneration.In cats sacrificed 48 hours after section of lumbo‐sacral dorsal roots a high number of “dark” boutons are observed in various stages of degeneration. These terminals are identifiable with the large boutons containing rounded vesicles and postsynaptic to the smaller boutons with flattened vesicles. The morphology of dorsal root terminals in the nucleus gracilis is discussed in relation to that of primary afferent terminals in other central structures and to the functional aspects of axo‐axonic contacts.The sensori‐motor cortex was removed in another series of animals which were sacrificed after one to four days. As a consequence of such lesions cortical fiber terminals in the nucleus gracilis may undergo either the “dark” or the “light” type of degeneration. These terminals are of smaller size than those of primary afferents, they usually synapse on dendritic profiles of small diameter, are not involved in axo‐axonic contacts and seem to contain rounded vesicles. Therefore they can be identified with at least some of the small and medium‐sized boutons of the “isolated” type.