Caspr2-reactive antibody cloned from a mother of an ASD child mediates an ASD-like phenotype in mice.

Autism spectrum disorder (ASD) occurs in 1 in 68 births, preferentially affecting males. It encompasses a group of neurodevelopmental abnormalities characterized by impaired social interaction and communication, stereotypic behaviors and motor dysfunction. Although recent advances implicate maternal brain-reactive antibodies in a causative role in ASD, a definitive assessment of their pathogenic potential requires cloning of such antibodies. Here, we describe the isolation and characterization of monoclonal brain-reactive antibodies from blood of women with brain-reactive serology and a child with ASD. We further demonstrate that male but not female mice exposed in utero to the C6 monoclonal antibody, binding to contactin-associated protein-like 2 (Caspr2), display abnormal cortical development, decreased dendritic complexity of excitatory neurons and reduced numbers of inhibitory neurons in the hippocampus, as well as impairments in sociability, flexible learning and repetitive behavior. Anti-Caspr2 antibodies are frequent in women with brain-reactive serology and a child with ASD. Together these studies provide a methodology for obtaining monclonal brain-reactive antibodies from blood B cells, demonstrate that ASD can result from in utero exposure to maternal brain-reactive antibodies of single specificity and point toward the exciting possibility of prognostic and protective strategies.

Periodontal status in the Down's syndrome subjects living in central-eastern Italy: the effects of place of living.

AIM: The purpose of this study was to determine the influence of the place of living on periodontal status of 62 Down's syndrome (DS) subjects resident at home (DSH) or in specialized institutes (DSI) in central-eastern Italy. METHODS: The demographic characteristics of the subjects and the periodontal variables were evaluated according to their living conditions. Descriptive analyses were conducted by stratifying subjects into three age groups (0-13; 14-22; >23 years), using medians and 25th-75th percentiles to summarized data. Comparisons between DSH and DSI subjects were performed using Wilcoxon rank sum test. The effect of demographic and clinical variables on periodontal status was evaluated by means of quantile regression analysis. RESULTS: No significant differences resulted between DSH and DSI patients, when compared for gender, age and mental retardation. No significant differences were found in the periodontal variables for the subjects with 0-13 years, while DSI subjects between 14 and 22 years of age presented higher levels of plaque index, probing depth, clinical attachment loss and a lower number of surviving teeth compared to DSH subjects. When DSI and DSH groups ≥ 23 years of age were compared, no differences were observed in the periodontal conditions except for PI and the number of surviving teeth. Age, body mass index and severe mental retardation were found to be significant predictors of periodontal conditions. CONCLUSIONS: Institutionalization has a negative effect on surviving teeth number of Down's syndrome subjects. Furthermore, the home care seems to produce benefits on the periodontal conditions of DSH 14-22 years of age.

Effect of colchicine and vinblastine on the topographical separation of membrane functions.

The topographical separation of membrane functions into phagocytic and transport areas, inferred from physiological studies, is not demonstrable in cells treated with colchicine and vinblastine, alkaloids which bind to microtubular proteins.

Effect of phagocytosis on membrane transport of nonelectrolytes.

The activities of specific transport systems were determined before and after large portions of the surface membrane had been interiorized by phagocytosis of inert particles. In five separate transport systems in rabbit polymorphonuclear leukocytes (adenosine and two adenine transport systems) and alveolar macrophages (adenosine and lysine transport systems), the rate of transport was unaffected even after an estimated 35-50% of the membrane had been internalized. Studies of the kinetics of lysine and adenosine transport, exchange diffusion of lysine transport in alveolar macrophages, and the specificities of adenine transport in polymorphonuclear leukocytes indicate that the nature of the membrane transport systems is not altered by phagocytosis. Therefore the constancy of transport indicates that the number of carriers remains the same before and after phagocytosis. It was also shown that this constancy of transport did not depend on the introduction into the surface of new transport sites during phagocytosis. Therefore transport sites are preserved on the surface during the internalization of membrane which accompanies phagocytosis. The results are best explained by the concept that the membrane is mosaic in character with geographically separate transport and phagocytic sites.

Effects of serum on membrane transport. I. Separation and preliminary characterization of factors which depress lysine or stimulate adenosine transport in rabbit alveolar macrophages.

The effects of normal rabbit serum (NRS) on two transport systems in rabbit lung macrophages have been examined. A 20 min preincubation with serum was required for the effects, which were retained for at least 40 min after serum was removed. No serum was present during the transport studies. (a) Preincubation with 0.5 or 1.0% NRS resulted in depression of lysine transport to 59 +/- 2.6% (SE, 31 observations) of control levels. The activity was heat stable to 100 degrees C for 30 min and lost after dialysis. Pretreatment with serum did not alter the intracellular concentration of lysine attained when cells were then incubated with 10 mM lysine for 30 min. The relative depression of lysine transport by serum was unaltered by preloading with such high concentrations of lysine. (b) Preincubation with 5% NRS resulted in enhancement of adenosine transport by 35 +/- 2.3% (SE, 60 observations). Activity was stable to heating at 65 degrees C for 40 min but lost at 100 degrees C for 20 min. It was nondialyzable. Total radioactivity accumulated after 30 min incubation with 1 mM adenosine was unaffected by serum pretreatment. The two activities were separable by passage over Sephadex G25.

Studies on the pathogenesis of fever. 18. Activation of leukocytes for pyrogen production.

Blood leukocytes, in contrast to exudate leukocytes, release little or no pyrogen when incubated in 0.15 M NaCl unless previously activated by exposure to endotoxin or to a protein activator that is present in acute exudate fluid. The activation process, which also occurs during phagocytosis, involves the synthesis of cellular protein, presumably related to the pyrogen molecule. Evidence is presented that generation of pyrogen in sterile inflammatory lesions depends on both the activator and the anaerobic conditions in the exudate fluid.

Intracellular elevations of free calcium induced by activation of histamine H1 receptors in interphase and mitotic HeLa cells: hormone signal transduction is altered during mitosis.

A broad range of membrane functions, including endocytosis and exocytosis, are strongly inhibited during mitosis. The underlying mechanisms are unclear, however, but will probably be important in relation to the mitotic cycle and the regulation of surface phenomena generally. A major unanswered question is whether membrane signal transduction is altered during mitosis; suppression of an intracellular calcium [( Ca2+]i) transient could inhibit exocytosis; [Ca2+]i elevation could disassemble the mitotic spindle. Activation of the histamine H1 receptor interphase in HeLa cells is shown here by Indo-1 fluorescence to produce a transient elevation of [Ca2+]i. The [Ca2+]i transient consists of an initial sharp rise that is at least partially dependent on intracellular calcium followed by an elevated plateau that is absolutely dependent on extracellular calcium. The [Ca2+]i transient is completely suppressed by preincubation with the tumor promoter, phorbol myristate acetate, but is unaffected by preincubation with pertussis toxin (islet-activating protein). In mitotic (metaphase-arrested) HeLa cells, the [Ca2+]i transient is largely limited to the initial peak. Measurement of 45Ca2+ uptake shows that it is stimulated by histamine in interphase cells, but not in mitotics. We conclude that the histamine-stimulated generation of the second messenger, [Ca2+]i, in mitotic cells is limited by failure to activate a sustained calcium influx. The initial phase of calcium mobilization from intracellular stores is comparable to that in interphase cells. Hormone signal transduction thus appears to be altered during mitosis.

Packing volume of sedimented microtubules: regulation and potential relationship to an intracellular matrix.

To determine the contribution of microtubules to a hypothetical intracellular matrix, we have analyzed the space occupied by microtubules in vitro. Taxol-stabilized microtubules assembled from purified (three-times-cycled) bovine brain microtubule protein were pelleted by centrifugation under standardized conditions. The specific volume of the pellet, defined as the microliter volume per milligram protein, was 22.4. As suggested by others, this volume was strongly dependent on microtubule-associated proteins (MAPs), as shown by quantitation of the effects of purified MAP supplementation on specific volume. The specific volumes of microtubule pellets stripped of MAPs by high salt or chymotryptic digestion approached the mathematically optimal (least occupied space) and increased 14-fold with the highest MAP concentrations employed. Packing was also dependent on pH. Specific volumes comparable to those of MAP-depleted microtubules were attainable at pH's from 5.5 to 6.0, and specific volumes more than doubled at pH 7.5. MAP content was unaffected by pH. We present a theoretical analysis that suggests that as microtubules are centrifuged the mixture behaves as a liquid crystal. With packing, the mixture undergoes an isotropic-nematic phase transition in which the microtubules become oriented principally as parallel rods, mimicking their orientation in vivo. From the known concentration of microtubules in vivo, it can be inferred from our measurements that in some cells a large fraction, perhaps 40-50% of the cytosolic volume, is occupied by microtubules that form a mechanically irreducible space. Further theoretical analysis employing Ogston's formulation of the penetrability of fibrous networks suggests that the space between microtubules (in contrast to the extracellular matrix) imposes little barrier to the diffusion of macromolecules. A microtubule array thus achieves mechanical stability without affecting transport by diffusion. The space can accommodate other fibrous networks that could then affect transport, and, as we show, the space itself may be regulated by MAP content and intracellular pH.

Interaction of microtubule proteins with phospholipid vesicles.

We have examined the interaction of unilamellar dimyristoyl phosphatidylcholine liposomes with the high-speed supernate of brain homogenate and with tubulin purified through one or two cycles of microtubule assembly-disassembly. Tubulin and certian high molecular weight proteins are selectively adsorbed from these mixtures onto liposomes. The composition of adsorbed proteins is similar to that obtained during corresponding cycles of microtubule assembly, suggesting the equivalency of these processes. Adsorption induces stacking and/or fusion of liposomes into multilamellar structures indicating strong protein-lipid interaction. In addition, liposome-adsorbed tubulin forms extensive intermolecular disulfide bridges that are inert to reducing agents in the aqueous medium. The observations form a basis for further study of the distribution, function, and properties of membrane-bound tubulin.

Analogous ultrastructure and surface properties during capping and phagocytosis in leukocytes.

Ultrastructural analyses have revealed striking similarities between Concanavalin A capping and phagocytosis in leukocytes. Both processes involve extensive membrane movement to form a protuberance or pseudopods; a dense network of microfilaments is recruited into both the protuberance and the pseudopods; microtubules are disassembled either generally (capping) or in the local region of the pseudopods (phagocytosis); and cells generally depleted of microtubules by colchicine show polarized phagocytosis via the microfilament-rich protuberance rather than uniform peripheral ingestion of particles via individual pseudopods. Cap formation can thus be viewed as occurring as an exaggeration of the same ultrastructural events that mediate phagocytosis. Similar changes in cell surface topography also accompany capping and phagocytosis. Thus, in nonfixed cells, Concanavalin A-receptor complexes aggregate into the region of the protuberance in colchicine-treated leukocytes (conventional capping) or into the region of pseudopod formation in phagocytizing leukocytes. In the latter case, the movement of lectin-receptor complexes occurs from membrane overlying peripheral microtubules into filament-rich pseudopods that exclude microtubules. These data provide evidence against a role for microtubules as "anchors" for lectin receptors. Rather, they indicate a preferential movement of cell surface Concanavalin A-receptor complexes towards areas of extensive (the protuberance) or localized (pseudopods) microfilament concentration. In conventional capping, Concanavalin A must be added to the colchicine-treated cells before fixation in order to demonstrate movement of receptors from a diffuse distribution into the protuberance. However, Convanavalin A receptors are enriched in the membrane associated with phagocytic particles as compared to the remaining membrane. This particle-induced redistribution of receptors is particularly prominent in colchicine-treated cells that phagocytize and are then fixed and Concanavalin A labeled; both lectin receptors and beads are concentrated over the protuberance. Thus, the final analogy between conventionally capped and phagocytic cells is that in both cases the properties of the plasma membrane in regions of microfilament concentration are modified by Concanavalin A itself (capping) or by the phagocytized particle, to limit locally the diffusion of Concanavalin A receptors.

Surface functions during mitosis. II. Quantitation of pinocytosis and kinetic characterization of the mitotic cycle with a new fluorescence technique.

The profound depression of fluid pinocytosis observed in mitotic cells (Berlin, R. D., et al. 1978. Cell. 15:327--341) is documented by quantitative microspectrofluorimetry of fluorescein-labeled dextran uptake in single cells. In J774.2 macrophages, fluid pinocytosis is reduced 30-fold during mitosis. The depression develops within 30 s of entry into prophase and recovers with equal rapidity upon emergence from telophase into G1. This characteristic pattern of fluid pinocytosis forms the basis of a new method for detailed kinetic analysis of the duration of mitosis and its phases. The analysis is applied to the J774.2 macrophage cell line but should be generally applicable to other lines. Effects of ouabain and colchicine on the length of mitosis and its phases are evaluated, revealing a selective prolongation of metaphase by ouabain and suggesting a role for microtubules in the transition from G2 into mitosis.

A reevaluation of the structure of purified tubulin in solution: evidence for the prevalence of oligomers over dimers at room temperature.

We studied the molecular form of tubulin in solution by ultrafiltration, nondenaturing electrophoresis, and chemical cross-linking. Our results are not consistent with the generally-held belief that tubulin in solution is a 110,000-mol-wt dimer. Rather, tubulin in solution consists of small oligomers; dimers are a minority species. The small proportion of dimers was readily apparent from ultrafiltration experiments. We first compared the filterability (defined as the ratio of protein concentration in filtrate to that applied to the filter) of phosphocellulose-purified tubulin (PC-tubulin) with aldolase (142,000 mol wt). Using an Amicon XM 300 filter, the filterability of PC-tubulin at room temperature and at a concentration of 0.5 mg/ml was only 0.12, whereas under the same conditions the filterability of aldolase was 0.60. We determined the average effective molecular weight of tubulin from its filterability on XM 300 filters calibrated with standard proteins. At room temperature, PC-tubulin at 0.5 mg/ml had an effective molecular weight of approximately 300,000. This molecular weight was significantly reduced at 10 degrees C, indicating that oligomers dissociated at low temperatures. Oligomers were also demonstrated by chemical cross-linking using glutaraldehyde, dimethyl suberimidate, and bis[2-(succinimidooxycarbonyoxy)ethyl] sulfone. In addition, PC-tubulin ran as a series of discrete bands in a nondenaturing PAGE system at alkaline pH. Quantitative examination of the mobilities of these bands and of standard proteins revealed that the bands represented a series of oligomeric forms. Similar electrophoretic patterns were observed in solutions of tubulin containing microtubule-associated proteins (MAPs) but with a shift to a greater proportion of higher oligomers. Nondenaturing PAGE at pH 8.3 showed that a shift towards higher oligomers also occurred in the absence of MAPs as the concentration of tubulin was increased. This concentration-dependence of oligomerization at room temperature was further demonstrated by ultrafiltration. When solutions of PC-tubulin at concentrations less than 0.25 mg/ml were ultrafiltered, filterability increased as concentration decreased. Quantitative studies of filterability following progressive dilution or concentration showed that this process was completely and rapidly reversible. A diffuse pattern of PC-tubulin on nondenaturing PAGE at pH 7 was observed and is consistent with a mixture of oligomers in rapid equilibrium.(ABSTRACT TRUNCATED AT 400 WORDS)

Microtubule assembly and disassembly at alkaline pH.

Although it is now apparent that the intracellular pH may rise considerably above neutrality under physiological conditions, information on the effect of alkaline pH on microtubule assembly and disassembly is still quite fragmentay. We have studied the assembly/disassembly of bovine brain microtubule protein at alkaline pH in vitro. When microtubules are assembled to a new steady state at pH less than 7 and pH is then made more alkaline, they undergo a rapid disassembly to a new steady state. This disassembly is reversed by acidification. The degree of disassembly is determined largely by the pH- dependence of the critical concentration, which increases five to eight times, from pH 7 to 8. A fraction of assembly-incompetent tubulin is identified that increases with pH, but its incompetency is largely reversed with acidification. Measurements of microtubule lengths are used to indicate that disassembly occurs by uniform shortening of microtubules. A comparison of shortening by alkalinization with dilution suggests that the intrinsic rate of disassembly is accelerated by increasing pH. The capacity for initiating assembly is progressively lost with incubation at alkaline pH (although some protection is afforded by sulfhydryl-reducing agents). However, direct assembly from depolymerized mixtures is possible at least up to pH 8.3, and the steady state achieved at these alkaline pH values is stable. Such preparations are readily disassembled by cold and podophyllotoxin (PLN). Disassembly induced by PLN is also markedly enhanced at alkaline pH, suggesting a corresponding enhancement of "treadmilling." The implications of physiological events leading to alkaline shifts of pH for microtubule assembly/disassembly are discussed, particularly in the light of recent hypotheses regarding treadmilling and its role in controlling the distribution of microtubules in vivo.

Control of cell volume in the J774 macrophage by microtubule disassembly and cyclic AMP.

We have explored the possibilities that cell volume is regulated by the status of microtubule assembly and cyclic AMP metabolism and may be coordinated with shape change. Treatment of J774.2 mouse macrophages with colchicine caused rapid microtubule disassembly and was associated with a striking increase (from 15-20 to more than 90 percent) in the proportion of cells with a large protuberance at one pole. This provided a simple experimental system in which shape changes occurred in virtually an entire cell population in suspension. Parallel changes in cell volume could then be quantified by isotope dilution techniques. We found that the shape change caused by colchicine was accompanied by a decrease in cell volume of approximately 20 percent. Nocodozole, but not lumicolchicine, caused identical changes in both cell shape and cell volume. The volume loss was not due to cell lysis nor to inhibition of pinocytosis. The mechanism of volume loss was also examined. Colchicine induced a small but reproducible increase in activity of the ouabain-sensitive Na(+), K(+)-dependent ATPase. However, inhibition of this enzyme/transport system by ouabain did not change cell volume nor did it block the colchicines-induced decrease in volume. One the other hand, SITS (4'acetamido, 4-isothiocyano 2,2' disulfonic acid stilbene), an inhibitor of anion transport, inhibited the effects of colchicines, thus suggesting a role for an anion transport system in cell volume regulation. Because colchicine is known to activate adenylate cyclase in several systems and because cell shape changes are often induced by hormones that elevate cyclic AMP, we also examined the effects of cyclic AMP on cell volume. Agents that act to increase syclic AMP (cholera toxin, which activates adenylate cyclase; IBMX, and inhibitor of phosphodiesterase; and dibutyryl cyclic AMP) all caused a volume decrease comparable to that of colchicine. To define the effective metabolic pathway, we studied two mutants of J774.2, one deficient in adenylate cyclase and the other exhibiting markedly reduced activity of cyclic AMP-dependent protein kinase. Cholera toxin did not produce a volume change in either mutant. Cyclic AMP produced a decrease in the cyclase-deficient line comparable to that in wild type, but did not cause a volume change in the kinase- deficient line. This analysis established separate roles for cyclic AMP and colchicine. The volume decrease induced by cyclic AMP requires the action of a cyclic AMP-dependent protein kinase. Colchicine, on the other hand, induced a comparable volume change in both mutants and wild type, and thus does not require the kinase.

Surface functions during mitosis. III. Quantitative analysis of ligand-receptor movement into the cleavage furrow: diffusion vs. flow.

The surface distribution of concanavalin A (Con A) bound to cell membrane receptors varies dramatically as a function of mitotic phase. The lectin is distributed diffusely on cells labeled and observed between mid-prophase and early anaphase, whereas cells observed in late anaphase or telophase demonstrate a marked accumulation of Con A-receptor complexes over the developing cleavage furrow (Berlin, Oliver, and Walter. 1978. Cell. 15:327-341). In this report, we first use a system based on video intensification fluorescence microscopy to describe the simultaneous changes in cell shape and in lectin-receptor complex topography during progression of single cells through the mitotic cycle. The video analysis establishes that fluorescein succinyl Con A (F-S Con A)-receptor complex redistribution begins coincident with the first appearance of the cleavage furrow and is essentially complete within 2-3 min. This remarkable redistribution of surface fluorescence occurs during only a modest change in cell shape from a sphere to a belted cylinder. It reflects the translocation of complexes and not the accumulation of excess labeled membrane in the cleavage furrow: first, bound fluorescent cholera toxin which faithfully outlines the plasma membrane is not accumulated in the cleavage furrow, and, second, electron microscopy of peroxidase-Con A labeled cells undergoing cleavage shows that there is a high linear density of lectin within the furrow while Con A is virtually eliminated from the poles. The rate of surface movement of F-S Con A was quantitated by photon counting during a repetitive series of laser-excited fluorescence scans across dividing cells. Results were analyzed in terms of two alternative models of movement: a flow model in which complexes moved unidirectionally at constant velocity, and a diffusion model in which complexes could diffuse freely but were trapped at the cleavage furrow. According to these models, the observed rates of accumulation were attainable at either an effective flow velocity of approximately 1 micron/min, or an effective diffusion coefficient of approximately 10(-9) cm2/s. However, in separate experiments the lectin-receptor diffusion rate measured directly by the method of fluorescence recovery after photobleaching (FRAP) on metaphase cells was only approximately 10(-10) cm2/s. Most importantly, photobleaching experiments during the actual period of F-S Con A accumulation showed that lectin-receptor movement during cleavage occurs unidirectionally. These results rule out diffusion and make a process of oriented flow of ligand-receptor complexes the most likely mechanism for ligand-receptor accumulation in the cleavage furrow.

Carbamycholine prevents giant granule-formation in cultured fibroblasts from beige (Chediak-Higashi) mice.

Primary embryonic fibroblasts isolated from beige (Chediak-Higashi) mice develop pathognomonic giant granules in vitro. Inclusion in the culture medium of carbamylcholine (carbachol) or phorbol myristate acetate (PMA) results in the generation of morphologically normally granules. Chediak-Higashi fibroblasts are highly susceptible to shape changes induced by colchicine. This abnormal property is also corrected by carbachol and PMA.

Effects of glutathione-oxidizing agents on microtubule assembly and microtubule-dependent surface properties of human neutrophils.

In human peripheral blood polymorphonuclear leukocytes and lymphocytes, GSH-oxidizing agents promote the movement of surface-bound concanavalin A (Con A) into caps and inhibit the assembly of microtubules (MT) that is normally induced by Con A binding. Con A capping and inhibition of MT assembly occur when GSH levels in cell suspensions are decreased by 30-70%, and return to GSH to control levels is accompanied by the appearance of cytoplasmic MT and by inhibition of the capping response with Con A. Oxidation of GSH markedly stimulates the hexose monophosphate shunt, and regeneration of GSH occurs rapidly. The data indicate that MT cannot be assembled or maintained in the face of decreased GSH levels. Thus, GSH homeostasis becomes critical during physiological events such as phagocytosis which simultaneously induce the assembly of MT and the production of agents like H2O2 that can oxidize GSH.

Polarization of endocytosis and receptor topography on cultured macrophages.

In the 1774.2 macrophage cell line, microtubule disassembly by colchicine causes the polarization of membrane functions ane structure. Colchicine-treated cells develop a bulge or protuberance that is bordered by microvillous membrane. The protuberance is the site of concanavalin A cap formation. The fluid pinocytosis of horseradish peroxidase and of fluorescein- and rhodamine-conjugated high molecular-weight dextrans, the adsorptive pinocytosis of concanavalin A, and the concentration and phagocytosis at 37 degrees C of a range of phagocytic particles (IgG- and complement-opsonized erythrocytes, complement-opsonized zymosan, latex shpres, albumin-stabilized oil droplets) are all similarly restricted to the protuberance. A reduction in the rate of dextran pinocytosis, determined by fluorimetry, and reductions in phagocytic rates for oil emulsion and IgG-opsonized erythrocytes accompany the polarization of endocytic activity in colchicine-trated 1774.2 macrophages. Membrane receptors for phagocytic particles are not confined to the protuberance but rather may display their own unique topographical asymmetry. The inherent topography of receptors was inferred from particle distribution under conditions that limit particle-receptor redistribution (after labeling at 4 degrees C or a very brief incubation at 37 degrees C). Under these restrictive conditions, latex binding sites were detected over the whole membrane whereas receptors for IgG-opsonized erythrocytes, aggregated IgG, complement-opsonized erythrocytes, and complement-opsonized zymosan were excluded from the protuberance. Thus, functional (endocytosis) and structural (inherent receptor distribution) analyses of membrane topography define different patterns of asymmetry in protuberant cells. The asymmetry induced in 1774.2 macrophages by colchicine is highly analogous to the functional and structural polarity of epithelial cells. Exploration of this analogy may provide insight into the development of polarized epithelia and, more generally, into mechanisms by which specialized areas of membrane are established.

A novel mitotic spindle pole component that originates from the cytoplasm during prophase.

Several unique aspects of mitotic spindle formation have been revealed by investigation of an autoantibody present in the serum of a patient with the CREST (calcinosis, Raynaud's phenomenon, esophageal dysmotility, schlerodacytyly, and telangiectasias) syndrome. This antibody was previously shown to label at the spindle poles of metaphase and anaphase cells and to be absent from interphase cells. We show here that the serum stained discrete cytoplasmic foci in early prophase cells and only later localized to the spindle poles. The cytoplasmic distribution of the antigen was also seen in nocodazole-arrested cells and prophase cells in populations treated with taxol. In normal and taxol-treated cells, the microtubules appeared to emanate from the cytoplasmic foci and polar stain, and in cells released from nocodazole block, microtubules regrew from antigen-containing centers. This characteristic distribution suggests that the antigen is part of a microtubule organizing center. Thus, we propose that a prophase originating polar antigen functions in spindle pole organization as a coalescing microtubule organizing center that is present only during mitosis. Characterization of the serum showed reactions with multiple proteins at 115, 110, 50, 36, 30, and 28 kD. However, affinity-eluted antibody from the 115/110-kD bands was shown to specifically label the spindle pole and cytosolic foci in prophase cells.

Effects of colchicine, cytochalasin B, and 2-deoxyglucose on the topographical organization of surface-bound concanavalin A in normal and transformed fibroblasts.

The distribution of surface-bound concanavalin A on the membranes of 3T3, and simian virus 40-transformed 3T3 cultured mouse fibroblasts was examined using a shadow-cast replica technique with a hemocyanin marker. When cells were prefixed in paraformaldehyde, the binding site distribution was always random on both cell types. On the other hand, labeling of transformed cells with concanavalin A (Con A) and hemocyanin at 37 degrees C resulted in the organization of Con A binding sites (CABS) into clusters (primary organization) which were not present on the pseudopodia and other peripheral areas of the membrane (secondary organization). Treatment of transformed cells with colchicine, cytochalasin B, or 2-deoxyglucose did not alter the inherent random distribution of binding sites as determined by fixation before labeling. However, these drugs produced marked changes in the secondary (but not the primary) organization of CABS on transformed cells labeled at 37 degrees C. Colchicine treatment resulted in the formation of a caplike aggregation of binding site clusters near the center of the cell, whereas cytochalasin B and 2-deoxyglucose led to the formation of patches of CABS over the entire membrane, eliminating the inward displacement of patches observed on untreated cells. The distribution of bound Con A on normal cells (3T3) at 37 degrees C was always random, in both control and drug-treated preparations. Pretreatment of cells with Con A enhanced the effect of colchicine on cell morphology, but inhibited the morphological effects of cytochalasin B. The mechanisms that determine receptor movement and disposition are discussed.