Ecological and evolutionary drivers of phenotypic and genetic variation in the European crabapple [Malus sylvestris (L.) Mill.], a wild relative of the cultivated apple.

BACKGROUND AND AIMS: Studying the relationship between phenotypic and genetic variation in populations distributed across environmental gradients can help us to understand the ecological and evolutionary processes involved in population divergence. We investigated the patterns of genetic and phenotypic diversity in the European crabapple, Malus sylvestris, a wild relative of the cultivated apple (Malus domestica) that occurs naturally across Europe in areas subjected to different climatic conditions, to test for divergence among populations. METHODS: Growth rates and traits related to carbon uptake in seedlings collected across Europe were measured in controlled conditions and associated with the genetic status of the seedlings, which was assessed using 13 microsatellite loci and the Bayesian clustering method. Isolation-by-distance, isolation-by-climate and isolation-by-adaptation patterns, which can explain genetic and phenotypic differentiation among M. sylvestris populations, were also tested. KEY RESULTS: A total of 11.6 % of seedlings were introgressed by M. domestica, indicating that crop-wild gene flow is ongoing in Europe. The remaining seedlings (88.4 %) belonged to seven M. sylvestris populations. Significant phenotypic trait variation among M. sylvestris populations was observed. We did not observe significant isolation by adaptation; however, the significant association between genetic variation and the climate during the Last Glacial Maximum suggests that there has been local adaptation of M. sylvestris to past climates. CONCLUSIONS: This study provides insight into the phenotypic and genetic differentiation among populations of a wild relative of the cultivated apple. This might help us to make better use of its diversity and provide options for mitigating the impact of climate change on the cultivated apple through breeding.

Contrasted patterns of selection since maize domestication on duplicated genes encoding a starch pathway enzyme.

Maize domestication from teosinte (Zea mays ssp. parviglumis) was accompanied by an increase of kernel size in landraces. Subsequent breeding has led to a diversification of kernel size and starch content among major groups of inbred lines. We aim at investigating the effect of domestication on duplicated genes encoding a key enzyme of the starch pathway, the ADP-glucose pyrophosphorylase (AGPase). Three pairs of paralogs encode the AGPase small (SSU) and large (LSU) subunits mainly expressed in the endosperm, the embryo and the leaf. We first validated the putative sequence of LSU(leaf) through a comparative expression assay of the six genes. Second, we investigated the patterns of molecular evolution on a 2 kb coding region homologous among the six genes in three panels: teosintes, landraces, and inbred lines. We corrected for demographic effects by relying on empirical distributions built from 580 previously sequenced ESTs. We found contrasted patterns of selection among duplicates: three genes exhibit patterns of directional selection during domestication (SSU(end), LSU(emb)) or breeding (LSU(leaf)), two exhibit patterns consistent with diversifying (SSU(leaf)) and balancing selection (SSU(emb)) accompanying maize breeding. While patterns of linkage disequilibrium did not reveal sign of coevolution between genes expressed in the same organ, we detected an excess of non-synonymous substitutions in the small subunit functional domains highlighting their role in AGPase evolution. Our results offer a different picture on AGPase evolution than the one depicted at the Angiosperm level and reveal how genetic redundancy can provide flexibility in the response to selection.

Structural and functional effects of hydrostatic pressure on centrosomes from vertebrate cells.

In an attempt to better understand the role of centrioles in vertebrate centrosomes, hydrostatic pressure was applied to isolated centrosomes as a means to disassemble centriole microtubules. Treatments of the centrosomes were monitored by analyzing their protein composition, ultrastructure, their ability to nucleate microtubules from pure tubulin, and their capability to induce parthenogenetic development of Xenopus eggs. Moderate hydrostatic pressure (95 MPa) already affected the organization of centriole microtubules in isolated centrosomes, and also impaired microtubule nucleation. At higher pressure, the protein composition of the peri-centriolar matrix (PCM) was also altered and the capacity to nucleate microtubules severely impaired. Incubation of the treated centrosomes in Xenopus egg extract could restore their capacity to nucleate microtubules after treatment at 95 MPa, but not after higher pressure treatment. However, the centriole structure was in no case restored. It is noteworthy that centrosomes treated with mild pressure did not allow parthenogenetic development after injection into Xenopus eggs, even if they had recovered their capacity to nucleate microtubules. This suggested that, in agreement with previous results, centrosomes in which centriole architecture is impaired, could not direct the biogenesis of new centrioles in Xenopus eggs. Centriole structure could also be affected by applying mild hydrostatic pressure directly to living cells. Comparison of the effect of hydrostatic pressure on cells at the G1/S border or on the corresponding cytoplasts suggests that pro-centrioles are very sensitive to pressure. However, cells can regrow a centriole after pressure-induced disassembly. In that case, centrosomes eventually recover an apparently normal duplication cycle although with some delay.

Establishment of a human malignant fibrous histiocytoma cell line, COMA. Characterization By conventional cytogenetics, comparative genomic hybridization, and multiplex fluorescence In situ hybridization.

The human COMA cell line has been established from a storiform pleomorphic malignant fibrous histiocytoma (MFH). As expected for this tumor type, a very complex karyotype was observed after R-banding analysis. An extensive analysis by 24-color painting, comparative genomic hybridization (CGH), and fluorescence in situ hybridization (FISH) was performed. Twelve complex marker chromosomes recurrently observed were clearly identified; among them, three were systematically present in all analyzed metaphases. Amplifications detected by CGH were refined by FISH with probes specific for various candidate loci. A significant aneuploidy and numerous micronuclei were observed, which could be related to the anomalies of centriole numbers detected in a proportion of cells. Such an analysis, performed on a series of MFH cell lines, would allow the delineation of the genomic alterations specific for the oncogenesis or progression of this complex tumor type or both.

The nucleolar phosphoprotein B23 redistributes in part to the spindle poles during mitosis.

B23 is a major phosphoprotein in the interphasic nucleolus where it is involved in the assembly of pre-ribosomes. Using several cultured animal cells, we report that, in addition to the known redistribution of the protein during mitosis, B23 also becomes associated with mitotic spindle poles starting from early prometaphase onwards. Colocalization of B23 with the protein NuMA (Nuclear Mitotic Apparatus protein) was studied in mitotic cells and taxol-arrested cells. During the onset of mitosis, we observed that a fraction of B23 associates with, and dissociates from, the poles later than NuMA. At metaphase, both proteins are colocalized at the poles. The polar redistribution of both B23 and NuMA is mediated by microtubules. In taxol-treated cells, B23 is associated with the microtubule minus ends in the center of mitotic asters together with NuMA. Association of B23 with microtubule minus ends of mitotic asters was further confirmed with an in vitro assay, where B23 was found by western blotting to co-sediment with taxol-induced microtubule asters formed in a mitotic cell extract. Immunolabeling demonstrated that B23 and NuMA were both present at the center of the asters. Furthermore, an additional hyperphosphorylated form of B23 appeared when microtubule asters formed and associated with the asters. Immunodepletion of B23 from the mitotic extract revealed that taxol-induced microtubule asters were still observed in B23-immunodepleted mitotic extract, indicating that the presence of B23 at the poles is unlikely to be essential for spindle formation or stabilisation.

Aminopeptidase B: a processing enzyme secreted and associated with the plasma membrane of rat pheochromocytoma (PC12) cells.

Aminopeptidase B (Ap-B) is a Zn2+-dependent exopeptidase which selectively removes Arg and/or Lys residues from the N terminus of several peptide substrates. Isolated and characterized from rat testes, this ubiquitous enzyme may participate in the final stages of precursor processing mechanisms. To test this hypothesis, we have investigated the secretion and subcellular localization of this enzyme in a rat cell line of pheochromocytoma (PC12 cells). By using a combination of biochemical and immunocytochemical methods, the following observations were made: (i) the level of aminopeptidase B detectable in the cell culture medium increased with time; (ii) 8-bromo-adenosine 3'-5'-cyclic monophosphate and the Ca2+ ionophore A23187 both stimulated enzyme liberation in the culture medium; (iii) brefeldin A, an inhibitor of vesicular transport from the endoplasmic reticulum to the Golgi apparatus, decreased enzyme secretion in a time-dependent manner; (iv) whereas nocodazole, a microtubule depolymerizing agent, inhibited enzyme secretion, cytochalasin D, a microfilament disruption agent, had no effect on released aminopeptidase B level; (v) immunofluorescence demonstrated the presence of aminopeptidase B in the Golgi apparatus; (vi) immunofluorescence, electron microscopy and tests of enzyme activity on intact cells showed an association of the peptidase with the external face of the plasma membrane. Together these data strongly argued in favour of the enzyme secretion by PC12 cells. It is concluded that aminopeptidase B may participate in processing events occurring either during its intracellular transport along the secretory pathway or at the plasma membrane level, or both.

Interaction of a Golgi-associated kinesin-like protein with Rab6.

Rab guanosine triphosphatases regulate vesicular transport and membrane traffic within eukaryotic cells. Here, a kinesin-like protein that interacts with guanosine triphosphate (GTP)-bound forms of Rab6 was identified. This protein, termed Rabkinesin-6, was localized to the Golgi apparatus and shown to play a role in the dynamics of this organelle. The carboxyl-terminal domain of Rabkinesin-6, which contains the Rab6-interacting domain, inhibited the effects of Rab6-GTP on intracellular transport. Thus, a molecular motor is a potential effector of a Rab protein, and coordinated action between members of these two families of proteins could control membrane dynamics and directional vesicular traffic.

Entry and distribution of fluorescent antiproliferative heparin derivatives into rat vascular smooth muscle cells: comparison between heparin-sensitive and heparin-resistant cultures.

We studied the binding and entry of fluorescein (FITC)-labeled heparin derivatives into rat aortic smooth muscle cells (SMC) by confocal microscopy. FITC-labeled heparin fractions or FITC-labeled SR 80037A, a potent antiproliferative heparin derivative (Bârzu et al., Eur. J. Pharmacol., 219:225-233 1992), were prepared and their antiproliferative activity was confirmed. By incubating SMC with FITC-labeled heparins, a specific cell-associated fluorescence was found. Cellular fluorescence was mostly located around the nucleus and at the level of cell contacts or cell adhesion. The fluorescence was displaced neither by chasing with excess of unlabeled heparins nor by washing with 1 M NaCl, which proved that labeled heparins had been internalized by SMC. Kinetics of internalization of FITC-heparins suggested receptor-mediated endocytosis of heparins by SMC. Double labeling of SMC with biotinylated Concanavalin A and FITC-SR 80037A also indicated that heparin derivative enters the endocytic pathway. The process was accelerated when serum was present in the incubation medium. Treatment of cells with chloroquine (50 microM) induced accumulation of FITC-SR 80037A in the late endosomes, around the nucleus. No fluorescence labeling could be evidenced inside the nucleus. Neither electron microscopy nor cell fractionation experiments performed with SMC previously incubated with [3H]-heparin were able to ascertain nuclear uptake of heparin, as proposed by other workers (Busch et al., Cell Biol., 116:31-42; 1992; Sing et al., Drug Dev. Res., 29:129-136 1993). The cell-associated fluorescence was very weak in SMC resistant to the antiproliferative activity of heparin, selected by long-term heparin treatment (HT-SMC) as previously shown [Bârzu et al., J. Cell. Physiol., 160:239-248, 1994]. The HT-SMC differed from control SMC with regard to expression of extracellular matrix proteins. These cells exhibited very low expression of fibronectin and prevalent expression of laminin and synthesized less cell-associated glycosaminoglycans. From our results, the following conclusions can be drawn: (1) the antiproliferative heparins are bound and internalized by SMC without being taken up into the nucleus; (2) there is a correlation between the binding and/or the internalization process and the sensitivity of SMC to the antiproliferative activity of heparins; and (3) selection of heparin-resistant SMC by long treatment with heparin results in particular growth pattern of SMC (absence of focal overgrowth), associated with changes in the expression of the extracellular matrix components (fibronectin, laminin, and cell-bound glycosaminoglycans).

Defined glycosaminoglycan motifs have opposite effects on neuronal polarity in vitro.

We previously reported that heparan sulfates enhance axonal outgrowth and inhibit dendrite elongation, whereas dermatan sulfates favor the development of both axons and dendrites. The present study focuses on the activity of small synthetic heparan or dermatan sulfate-like compounds. We found three heparan sulfate-like and three dermatan sulfate-like sugars that mimic the morphological effects of the high-molecular-weight natural glycosaminoglycans. Indeed, heparan sulfate-like compounds enhance axonal maturation and inhibit dendrite growth whereas the active sugars from the dermatan sulfate series act primarily on the elongation of cortical dendrites. The effect of dermatan sulfate-like sugars on cortical dendrite growth is only observed on the subpopulation of neurons with an established axon. We also studied the effects of the synthetic sugars on motoneurons. We found that the response of motoneurons to heparan sulfate-like compounds is indistinguishable from that of cortical neurons but that dermatan sulfate-like sugars do not enhance the development of motoneuron dendrites. The distinct effects of the two types of sugars and the fact that their activity only requires a short period of contact with the cells suggest the existence of specific binding sites for dermatan-like and heparan-like compounds. This possibility is reinforced by the fact that the binding and internalization of natural heparin fragments by neurons in culture is competitively inhibited by synthetic heparan sulfate-like derivatives, but not by dermatan sulfate-like derivatives.

Production and characterization of polyclonal anti-idiotypic anti-TRH antibodies: application to the study of pituitary TRH receptor.

cDNA encoding the thyrotropin-releasing hormone receptor (TRH-R) was recently cloned in rat pituitary prolactin cells and in mouse thyrotropes. The molecular weights of the protein sequences obtained are 46.6 and 44.5 kD. However, TRH-R has not yet been purified to homogeneity and specific anti-TRH-R antibody could not yet be obtained by classical biochemical methods. We thus attempted to obtain antibodies specific for TRH-R using an anti-idiotypic approach. Rabbits of the same allotype were immunized using Igs (Ab1) extracted from rabbit polyclonal anti-TRH immune serum. Anti-idiotypic rabbit polyclonal anti-anti-TRH antibodies (Ab2) were obtained, as shown by their ability to inhibit the formation of TRH-anti-TRH complexes in a radioimmunoassay system. One of them, the polyclonal Ab2 R38/B12, was tested for its ability to recognize the TRH-R in rat pituitary, tumor-derived, GH3/B6 prolactin-secreting cells. Immunoreactive material was immunocytochemically detected in fixed and saponin-permeabilized GH3/B6 cells. The immunostaining was localized at the plasma membrane and on intracellular structures. It was not observed using non-anti-TRH Ab2 and was abolished in the presence of excess TRH. Furthermore, binding of [125I]R38/B12 on fixed and saponin-permeabilized GH3/B6 cells was partially inhibited by excess TRH. By immunoblot analyses of Triton X-114 cell extracts performed under reducing or nonreducing conditions, the polyclonal R38/B12 Igs revealed two main protein species of approximately 98 and approximately 76 kD as well as several proteins < or = 46 kD. In the presence of excess TRH, the approximately 98- and approximately 42-kD bands were abolished, whereas the intensity of the other bands was faintly attenuated only. The approximately 98-kD protein was also revealed in a two-dimensional PAGE analysis. Nevertheless, the effects of R38/B12 Igs on [3H]TRH binding by GH3/B6 cells and on basal or TRH-induced prolactin secretion were not markedly different from those elicited by control Ab2. These data suggest that we have characterized Ab2 antibodies which recognize a molecular entity that might be related to the TRH-R in GH3B6 cells.

Specific responses of axons and dendrites to cytoskeleton perturbations: an in vitro study.

Several factors can influence the development of axons and dendrites in vitro. Some of these factors modify the adhesion of neurons to their substratum. We have previously shown that the threshold of neuron-substratum adhesion necessary for initiation and elongation of dendrites is higher than that required for axonal growth. To explain this difference we propose that, in order to antagonize actin-driven surface tension, axons primarily rely on the compression forces of microtubules whereas dendrites rely on adhesion. This model was tested by seeding the cells in conditions allowing the development either of axons or of axons and dendrites, then adding cytochalasin B or nocodazole 1 hour or 24 hours after plating. The addition of cytochalasin B, which depolymerizes actin filaments and thus reduces actin-tensile forces, increases the length of both axons and dendrites, indicating that both axons and dendrites have to antagonize surface tension in order to elongate. The addition of nocodazole, which acts primarily on microtubules, slightly reduces dendrite elongation and totally abolishes axonal growth. Similar results are obtained when the drugs are added 1 or 24 hours after plating, suggesting that the same mechanisms are at work both in initiation and in elongation. Finally, we find that in the presence of cytochalasin B axons adopt a curly morphology, a fact that could be explained by the importance of tensile forces in antagonizing the asymmetry created by polarized microtubules presenting a uniform minus/plus orientation.

In vitro control of neuronal polarity by glycosaminoglycans.

We have studied the effects of proteoglycans (PGs) and glycosaminoglycans (GAGs) on the growth and morphology of neurons in culture. PGs from glial cells or Engelbreth-Holm-Swarm tumor cells (EHS), pure bovine kidney heparan sulfate (HS), shark cartilage type C chondroïtin sulfate (CSc) and bovine mucosa dermatan sulfate (DS) added to embryonic rat neurons strongly enhanced total neurite growth after 48 h in vitro. No trophic effects were seen when PGs treated with a mixture of glycanases were used. PGs, CSc and HS not only enhanced neurite growth but induced the appearance of a majority of neurons with a single long axon whereas, in contrast, DS increased dendrite growth. GAGs bound to the cell surface and were rapidly internalized, a feature that correlated well with the absence of neurotrophicity of GAGs previously immobilized on the culture substratum. Although the mechanisms involved in GAGs neurotrophic effects and in the separate regulation of neuronal polarity by HS and DS were not elucidated, we found that, as opposed to HS, DS was able to enhance neuronal adhesion and spreading and to maintain a high level of expression of microtubule-associated protein 2 (MAP2), a specific dendritic marker. This finding confirms and extends our previous observations on the role of adhesion in the regulation of dendrite growth.

In vitro regulation of neuronal morphogenesis and polarity by astrocyte-derived factors.

Mesencephalic neurons were cultured from 2 to 5 days in mesencephalic (CM Gmes) or striatal (CM Gstr) astrocyte conditioned media or in the soluble (S100) and insoluble (P100) fractions prepared from these media by ultracentrifugation. CM Gmes as well as all soluble fractions induced dendritic and axonal elongation, whereas CM Gstr and the insoluble fractions promoted axonal growth only. The study of the shape of the neuronal cell bodies and the measurement of their adhesion to the substratum revealed that axons elongated under low adhesion conditions, but that dendrite growth was highly dependent upon adhesion and spreading of the neuronal soma. This different dependency of axonal and dendritic elongation upon spreading is explained by a model in which we consider the respective viscosities of axons and dendrites. From these observations and speculations we propose that axons and dendrites have different modes of elongation and that the primary effect of the astrocyte-derived factors capable of regulating neuronal polarity is to modify the adhesion of the neurons to their culture substratum.

Rat mesencephalic neurons in culture exhibit different morphological traits in the presence of media conditioned on mesencephalic or striatal astroglia.

Embryonic rat mesencephalic neurons were plated at low density in a chemically defined medium (CDM) or in CDM conditioned on either mesencephalic or striatal astrocytes (CM Gmes and CM Gstr). It was found that "axon-like" neurites, in general long with few branching points, could be initiated in CM Gmes and CM Gstr, whereas "dendrite-like" neurites (shorter and with a high branching capacity) were preferentially initiated in CM Gmes. The effects of CM Gmes and CM Gstr on the morphology of mesencephalic neurons were abolished by protein denaturating treatments. Comparisons with basic FGF, laminin, or fibronectin demonstrated that these three molecules were also able to modify the morphological traits of the neurons. However the different morphologies observed in CM Gmes and CM Gstr could not be explained only by the presence of these proteins in the conditioned media. Our results therefore indicate that different factors may regulate the initiation of different categories of neurites and that in contrast to several molecules able to promote neurite elongation these "initiation" factors may show important regional specificity.

Electron microscopic observations of reconstituted proteoliposomes with the purified major intrinsic membrane protein of eye lens fibers.

The purified major intrinsic protein of the lens fiber plasma membrane (MP26) reconstituted into liposomes favored membrane-to-membrane close contacts as visualized by freeze fracture and immunoelectron microscopy. Reconstituted apposed unilamellar vesicles formed pentalaminar profiles, and multilamellar liposomes showed regions of stacked bilayers. Immunogold labeling, using antibody directed against MP26, demonstrated that this polypeptide is present in regions of membrane-to-membrane close interaction. Fracture faces displayed both randomly distributed clusters of 8-nm polygonal intramembrane particles and membrane domains where a bidimensional lattice of repeating subunits was present. The structural pleomorphism which characterized the MP26-reconstituted proteoliposomes seems quite comparable to that visualized in natural fiber plasma membrane domains.

Further studies on the role of astroglia in brain neurons maturation and morphogenesis.

Using in vitro cultures of dissociated brain neurons and astrocytes, we have compared the morphologies of mesencephalic and striatal neurons cultured for two days on mesencephalic and striatal astrocytes in the four possible combinations. From these comparisons, it appears that: 1. Neurons grown on co-regionalized (homotopic) astrocytes have more primary neurites and branching points than neurons grown on heterotopic astrocytes. 2. The total neuritic length is only slightly affected by the type of co-culture. 3. The branched arborization which develop faster on homotopic astrocytes present several dendritic features. Following these morphological observations, we have been able to demonstrate: 1. That mesencephalic astrocytes (but not striatal astrocytes) secrete trypsin sensitive factors different from laminin and FGF that increase the number of primary neurites and branching points but have no or little effect on total neuritic length. 2. That mesencephalic astrocytes (but not striatal astrocytes) present at their surface a 190 KD glycoprotein specifically recognized by the fucose-specific lectin UEA.

Protein rotational mobility in thylakoid membranes of different polypeptide composition in the wild type and mutant strains of Chlamydomonas reinhardtii.

The rotational mobility of thylakoid membrane proteins labeled with a paramagnetic analog of N-ethylmaleimide was investigated by saturation transfer electron spin resonance. In the wild type strain of Chlamydomonas reinhardtii two polypeptides are prominently labeled. They correspond to the 19-kDa subunit of the reaction center I protein and to the 30-kDa subunit of the light harvesting complex. Several polypeptides, most of which are either trypsin or alkaline sensitive, are also labeled. In order to circumvent the lack of specificity during the labeling, we have compared the rotational mobilities of labeled proteins in thylakoid membranes from several mutant strains which lack in photosystem I., ATPase or light harvesting complexes. Comparison of the saturation transfer electron spin resonance spectra obtained with these mutant membranes as well as with trypsin- and alkaline-treated membranes allowed us to characterize the rotational contribution of some of the labeled proteins to the overall protein dynamics observed in the wild type strain. The reaction center I protein undergoes slow rotation as compared to the other labeled proteins. The rotational characteristics of the labeled light harvesting complexes are those of a peptide fragment in the complex which is in rapid motion in unstacked membranes. Stacking of the thylakoid membranes upon Mg2+ addition is accompanied by a marked change in shape of the saturation transfer spectra, and corresponds to the appearance of highly immobilized nitroxides. We interpret these changes as arising mainly from the hindrance upon membrane appression, of the labeled fragment of the light harvesting complexes which protrude at the thylakoid outer surface.

Interaction of crotoxin and its isolated subunits with spin-labeled fatty acids.

We have investigated the interaction of crotoxin (component A-component B complex) and of its isolated phospholipase subunit (component B) with hydrophobic compounds by ESR, using spin-labeled fatty acids as probes. The phospholipase subunit alone (component B) binds more than three labeled fatty acid molecules/molecule with different affinities, the highest corresponding to a Kd of 10 microM in the case of 5-doxyl palmitic acid. In contrast, the noncatalytic subunit (component A) and the crotoxin complex do not bind fatty acids. ESR studies of the component B-fatty acid complex reveal a strong immobilization of the whole length of the fatty acid chain, strong spin-spin interactions between bound fatty acids, and nonaccessibility of the bound paramagnetic probe to Ni2+ ions. This suggests that the phospholipase component B possesses a hydrophobic cleft which may contain one or two fatty acids. This hydrophobic cleft is not accessible to spin-labeled fatty acids in the crotoxin complex. An overall rotational correlation time of about 200 ns of the phospholipase component B was determined by saturation transfer ESR. This high value is incompatible with the diffusion of a polypeptide of 14,500 molecular weight. The hydrodynamic analysis of the fatty acid-component B complex led us to estimate an apparent molecular weight of 95,000 which reveals that fatty acids induce the formation of polymers (most probably octamers) of component B. We propose a model in which the phospholipase component B exists in two conformational states which differ by their hydrophobicity.

The interaction of neurotoxin derivatives with either acetylcholine receptor or a monoclonal antibody. An electron-spin-resonance study.

Five derivatives of Naja nigricollis toxin alpha, spin-labeled on a single amino group, were prepared. The toxin derivatives were purified to homogeneity by ion-exchange and high-pressure liquid chromatographies. The modified amino groups are localized at residue 1 and lysines 15, 27, 47 and 51. Competition data show that incorporation of spin label at residues 27 or 47 reduces the affinity of the toxin for the nicotinic acetylcholine receptor (AcChR), while incorporation at residues 1 or 15 diminishes toxin affinity for a monoclonal toxin-specific immunoglobulin (M alpha 1). Classical and/or saturation transfer electron spin resonance (ESR) analysis was carried out on each derivative, either in the free state or bound to AcChR or M alpha 1. The data obtained give the following indications. In the free state, the nitroxides incorporated at residues 1, 15, 47 and 51 have their own rapid motion, while that at residue 27 had no residual mobility and reflects the toxin rotation. Binding of AcChR to the toxin reduces the motion of the nitroxide bound to Lys47. Binding of M alpha 1 to the toxin immobilizes the two nitroxides fixed on residues 1 and 15. ESR spectra show that Lys27-bound nitroxide remains immobilized upon binding of either AcChR or M alpha 1. The change in nitroxide immobilization observed upon AcChR or M alpha 1 binding correlates well with the variation of nitroxide accessibility to a water-soluble paramagnetic N2+i ion. Binding of the labeled Lys47 toxin derivative to AcChR yields a complex ESR signal, disclosing the existence of a physical difference between the two toxin binding sites on AcChR. All the data indicate that AcChR and M alpha 1 bind at two topographically distinct sites on the toxin surface.