Two sites for calcium action in compaction of the mouse embryo

Compact mouse morulae were decompacted in calcium-free medium and allowed to recompact in standard embryo culture medium. When the recompaction medium contained trifluoperazine (TFP)(0.05 mM), an inhibitor of the calcium-dependent protein calmodulin, the embryos failed to recompact. This effect could not be overcome by either db-cAMP (1.0 mM) or theophylline (0.75 mg/ml). When the recompaction medium contained less than standard calcium (0.085 or 0.17 mM) the embryos recompacted at a slower rate than in control medium (1.7 mM). The calcium ionophore A23187, at concentrations up to 1.5 X 10(-3) mM, had no significant stimulatory effect upon the recompaction rte of embryos in the reduced calcium medium. In addition, the calcium antagonist Verapamil (0.3 mM), which blocks calcium uptake by cells, significantly inhibited recompaction in standard culture medium. Large doses of diazepam inhibited recompaction only slightly in standard culture medium. Large doses of diazepam inhibited recompaction only slightly in standard culture medium. We conclude that calcium uptake into the cytoplasm is required for recompaction, but that cell surface calcium is also required and is rate-limiting under these experimental conditions.     

Calmodulin X (Ca2+)4 is the active calmodulin-calcium species activating the calcium-, calmodulin-dependent protein kinase of cardiac sarcoplasmic reticulum in the regulation of the calcium pump

Calcium-, calmodulin-dependent phosphorylation of cardiac sarcoplasmic reticulum increases the rate of calcium transport. The complex dependence of calmodulin-dependent phosphoester formation on free calcium and total calmodulin concentrations can be satisfactorily explained by assuming that CaM X (Ca2+)4 is the sole calmodulin-calcium species which activates the calcium-, calmodulin-dependent, membrane-bound protein kinase. The apparent dissociation constant of the E X CaM X (Ca2+)4 complex determined from the calcium dependence of calmodulin-dependent phosphoester formation over a 100-fold range of total calmodulin concentrations (0.01-1 microM) was 0.9 nM; the respective apparent dissociation constant at 0.8 mM free calcium, 1 mM free magnesium with low calmodulin concentrations (0.1-50 nM) was 2.60 nM. These results are in good agreement with the apparent dissociation constant of 2.54 nM of high affinity calmodulin binding determined by 125I-labelled calmodulin binding to sarcoplasmic reticulum fractions at 1 mM free calcium, 1 mM free magnesium and total calmodulin concentration ranging from 0.1 to 150 nM, i.e. conditions where approximately 98% of the total calmodulin is present as CaM X (Ca2+)4. The apparent dissociation constant of the calcium-free calmodulin-enzyme complex (E X CaM) is at least 100-fold greater than the apparent dissociation constant of the E X CaM X (Ca2+)4 complex, as judged from non-saturation 125I-labelled calmodulin binding at total calmodulin concentrations of up to 150 nM, in the absence of calcium.     

Calmodulin·(Ca2+)4 is the active calmodulin-calcium species activating the calcium-, calmodulin-dependent protein kinase of cardiac sarcoplasmic reticulum in the regulation of the calcium pump

Calcium-, calmodulin-dependent phosphorylation of cardiac sarcoplasmic reticulum increases the rate of calcium transport. The complex dependence of calmodulin-dependent phosphoester formation on free calcium and total calmodulin concentrations can be satisfactorily explained by assuming that CaM · (Ca²⁺)₄ is the sole calmodulin-calcium species which activates the calcium-, calmodulin-dependent, membrane-bound protein kinase. The apparent dissociation constant of the E · CaM · (Ca²⁺)₄ complex determined from the calcium dependence of calmodulin-dependent phosphoester formation over a 100-fold range of total calmodulin concentrations (0.01–1 μ M) was 0.9 nM; the respective apparent dissoclation constant at 0.8 mM free calcium, 1 mM free magnesium with low calmodulin concentrations (0.1–50 nM) was 2.60 nM. These results are in good agreement with the apparent dissociation constant of 2.54 nM of high affinity calmodulin binding determined by ¹²⁵I-labelled calmodulin binding to sarcoplasmic reticulum fractions at 1 mM free calcium, 1 mM free magnesium and total calmodulin concentration ranging from 0.1 to 150 nM, i.e. conditions where approximately 98% of the total calmodulin is present as CaM · (Ca²⁺)₄. The apparent dissociation constant of the calcium-free calmodulin-enzyme complex (E · CaM) is at least 100-fold greater than the apparent dissociation constant of the E · CaM · (Ca²⁺)₄ complex, as judged from non-saturation ¹²⁵I-labelled calmodulin binding at total calmodulin concentrations of up to 150 nM, in the absence of calcium.     

Calcium binding to calmodulin leads to an N-terminal shift in its binding site on the ryanodine Receptor

The skeletal muscle calcium release channel, ryanodine receptor, is activated by calcium-free calmodulin and inhibited by calcium-bound calmodulin. Previous biochemical studies from our laboratory have shown that calcium-free calmodulin and calcium bound calmodulin protect sites at amino acids 3630 and 3637 from trypsin cleavage (Moore, C. P., Rodney, G., Zhang, J. Z., Santacruz-Toloza, L., Strasburg, G., and Hamilton, S. L. (1999) Biochemistry 38, 8532-8537). We now demonstrate that both calcium-free calmodulin and calcium-bound calmodulin bind with nanomolar affinity to a synthetic peptide matching amino acids 3614-3643 of the ryanodine receptor. Deletion of the last nine amino acids (3635-3643) destroys the ability of the peptide to bind calcium-free calmodulin, but not calcium-bound calmodulin. We propose a novel mechanism for calmodulin's interaction with a target protein. Our data suggest that the binding sites for calcium-free calmodulin and calcium-bound calmodulin are overlapping and, when calcium binds to calmodulin, the calmodulin molecule shifts to a more N-terminal location on the ryanodine receptor converting it from an activator to an inhibitor of the channel. This region of the ryanodine receptor has previously been identified as a site of intersubunit contact, suggesting the possibility that calmodulin regulates ryanodine receptor activity by regulating subunit-subunit interactions.     

Molecular dynamics simulations of calcium-free calmodulin in solution

A 4-ns molecular dynamics simulation of calcium-free calmodulin in solution has been performed, using Ewald summation to treat electrostatic interactions. Our simulation results were mostly consistent with solution experimental studies, including NMR, fluorescence and x-ray scattering. The secondary structures within the N- and C-terminal domains were conserved in the simulation, with trajectory structures similar to the NMR-derived model structure 1CFD. However, the relative orientations of the domains, for which there are no NMR restraints, differed in details between the simulation and the 1CFD model. The most interesting information provided by the simulations is that the dynamics of calcium-free calmodulin in solution is dominated by slow rigid body reorientations of the domains. The interdomain distance fluctuated between 29 and 39 A, and interdomain orientation angle, defined as the pseudo-dihedral formed by the four calcium binding sites, varied between -2 degrees and 108 degrees. Similarly, the domain linker region also exhibited significant fluctuations, with its length varying in the 34-45 A range and its bend angle in the 10-100 degrees range. The simulations are in accord with fluorescence results suggesting that calcium-free calmodulin is more compact and more flexible than the calcium activated form. Surprisingly, quite similar solvent accessibilities of the hydrophobic patches were seen in the calcium-free trajectory described in this work and previously generated calcium-loaded calmodulin simulations. Thus, our simulations suggest a reexamination of the standard model of the structural change of calmodulin upon calcium binding, involving exposure of the hydrophobic patches to solvent.     

Induction of Actin-Based Cytoplasmic Contraction in the Siphonous Green Alga Acetabularia (Chlorophyceae) by Locally Restricted Calcium Influx

Perfused cell segments dissected from the stalk or from detached cap ray chambers of Acetabularia were used as an experimental system to study the induction of cytoplasmic contractions and concurrent cytoskeletal changes in plant cells. Immunofluorescence microscopy revealed that the actin cytoskeleton quickly rearranges upon induction of contraction by forming bundles oriented circumferentially around the affected area, whereas microtubules were not detected. Contraction is blocked by cytochalasin D or N-ethylmaleimide but is unaffected by microtubule specific inhibitors. Contraction requires external Ca²⁺ at concentrations of 1 μM or more, but fails to occur below 0.1 μM. Higher concentrations of Ca²⁺ up to 10 mM have no adverse effect. Contraction is prevented in the presence of micromolar Ca²⁺ by either 1 mM of the calcium channel blocker LaCl₃ or 10 μM of the calmodulin inhibitor fluphenazine. Calcium ionophore A 23187 (1 μM) does not perturb wound contraction per se but causes the entire cytoplasm of wounded or unwounded cells to contract slowly. These data suggest that a localized influx of calcium ions at the wound edge causes major rearrangements in the distribution of cytoskeletal actin prior to contraction in Acetabularia. An involvement of calmodulin in calcium signaling is proposed.     

Immunocytochemical localization of calmodulin and a heat-labile calmodulin-binding protein (CaM-BP80) in basal ganglia of mouse brain

Antisera to calmodulin, a Ca2%-dependent modulator protein, and a heat- labile calmodulin-binding protein have been used to localize these proteins in mouse caudate-putamen. The two proteins appear to be located at identical sites in this brain area. At the light microscopic level, calmodulin and calmodulin-binding protein are found within the cytoplasm and processes of large cells. At the electron microscopic level the proteins are associated with neuronal elements only, primarily at postsynaptic sites within neuronal somata and dendrites. Within the dendrites the immunocytochemical label is associated predominantly with the postsynaptic density and dendritic microtubules. These results are in accord with recent biochemical and immunihistochemical studies of calmodulin in brain and in dividing cells. Thus, calmodulin and the heat-labile calmodulin-binding protein may play a role in the nervous system at the site of neurotransmitter action and at the level of microtubular function.     

Molecular Dynamics Simulations of Calcium-Free Calmodulin in Solution

Abstract

A 4-ns molecular dynamics simulation of calcium-free calmodulin in solution has been performed, using Ewald summation to treat electrostatic interactions. Our simulation results were mostly consistent with solution experimental studies, including NMR, fluorescence and x-ray scattering. The secondary structures within the N- and C-terminal domains were conserved in the simulation, with trajectory structures similar to the NMR-derived model structure 1CFD. However, the relative orientations of the domains, for which there are no NMR restraints, differed in details between the simulation and the 1CFD model. The most interesting information provided by the simulations is that the dynamics of calcium-free calmod- ulin in solution is dominated by slow rigid body reorientations of the domains. The interdomain distance fluctuated between 29 and 39 Å, and interdomain orientation angle, defined as the pseudo-dihedral formed by the four calcium binding sites, varied between ?2° and 108°. Similarly, the domain linker region also exhibited significant fluctuations, with its length varying in the 34–45 Å range and its bend angle in the 10–100° range. The simulations are in accord with fluorescence results suggesting that calcium-free calmodulin is more compact and more flexible than the calcium activated form. Surprisingly, quite similar solvent accessibilities of the hydrophobic patches were seen in the calcium-free trajectory described in this work and previously generated calcium-loaded calmodulin simulations. Thus, our simulations suggest a reexamination of the standard model of the structural change of calmodulin upon calcium binding, involving exposure of the hydrophobic patches to solvent.     

Expression of the calcium binding protein calretinin in WiDr cells and its correlation to their cell cycle

Ca2+ ions intervene during different phases of the progression of the cell cycle, but only one calcium-binding protein, calmodulin, has been shown to be associated with dividing cells. We therefore screened cancer cells for the presence of other related calcium-binding proteins. Using molecular biological and immunohistochemical techniques we show that human tumor cells of epithelial origin, express calretinin. Calretinin immunoreactivity can be demonstrated at precise moments of the cell cycle and, in particular, in phase G1 and during mitosis. During mitosis calretinin is localized both in the cytoplasm and in the mitotic spindle. In the cytoplasm we find calretinin after prophase and until telophase. In the spindle apparatus, calretinin is already present in cells in prometaphase and persists in all the succeeding mitotic phases. It is associated with the kinetochore microtubules but, in contrast to calmodulin, also with the polar microtubules. The role that calretinin plays in well-defined moments of the cell cycle of these cells is as yet unknown, but our results strongly suggest that, in collaboration with other molecules, calretinin intervenes in the dynamic phenomena regulating the separation of the chromosomes.     

Increased covalent binding of acetaldehyde to calmodulin in the presence of calcium

The regulatory protein, calmodulin, undergoes major conformational changes in response to changes in intracellular calcium concentration. Furthermore, calmodulin has been reported to have lysine residues which markedly increase their reactivity toward electrophilic substances in the calcium-loaded state. We found that calmodulin formed two to three times more stable adducts with acetaldehyde in the calcium-loaded state as compared to the calcium-free state. Competition-binding studies showed that calmodulin could preferentially compete with albumin for acetaldehyde in the presence, but not in the absence, of calcium. When calmodulin was in the calcium-loaded state, trifluoperazine, an inhibitor of calmodulin activity, significantly decreased the stable binding of acetaldehyde to the protein, whereas in the calcium-free state, minimal effects on binding were observed. Since calmodulin is involved in regulation of multiple important processes in the cell, it is possible that acetaldehyde-calmodulin adducts could contribute to liver injury by perturbation of calcium-dependent homeostatic mechanisms within the hepatocyte.     

Calmodulin immunolocalization in outer segments of Xenopus laevis photoreceptors

Because adaptation of vertebrate photoreceptors to light is mediated by changes in the level of calcium in their outer segments (OS), proteins that bind calcium are important in phototransduction. This study has used immunofluorescence to investigate the distribution of the calcium-binding protein calmodulin within photoreceptor OS dissociated from amphibian ( Xenopus laevis) retinas. The OS of rods and cones had a streak of fluorescence to calmodulin at the ciliary axoneme. The OS of rods (but not cones) also displayed regularly spaced puncta of anti-calmodulin fluorescence along longitudinal lines coinciding with their multiple incisures. This location of calmodulin immunofluorescence closely matches the known location of microtubules within the OS of amphibian rods and cones. These findings provide evidence that calmodulin is closely associated with the microtubules of both the axonemal and incisural cytoskeletal systems in OS, and suggest that this association is important for calmodulin function in photoreceptors.     

Activation of the calcium permeability of erythrocyte membranes by perfringolysin O

Calcium ions inhibited perfringolysin O-induced hemolysis at a concentration lower than 1 mM, but not the hemolysis by digitonin at 10 mM. The introduction of calcium ions into ghosts inhibited the lysis more strongly than the addition of calcium ions outside ghosts. When erythrocytes were treated with perfringolysin O in the presence of 1 mM CaCl2 containing 45CaCl2, the radioactivities inside cells rapidly increased during incubation. On the other hand, when perfringolysin O-treated erythrocytes were incubated in a calcium-free medium, the erythrocytes released calcium ions at a 3.3-fold higher rate than untreated cells. These results suggested that perfringolysin O accelerated both the calcium influx into and efflux from erythrocytes.     

Characterization and immunocytochemical distribution of calmodulin in higher plant endosperm cells: localization in the mitotic apparatus

In this study we have examined the immunocytochemical distribution of calmodulin during mitosis of higher plant endosperm cells. Spindle development in these cells occurs without centrioles. Instead, asterlike microtubule converging centers appear to be involved in establishing spindle polarity. By indirect immunofluorescence and immunogold staining methods with anti-calmodulin antibodies, we found endosperm calmodulin to be associated with the mitotic apparatus, particularly with asterlike and/or polar microtubule converging centers and kinetochore microtubules, in an immunocytochemical pattern distinct from that of tubulin. In addition, endosperm calmodulin and calcium showed analogous distribution profiles during mitosis. Previous reports have demonstrated that calmodulin is associated with the mitotic apparatus in animal cells. The present observation that calmodulin is also associated with the mitotic apparatus in acentriolar, higher plant endosperm cells suggests that some of the regulatory mechanisms involved in spindle formation, microtubule disassembly, and chromosome movement in plant cells may be similar to those in animal cells. However, unlike animal cell calmodulin, endosperm calmodulin appears to associate with kinetochore microtubules throughout mitosis, which suggests a specialized role for higher plant calmodulin in the regulation of kinetochore microtubule dynamics.     

db—cAMP对转化细胞钙调素基因表达与细胞骨架的影响

We have demonstrated that the distribution of microtubules (MT), microfilaments (MF) and fibronectin (FN) were diminished, while the gene expression of the calmodulin and c-fos enhanced in the transformed C3 H10 T1/2 cells. After treatment with 1 mM db-cAMP for 1 hr. and 2 hrs., there was an early and rapidly reduced in gene expression of calmodulin and c-fos respectively. After db-cAMP treatment for 4-5 days, the number of Capping cells of ConA binding decreased significantly and the cell surface microvilli decreased also. The growth of treated cells was inhibited markedly. By using 4F1 cDNA probe, which is preferentially expressed in G1 phase, we have found that the db-cAMP treated cells were accumulated at G1 phase. Of particular interest is the fact that the distribution of microtubules, microfilaments and fibronectin were recovered after treatment with 1 mM db-cAMP for 6 days. It is suggested that the inhibition of proliferation, alteration of phenotype and recovery of cytoskeleton in transformed cells after treatment with db-cAMP are related to the inhibition of gene expression of calmodulin.     

Calcium-dependent stimulation of BALB/c 3T3 mouse cell DNA synthesis by a tumor-promoting phorbol ester (PMA).

During the first six passages after their arrival in this laboratory, BALB/c 3T3 mouse cells did not proliferate in serum containing-medium having an ionic calcium concentration of 0.05 mM or less, but by the ninth passage they had become able to multiply in the presence of these lower calcium levels. In low calcium (e.g., 0.02 mM) medium, passage 1-6, cells in sparse cultures were blocked at the Gl/S boundary of their cycle. These blocked cells could be induced to start making DNA within only one hour either by returning the ionic calcium level to a normal range of values (1.25 mM), or by adding 0.05 mug/ml of PMA (12-O-tetradecanoyl-phorbol-13-acetate). PMA probably acted by sensitizing the blocked cells to calcium rather than replacing the ion, because it was ineffective in ionic calcium-free medium. Finally, PMA did not by itself induce proliferation of cells (regardless of the number of passages) which had been proliferatively inactivated by density-dependent factors in confluent cultures. However, PMA did promote DNA synthesis by these cells during their brief transition to the "cycling" state caused by exposure to fresh serum-containing medium.     

Characteristics and regulation of active calcium transport in inside-out red cell membrane vesicles

Sealed, inside-out human red cell membrane vesicles, prepared by a modified method of Steck (Steck T.L. (1974) in Methods in Membrane Biology (Korn, E.D., ed.), Vol 2, pp. 245–281, Plenum Press, New York), accomplish an ATP and Mg²⁺-dependent uphill calcium uptake with a reproducible maximum rate of 12–15 nmol/mg vesicle protein per min under physiological conditions. This maximum rate is increased by about 60–70% in the presence of a heatstable cytoplasmic activator protein (calmodulin) obtained from red cells. Calcium efflux from inside-out vesicles is smaller than 0.01 nmol/mg vesicle protein per min at intravesicular calcium concentrations between 0.1 and 20.0 mM.In the presence of Mg²⁺, active calcium uptake is supported by ATP, ITP, or UTP, but not by ADP, AMP, or p-nitrophenyl phosphate. The optimum pH for the process is 7.4–7.6, and the activation energy is 19–20 kcal/mol, irrespective of the presence or absence of calmodulin. Calcium uptake in inside-out vesicles is unaffected by ouabain or oligomycin, but blocked by low concentrations of lanthanum, ruthenium red, quercetin and phloretin. K⁺ and Na⁺, when compared to choline⁺ or Li⁺, significantly increase active calcium uptake. This stimulation by K⁺ and Na⁺ is independent of that by calmodulin.Concentrated red cell cytoplasm activates calcium uptake at low soluble protein:membrane protein ratios, while a ‘deactivation’ of the transport occurs at high cytoplasm: membrane protein ratios. A heat-labile cytoplasmic protein fraction antagonizing calmodulin activation, can be separated by DEAE-Sephadex chromatography. Based on these findings the regulation of active calcium transport in human red cells is discussed.     

Inhibition of fast axonal transport in bullfrog nerves by dibenzazepine and dibenzocycloheptadiene calmodulin inhibitors

The effects of the calmodulin inhibitors amitriptyline, desipramine, imipramine, and clomipramine on fast axonal transport, oxidative metabolism, and density of axonal microtubules were measured in bullfrog spinal nerves in vitro. The four drugs tested inhibited the fast orthograde transport of [3H]leucine-labelled proteins and the fast retrograde transport of acetylcholinesterase at a concentration of 0.2 mM. Amitriptyline, desipramine, and imipramine were equipotent inhibitors of transport, and clomipramine was a more potent inhibitor than imipramine. The adenosine triphosphate content of the nerves was reduced by at most 19% by the compounds under study; such a reduction cannot account for the inhibition of fast axonal transport. Desipramine and imipramine had no significant effect on the density of microtubules in unmyelinated axons, whereas amitriptyline only reduced it by 18%; the inhibition of axonal transport by these three drugs can therefore not be explained by microtubule disruption. Clomipramine reduced microtubular density by 40%, and this effect may have contributed to the inhibition of fast axonal transport. The inhibition of fast axonal transport by desipramine, imipramine, and amitriptyline may be related to the inhibition of calmodulin function by these drugs. The similar potency of these three drugs as inhibitors of fast axonal transport goes in parallel with their known similar potency as calmodulin antagonists.     

Ionophore-induced disassembly of blood platelet microtubules: effect of cyclic AMP and indomethacin

Cytoplasmic calcium levels are believed to be important in blood platelet activation. Upon activation, the discrete marginal microtubule band, which maintains the discoid shape of non-activated platelets, becomes disrupted. Present studies demonstrate that the extent of assembly of the marginal microtubule band is related to cytoplasmic calcium levels. The divalent cationophore, A23187, causes platelet aggregation, secretion, and contraction by promoting calcium transport from intraplatelet storage sites into the cytoplasm. A23187 caused disassembly of platelet microtubules. Quantitation of electron micrographs revealed that numbers of microtubules were reduced by approximately 80% after A23187 treatment. Secondly, assembled microtubules in homogenates of platelets, in which microtubules were stabilized prior to homogenization, were decreased in favor of free tubulin in A23187-treated platelets. Thirdly, A23187 increased 14C-colchicine binding by intact platelets; this also indicated a shift in the microtubule subunit equilibrium to favor free, colchicine-binding tubulin subunits. In control experiments, A23187 did not affect the stability of platelet tubulin, the colchicine binding reaction, or the total tubulin content of platelets. Stimulation of colchicine binding depended on A23187 concentration (0.05-0.5 microM) and did not require extracellular calcium. A23187-stimulation of colchicine binding was blocked by dibutyryl cyclic AMP (0.80 mM) and/or 3-isobutyl-1-methylxanthine (50 microM) and by indomethacin (10 microM). Cyclic AMP or indomethacin also interferes with A23187-induced platelet activation, but indomethacin is not likely to completely inhibit the perturbation of intraplatelet calcium gradients by A23187. It is suggested that A23187-induced microtubule disassembly may be an indirect effect of calcium on microtubules.     

Evidence that calcium may control neurite outgrowth by regulating the stability of actin filaments

We investigated the effects of calcium removal and calcium ionophores on the behavior and ultrastructure of cultured chick dorsal root ganglia (DRG) neurons to identify possible mechanisms by which calcium might regulate neurite outgrowth. Both calcium removal and the addition of calcium ionophores A23187 or ionomycin blocked outgrowth in previously elongating neurites, although in the case of calcium ionophores, changes in growth cone shape and retraction of neurites were also observed. Treatment with calcium ionophores significantly increased growth cone calcium. The ability of the microtubule stabilizing agent taxol to block A23187-induced neurite retraction and the ability of the actin stabilizing agent phalloidin to reverse both A23187-induced growth cone collapse and neurite retraction suggested that calcium acted on the cytoskeleton. Whole mount electron micrographs revealed an apparent disruption of actin filaments in the periphery (but not filopodia) of growth cones that were exposed to calcium ionophores in medium with normal calcium concentrations. This effect was not seen in cells treated with calcium ionophores in calcium-free medium or cells treated with the monovalent cation ionophore monensin, indicating that these effects were calcium specific. Ultrastructure of Triton X-100 extracted whole mounts further indicated that both microtubules and microfilaments may be more stable or extraction resistant after treatments which lower intracellular calcium. Taken together, the data suggest that calcium may control neurite elongation at least in part by regulating actin filament stability, and support a model for neurite outgrowth involving a balance between assembly and disassembly of the cytoskeleton.     

水稻雄性不育系珍汕97A小孢子发育过程中的微管骨架

水稻（Oryza sativaL.)雄性不育系珍汕97A,保持系珍汕97B和恢复系测64三系小孢子发生过程的研究表明;恢复系测64小孢子母细胞细胞质浓,有明显的微管荧光围绕着细胞核。小孢子母细胞经两次减数分裂形成四分体。四分体和小孢子的微管从细胞核表面向胞质周缘延伸,形成放射性排列格局,花粉发育正常。细胞质中有少量点状微管荧光,保持系珍汕97B小孢子发生过程的细胞形态和微管结构与恢复系测64相似。但细胞质中的点状微管荧光多一些。雄性不育系珍汕97A小孢子发生早期,小孢子母细胞内出现液泡,核中染色质凝集,微管荧光很弱,没有清晰的微管丝结构。细胞质中有许多点状微管荧光等不正常现象。小孢子母细胞经过减数分裂形成的四分体也没有清晰的丝状微管结构。随后,所有的小孢子迅速败育,雄性不育系珍汕97A在小孢子母细胞发生的很早时期,微管结构就明显不正常。