百合花粉及花粉管内微丝和微管的分布

利用免疫荧光定位及荧光定位方法,结合共焦激光扫描显微镜,对百合（ＬｉｌｉｕｍｄａｖｉｄｉＤｕｃｈ．）花粉及花粉管内微丝及微管的分布进行了观察,得出了一些新的结果：１化学固定方法可以较好地保存花粉和花粉管内的微丝,从而可以在此条件下较好地进行微管和微丝的双标记,并进行两者相互关系的研究;２在距花粉管顶端１０～２０μｍ的范围内,用化学固定及ＴＲＩＴＣ鬼笔碱标记显示微丝的存在是很微弱的,基本上无法看到明显的微丝束,而同时用免疫荧光法标记却发现此部位微管很丰富,在花粉管顶端微管形成浓密的网状,而且其末端与花粉管顶端质膜相连;３在花粉管中,只有少数微丝与微管相互平行排列,而其中大多数微丝骨架与微管骨架并不存在共分布现象。为了解花粉管内微管和微丝的功能及相互关系提供了新的证据。     

Circular F-actin bundles and a G-actin gradient in pollen and pollen tubes of Lilium davidii

The distribution of and relationship between F-actin and G-actin were investigated in pollen grains and pollen tubes of Lilium davidii Duch. using a confocal laser scanning microscope after fluorescence and immunofluorescence labeling. Circular F-actin bundles were found to be the main form of microfilament cytoskeleton in pollen grains and pollen tubes. Consistent with cytoplasmic streaming in pollen tubes, there were no obvious F-actin bundles in the 10- to 20-microm tip region of long pollen tubes, only a few short F-actin fragments. Labeling with fluorescein isothiocyanate (FITC)-DNase I at first established the presence of a tip-focused gradient of intracellular G-actin concentration at the extreme apex of the tube, the concentration of G-actin being about twice as high in the 10- to 20-microm region of the tip as in other regions of the pollen tube. We also found that the distribution of G-actin was related negatively to that of the F-actin in pollen tubes of L. davidii. Caffeine treatment caused the G-actin tip-focused gradient to disappear, and F-actin to extend into the pollen tube tip. Based on these results, we speculate that the circular F-actin bundles may be the track for bidirectional cytoplasmic streaming in pollen tubes, and that in the pollen tube tip most of the F-actin is depolymerized into G-actin, leading to the absence of F-actin bundles in this region.     

Cytoskeleton in Pollen and Pollen Tubes of Ginkgo biloba L.

The distribution of F-actin and microtubules was investigated in pollen and pollen tubes of Ginkgo biloba L. using a confocal laser scanning microscope after fluorescence and immunofluorescence labeling. A dense F-actin network was found in hydrated Ginkgo pollen. When Ginkgo pollen was germinating,F-actin mesh was found under the plasma membrane from which the pollen tube would emerge. After pollen germination, F-actin bundles were distributed axially in long pollen tubes of G. biloba. Thick F-actin bundles and network were found in the tip of the Ginkgo pollen tube, which is opposite to the results reported for the pollen tubes of some angiosperms and conifers. In addition, a few circular F-actin bundles were found in Ginkgo pollen tubes. Using immunofluorescence labeling, a dense microtubule network was found in hydrated Ginkgo pollen under confocal microscope. In the Ginkgo pollen tube, the microtubules were distributed along the longitudinal axis and extended to the tip. These results suggest that the cytoskeleton may have an essential role in the germination of Ginkgo pollen and tube growth.     

Comparison of F-actin fluorescent labeling methods in pollen tubes of Lilium davidii

Fluorescence labeling of F-actin in pollen tubes by various methods has produced inconsistent results in the literature. Here, we report that EGTA, which was always used in fixative buffers in the past and thought to help cytoskeleton stabilization, can significantly affect F-actin distribution and lead to the formation of thick F-actin bundles at the tip of the pollen tube. We also found that vacuum-infiltration for the first 5 min during pollen tube fixation can better preserve normal cytoplasm structure and F-actin distribution. In contrast, m-maleimidobenzoic acid N-hydroxysuccinimide ester (MBS) treatment before chemical fixation resulted in a shortening of the free zone of thick F-actin bundles in the pollen tube tip. Taken together, our results suggest that exclusion of EGTA and MBS from the fixative buffer, in combination with vacuum-infiltration in the first 5 min of fixation, can improve F-actin fluorescence labeling in pollen tubes of Lilium davidii.Li Wang and Yi-Min Liu are considered joint first authors     

Rhodamine-phalloidin staining of F-actin in pollen after dimethylsulphoxide permeabilization

Summary Bundles of actin filaments were observed in in vitro cultured pollen of Lilium longiflorum and Nicotiana tabacum which had been permeabilized in a buffered medium containing 5% dimethylsulphoxide (DMSO), EGTA, rhodaminephalloidin for F-actin staining, and sucrose as an osmoticum. In imbibed pollen grains, especially those of lily, numerous short bundles and small foci of F-actin were clearly visible. In germinated pollen grains, fine bundles of F-actin could be seen to converge at the aperture of the pollen grain. Along the entire length of the pollen tube, including the extreme tip, a dense three-dimensional netaxial distribution of actin filaments was observed. The F-actin patterns visualized after permeabilization with DMSO are much finer and more detailed than those observed after conventional fixation with formaldehyde.     

川百合精细胞的超微结构(英文)

川百合与朱顶红花粉管中的生殖细胞分裂行为非常不同。诸如：染色体行为、微管的组织形式和分布、包括着丝点、微管形成的时间,纺锤体的形状及间期周质微管网络在生殖细胞分裂过程中消失与否等。但这两种细胞具有某些共性,包括在有丝分裂前期缺乏早前期带微管（ＰＰＢ）,末期形成细胞板等。这两种植物精细胞的结构应有较大差异。我们曾报道了朱顶红精细胞的超微结构,本文详细从超微结构方面描述了川百合精细胞的特征。川百合花粉管的萌发采用半离体活体培养方式。１１～１８小时后,ＤＮＡ荧光染料Ｈｏｅｃｈｓｔ３３２５８和醋酸地衣红染色检查花粉管中生殖细胞和精细胞发育时期。切取含有分裂的生殖细胞和精细胞的花柱部分,按曾报道的方法固定、包埋、切片、染色及观察。在所有检查的花粉管中,两精子均前后排列（Ｆｉｇ．１～３）,营养核前导并靠近花粉管顶端（Ｆｉｇ．２,３）。Ｈ３３２５８染色可见两精核间以ＤＮＡ联系（Ｆｉｇ．３）。两个新形成的精核彼此分离（Ｆｉｇ．１）,后来又相互靠近,并维持一定距离（Ｆｉｇ．３）。偶尔一对精子与营养核靠近（Ｆｉｇ．２）。两精细胞被一共同的细胞壁连接,他们不仅被自己的质膜也被营养细胞的质膜包围构成周质。周质平坦光滑。共同壁横向     

Identification and characterization of a Ca2+-dependent actin filament-severing protein from lily pollen

It is well known that a tip-focused intracellular Ca2+ gradient and the meshwork of short actin filaments at the tip region are necessary for pollen tube growth. However, little is known about the connections between the two factors. Here, a novel Ca2+-dependent actin-binding protein with molecular mass of 41 kD from lily (Lilium davidii) pollen (LdABP41) was isolated and purified with DNase I chromatography. Our purification procedure yielded about 0.6 mg of LdABP41 with >98% purity from 10 g of lily pollen. At least two isoforms with isoelectric points of 5.8 and 6.0 were detected on two-dimensional gels. The results of N-terminal sequencing and mass-spectrometry analysis of LdABP41 showed that both isoforms shared substantial similarity with trumpet lily (Lilium longiflorum) villin and other members of the gelsolin superfamily. Negative-stained electron microscope images showed that LdABP41 severed in vitro-polymerized lily pollen F-actin into short actin filaments in a Ca2+-sensitive manner. Microinjection of the anti-LdABP41 antibody into germinated lily pollen demonstrated that the protein was required for pollen tube growth. The results of immunolocalization of the protein showed that it existed in the cytoplasm of the pollen tube, especially focused in the tip region. Our results suggest that LdABP41 belongs to the gelsolin superfamily and may play an important role in controlling actin organization in the pollen tube tip by responding to the oscillatory, tip-focused Ca2+ gradient.     

植物花粉管中类整联蛋白的免疫荧光定位研究

The Strong fluorescence signals were obtained in pollen tube of Lilium davidii Duch with labeled anti-VnR integrin serum, and anti-beta 3, alpha v integrin subunit cytoplasmic domain serum separately. The highest density of immunolabel was in the tip of pollen tube. There was little or no immunolabel in control experiment using non-immune serum, second antibody alone and anti-FnR, LnR integrin serum separately. In pollen of Prunus persica f. rubro-piena Schneid, fluoresence signals were also obtained in tube using labeled anti-beta 1, beta 3 integrin subunit cytoplasmic domain serum separately and in apertures using anti-beta 1 serum. Preliminary results show that during the germination of pollen and the growth of pollen tube, there may be integrin-like proteins in pollen tube, consisting of alpha v and beta s integria subunits in the pollen tube of Lilium davidii Duch, which is the receptor of vitronectin-like protein.     

Confocal Microscopy Observations on Actin Cytoskeleton in the Pollen and Pollen Protoplast of Narcissus

Actin cytoskeleton was localized in the pollen and pollen protoplast of Narcissus cyclamineus using fluorescence labelled phalloidin andconfocal microscopy. In the hydrated pollen (before germination) actin filamem bundles were arranged in a parallel array and at right angles to the long axis of the pollen grain in the cortex. But at the germination pore region(or fur row) the actin filament bundles formed a reticulate network. In the centre of the grain there was also an actin filament network which was more open and had less bundles associated with it than the network underneath the furrow. When the pollen grain started to produce pollen tube, most(if not all) of the actin filament bundles in the pollen grain rearranged into a parallel array pointing towards the tube. The bundles in the array later elongated and extended into the pollen tube. In the pollen protoplast a very tightly-packed actin bundle network was present. Numerous branches and jonts of actin filament bundles could be seen in the network. If the protoplasts were fixed before staining, the bundles aggregated and the branches and joints became less obvious indicating that fixation had affected the nature and arrangement of the actin filament bundles. If the pollen protoplasts were bursted (using the osmotic shock technique) or extracted (using Triton X-100), fragments of actin filament bundles could still be found associated with the membrane ghost indicating that some of the actin filament bundles in the cortex were tightly attached to the membrane. Using a double staining technique, actin filaments and microtubules were co-localized in the pollen protoplast. The co-alignment of some of the actin filament bundles with the microtubule bundles suggested that the actin cytoskeleton and the microtubule cytoskeleton were not distributed at random but in a well organized and orchestrated manner [possibly under the control of a yet undiscovered structure(s). The actin filament cytoskeleton in the generative cells failed to stain either in pollen or pollen tube, but they became stained in the pollen protoplast. The actin cytoskeleton in the generative cell appeared as a loosely organized network made up of short and long actin filament bundles.     

An actin-binding protein, LlLIM1, mediates calcium and hydrogen regulation of actin dynamics in pollen tubes

Actin microfilaments are crucial for polar cell tip growth, and their configurations and dynamics are regulated by the actions of various actin-binding proteins (ABPs). We explored the function of a lily (Lilium longiflorum) pollen-enriched LIM domain-containing protein, LlLIM1, in regulating the actin dynamics in elongating pollen tube. Cytological and biochemical assays verified LlLIM1 functioning as an ABP, promoting filamentous actin (F-actin) bundle assembly and protecting F-actin against latrunculin B-mediated depolymerization. Overexpressed LlLIM1 significantly disturbed pollen tube growth and morphology, with multiple tubes protruding from one pollen grain and coaggregation of FM4-64-labeled vesicles and Golgi apparatuses at the subapex of the tube tip. Moderate expression of LlLIM1 induced an oscillatory formation of asterisk-shaped F-actin aggregates that oscillated with growth period but in different phases at the subapical region. These results suggest that the formation of LlLIM1-mediated overstabilized F-actin bundles interfered with endomembrane trafficking to result in growth retardation. Cosedimentation assays revealed that the binding affinity of LlLIM1 to F-actin was simultaneously regulated by both pH and Ca(2+): LlLIM1 showed a preference for F-actin binding under low pH and low Ca(2+) concentration. The potential functions of LlLIM1 as an ABP sensitive to pH and calcium in integrating endomembrane trafficking, oscillatory pH, and calcium circumstances to regulate tip-focused pollen tube growth are discussed.     

The Involvement of PLC-IP3 Signaling Pathway in Pollen Tube Growth

The effect of phospholipase C (PLC) signaling pathway on lily (Lilium davidii Duch.) pollen tube elongation was examined by means of microinjection. Pollen tube elongation was inhibited by microinjecting antibodies against animal PLCβ1-3 or inositol-1,4,5-triphosphate receptor （IP3R2, 3）, but was not affected by antibodies against animal PLCβ4 or IP3R1. Pollen tube elongation was also stimulated significantly by microinjecting IP3. The results suggest that PLC-IP3 signaling pathway might present in pollen system and be involved in pollen tube growth.     

Integrin-like proteins in the pollen tube: detection, localization and function

The distribution of integrin-like proteins in the pollen tube was examined by immunofluorescent labeling and western blotting techniques using antibodies against human placenta integrin vitronectin receptor (VnR), and alpha(v), beta3 and beta1 integrin subunits. Pseudocolor-coded confocal images showed intense immunostaining within 10 and 5 microm of the tip of the pollen tube in Lilium davidii and Nicotiana tabacum respectively. In both segments the site near the plasma membrane was labeled. Western blotting analyses revealed cross-reaction of anti-beta3, anti-alpha(v) and anti-VnR with the proteins in the plasma membrane preparation of L. davidii and Hemerocallis citrina pollen tube. These studies provide evidence for the first time that the integrin-like protein is present in pollen tubes, and it may be mainly composed of alpha(v) and beta3 subunits in lily pollen tubes. In a functional assay, neither anti-VnR antibody nor the Arg-Gly-Asp-Ser tetrapeptide inhibited pollen tube growth of N. tabacum in vitro, but both of them depressed tube growth on the stigma and in style under quasi in vivo culture conditions. The integrin-like proteins localized in the tip and periphery of the pollen tube appeared to play roles in growth of the pollen tube tip and interaction with the extracellular matrix of the style.     

Reversible protein phosphorylation regulates the dynamic organization of the pollen tube cytoskeleton: effects of calyculin A and okadaic acid

We investigated the cytoskeleton of Lilium longiflorum pollen tubes and examined the effects of the type 2A protein phosphatase (PP2A) inhibitors calyculin A and okadaic acid. An improved method for actin visualization, the simultaneous fixation and staining with rhodamine-labelled phalloidin during microscopical observation, revealed abundant actin filaments of no preferential orientation in the apical clear zone. Microtubules, visualized by indirect immunofluorescence, were mostly absent from the apices of straight-growing pollen tubes but present in those with irregular shape. Double labelling showed that both actin bundles and microtubules had a similar longitudinal or slightly helical orientation in the pollen tube shaft. In the presence of 30 nM calyculin A or okadaic acid, pollen tubes grew very slowly, branched frequently, and contained isolated, randomly oriented, curved actin bundles and microtubules. Treating pollen tubes with calyculin A or okadaic acid after germination arrested growth immediately, reversibly altered the alignment of actin bundles from axial to transverse, and disassembled microtubules. The changes in actin organization caused by the PP2A inhibitors were similar to those observed upon overexpression of AtRop1 (Y. Fu, G. Wu, Z. Yang, Journal of Cell Biology 152: 1019-1032, 2001), suggesting that hyperphosphorylation interferes with the signalling pathway of small GTPases. The effects of the PP2A inhibitors could be ameliorated with nanomolar concentrations of latrunculin B.     

Rop1Ps promote actin cytoskeleton dynamics and control the tip growth of lily pollen tube

Rop, the small GTPase of the Rho family in plants, is believed to exert molecular control over dynamic changes in the actin cytoskeleton that affect pollen tube elongation characteristics. In the present study, microinjection of Rop1Ps was used to investigate its effects on tip growth and evidence of interaction with the actin cytoskeleton in lily pollen tubes. Microinjected wild type WT-Rop1Ps accelerated pollen tube elongation and induced actin bundles to form in the very tip region. In contrast, microinjected dominant negative DN-rop1Ps had no apparent effect on pollen tube growth or microfilament organization, whereas microinjection of constitutively active CA-rop1Ps induced depolarized growth and abnormal pollen tubes in which long actin bundles in the shank of the tube were distorted. Injection of phalloidin, a potent F-actin stabilizer that inhibits dynamic changes in the actin cytoskeleton, prevented abnormal growth of the tubes and suppressed formation of distorted actin bundles. These results indicate that Rop1Ps exert control over important aspects of tip morphology involving dynamics of the actin cytoskeleton that affect pollen tube elongation. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

FIMBRIN1 Is Involved in Lily Pollen Tube Growth by Stabilizing the Actin Fringe

An actin fringe structure in the subapex plays an important role in pollen tube tip growth. However, the precise mechanism by which the actin fringe is generated and maintained remains largely unknown. Here, we cloned a 2606-bp full-length cDNA encoding a deduced 77-kD fimbrin-like protein from lily (Lilium longiflorum), named FIMBRIN1 (FIM1). Ll-FIM1 was preferentially expressed in pollen and concentrated at actin fringe in the subapical region, as well as in longitudinal actin-filament bundles in the shank of pollen tubes. Microinjection of Ll-FIM1 antibody into lily pollen tubes inhibited tip growth and disrupted the actin fringe. Furthermore, we verified the function of Ll-FIM1 in the fim5 mutant of its closest relative, Arabidopsis thaliana. Pollen tubes of fim5 mutants grew with a larger diameter in early stages but could recover into normal forms in later stages, despite significantly slower growth rates. The actin fringe of the fim5 mutants, however, was impaired during both early and late stages. Impressively, stable expression of fim5pro:GFP:Ll-FIM1 rescued the actin fringe and the growth rate of Arabidopsis fim5 pollen tubes. In vitro biochemical analysis showed that Ll-FIM1 could bundle actin filaments. Thus, our study has identified a fimbrin that may stabilize the actin fringe by cross-linking actin filaments into bundles, which is important for proper tip growth of lily pollen tubes.     

Actin microfilaments do not form a dense meshwork inLilium longiflorum pollen tube tips

Summary Controversy over whether the apical region of a growing pollen tube contains a dense array of actin microfilaments (MFs) was the impetus for the present study. Microinjection of small amounts of fluorescently labeled phalloidin allowed the observation of MF bundles inLilium longiflorum pollen tubes that were growing and functioning normally. The results show that while the pollen tube contains numerous MF bundles arranged axially, the apical region is essentially devoid of them. The MF bundles could be seen shifting and changing in distribution as the cells grew, but they always remained out of the apical regions. Perturbation of normal growth and function by caffeine causes a change in the MF distribution, which returns to normal upon removal of caffeine from the growth medium. The lack of MFs in the apex is confirmed by careful immunogold electron microscopic analysis of thin sections of rapidly frozen and freeze-substituted pollen tubes, in which very fine MF bundles could be seen somewhat closer to the tip than is discernible with fluorescence microscopy. Still, these are very few in number and are basically absent from the very tip. Thus a reassessment of current assumptions about the distribution of actin in the pollen tube apical region is required.Abbreviations MF microfilaments - FITC fluorescein isothiocyanate - RF-FS rapidly frozen and freeze-substituted - EM electron microscopy Dedicated to Professor Eldon H. Newcomb in recognition of his contributions to cell biology     

Cytological Studies of the Cytoplasmic Inheritance in Lilium regale and L. davidii

The distribution and characteristics of plastids and mitochondria in the generative and sperm cells of Lilium regale Wils. and L. davidii Duch. were described. In L. regale there were few plastids and abundant mitochondria in the newly formed generative cell. When the generative cell became free in the vegetative cytoplasm, the plastids degenerated completely within the generative cell. It was further proved by DAPI fluorescent technique that there was no organell DNA in the generative cell within the mature pollen grain or the pollen tube. However, distribution of the plastids was strictly polarizable during the division of the micmspore in L. davidii, resulting the lack of plastids in the newly formed generative cell. Data of RFLP analysis comparable between L. davidii, L. longifiorum and their interspecific hybrid have also proved the plastid inheritance in L. davidii to be of uniparental maternal transmission. Although the mitoehondria were observed both in the generative and sperm cells of L. regale and L. davidii but their DNA was decomposed in the male gametophyte stage. Therefore the mitochondda in the sperm cell could not be transmitted into the offspring. The results provided the detail, cytological evidence that organelles in the microgametophyte are incapable of genetic transmission in the two species of Lilium.     

Sites of Origin of the Peripheral Microtubule System of the Vegetative Cell of the Angiosperm Pollen Tube

From earlier published work it is known that microtubules inthe vegetative cell of angiosperm pollen tubes mainly occurin the form of longitudinally disposed strands closely associatedwith the plasmalemma. This peripheral cytoskeleton is extendedapically at a speed matching the growth rate of the tube. Immunofluorescencelocalization shows that in the actively elongating tube it originatesin the sub-apical zone in bands or ribbons up to 2 µmwide, interpreted here as comprising aggregates of apposed,axially oriented microtubules. These appear first in the corticalcytoplasm in close association with the wall in the part ofthe tube where the callose inner lining can first be detected.The bands do not extend apically into the region of the pecticsheath of the extreme tip. In the course of normal growth, theperipheral microtubule investment remains in the older partsof the tube from which the bulk of the cytoplasm has been withdrawn,indicating that tubulin is probably not recycled. If the growth of the tube is retarded, the inner callosic layerextends apically. The acropetal movement of callose is accompaniedby a migration of the limit of detectable tubulin towards theextreme tip, and the axially oriented bands are replaced bya confused mass of granules and short spicules. It is suggested that the bands represent nucleation zones associatedwith the stabilizing plasmalemma in the sub-apical stretch ofthe tube where the insertion of wall-precursor material is diminishing,and that it is from these zones that the microtubule cytoskeletonof the pollen tube originates. Since during growth the nucleationzones progress rapidly forward into association with new membrane,it is considered unlikely that their sites are determined bylocal differentiations of the plasmalemma. An alternative possibilityis that the distribution of the zones is related to the calciumion gradient known to be present in the apical stretch of theextending pollen tube. Microtubules, pollen tube growth, Lilium auratum     

Calcium-calmodulin suppresses the filamentous actin-binding activity of a 135-kilodalton actin-bundling protein isolated from lily pollen tubes

We have isolated a 135-kD actin-bundling protein (P-135-ABP) from lily (Lilium longiflorum) pollen tubes and have shown that this protein is responsible for bundling actin filaments in lily pollen tubes (E. Yokota, K. Takahara, T. Shimmen [1998] Plant Physiol 116: 1421-1429). However, only a few thin actin-filament bundles are present in random orientation in the tip region of pollen tubes, where high concentrations of Ca(2+) have also been found. To elucidate the molecular mechanism for the temporal and spatial regulation of actin-filament organization in the tip region of pollen tubes, we explored the possible presence of factors modulating the filamentous actin (F-actin)-binding activity of P-135-ABP. The F-actin-binding activity of P-135-ABP in vitro was appreciably reduced by Ca(2+) and calmodulin (CaM), although neither Ca(2+) alone nor CaM in the presence of low concentrations of Ca(2+) affects the activity of P-135-ABP. A micromolar order of Ca(2+) and CaM were needed to induce the inhibition of the binding activity of P-135-ABP to F-actin. An antagonist for CaM, W-7, cancelled this inhibition. W-5 also alleviated the inhibition effect of Ca(2+)-CaM, however, more weakly than W-7. These results suggest the specific interaction of P-135-ABP with Ca(2+)-CaM. In the presence of both Ca(2+) and CaM, P-135-ABP organized F-actin into thin bundles, instead of the thick bundles observed in the absence of CaM. These results suggest that the inhibition of the P-135-ABP activity by Ca(2+)-CaM is an important regulatory mechanism for organizing actin filaments in the tip region of lily pollen tubes.     

Effect of Extracellular Calmodulin on the Cytosolic Ca2+ Concentration in Lily Pollen Grains

Using Lilium davidii Duchartre pollen as material, the calcium ion-fluorescence indicator fluo-3AM was loaded successfully into the pollen grains by low temperature loading method. Laser confocal scanning microscopy was used to study the effect of extracellular calmodulin on intracellular calcium. It is found that the purified exogenous calmodulin could elevate the intracellular calcium ion concentration, and the effect was correlated with the concentration of exogenous calmodulin to a certain extent. Cell membrane nonpermeable inhibitor of calmodulin, W 7-agarose, and the anti-serum of calmodulin could decrease the cytosolic calcium level. The results show that the endogenous extracellular calmodulin may play an important role in maintaining and increasing the cytosolic calcium level in pollen grain cell.