Galectin-3 expression and subcellular localization in senescent human fibroblasts

Galectin-3 is a galactose/lactose-binding protein (M(r) approximately 30,000), identified as a required factor in the splicing of pre-mRNA. In the LG1 strain of human diploid fibroblasts, galectin-3 could be found in both the nucleus and the cytoplasm of young, proliferating cells. In contrast, the protein was predominantly cytoplasmic in senescent LG1 cells that have lost replicative competence through in vitro culture. Incubation of young cells with leptomycin B, a drug that disrupts the interaction between the leucine-rich nuclear export signal and its receptor, resulted in the accumulation of galectin-3 inside the nucleus. In senescent cells, galectin-3 staining remained cytoplasmic even in the presence of the drug, thus suggesting that the observed localization in the cytoplasm was due to a lack of nuclear import. In heterodikaryons derived from fusion of young and senescent LG1 cells, the predominant phenotype was galectin-3 in both nuclei. These results suggest that senescent LG1 cells might lack a factor(s) specifically required for galectin-3 nuclear import.     

Iron containing nuclear inclusions in human pigmentary cirrhosis (author's transl)]

In human pigmentary cirrhosis nuclear (pseudo-)inclusions of cytoplasmic material, containing less or more degenerated and therefore faintly stained hemosiderin granules, are to be observed. But sometimes there are also finely fibrillar or granular proteinaceous materials, stainable by the Prussian-blue reaction, lying between the chromatin-strands or occupying the whole nucleus and displacing the chromatin to the nuclear envelope (margination of chromatin). Such uncoloured substances may condense into homogeneous masses and nearly hexagonal (0r related) crystals with a diameter up to 14 micron and a yellow-brownish colour, giving a strongly positive PERL's reaction. In contrast to the preceding stages intranuclear crystals of this kind have been observed in one case only. After destruction of the nuclear envelope and the marginated chromatin the crystals are lying free in the cytoplasm and later on, the cytoplasm being destroyed too, they may be ingested by von Kupffer cells. All the iron containing crystals, to be found in the cytoplasm, derive from former intranuclear inclusions. The intranuclear deposits of iron containing protein are interpreted as ferritin-aggregates. It is supposed that ferritin molecules, built up in the cytoplasm, do enter the nucleus via the pores of the nuclear envelope. Such an event not only signalizes a cytopathologic reaction but in turn may give rise to such additional cytopathologic lesions as cell shrinking and cell death.     

Changes in the level and distribution of Ku proteins during cellular senescence

Aging is associated with accumulation of genomic rearrangements consistent with aberrant repair of DNA breaks. We have shown previously that DNA repair by non-homologous end joining (NHEJ) becomes less efficient and more error-prone in senescent cells. Here, we show that the levels of Ku70 and Ku80 drop approximately twofold in replicatively senescent cells. Intracellular distribution of Ku also changes. In the young cells roughly half of Ku is located in the nucleus and half in the cytoplasm. In senescent cells the nuclear levels of Ku do not change, while the cytoplasmic Ku fraction disappears. Upon treatment with gamma-irradiation, in the young cells cytoplasmic Ku moved into the nuclear and membrane fractions, while no change in the Ku distribution occurred in senescent cells. Upon treatment with UVC Ku moved out of the nucleus in the young cells, while most Ku remained nuclear in senescent cells. This suggests that the nuclear Ku in senescent cells is unable to respond to DNA damage. We hypothesize that overall decline in Ku levels changes in Ku intracellular distribution, and the loss of appropriate response of Ku to DNA damage in senescent cells contribute to the decline of NHEJ and to age-related genomic instability.     

Reactivation of chick erythrocyte nuclei in young and senescent WI38 cells

The chromatin of the dormant chick nucleus is dispersed in the heterokaryons made by Sendai virus fusion of phase II WI38 cells with chick erythrocyte nuclei. The erythrocyte nucleus resumes RNA synthesis and enters into DNA synthesis with the host nucleus. In the heterokaryons of phase III WI38 cells and chick erythrocytes, the nuclear chromatin is not dispersed and RNA synthesis occurs at a reduced rate. The differences in the physiological state of the young and senescent cells measured by [³H]uridine incorporation into nuclear RNA is reflected in the extent of reactivation of the chick erythrocyte nuclei in the cytoplasm of these cells. The reactivation of the chick nucleus in enucleated fibroblasts parallels the nucleated cells. The results of these studies are interpreted as evidence that there is a specific loss of nuclear function in the senescent cells.     

A COMPARISON OF THE CHANGES IN FINE STRUCTURE OF L CELLS DURING SINGLE CYCLES OF VIRAL MULTIPLICATION, FOLLOWING THEIR INFECTION WITH THE VIRUSES OF MENGO AND ENCEPHALOMYOCARDITIS

The virus of encephalomyocarditis (EMC), examined by the negative-contrast method, is indistinguishable from the serologically related Mengovirus. The particles are 270 to 280 A in diameter. The surface of EMC is composed of an undetermined number of subunits. Frequent sampling of infected cells was carried out throughout one-step cycles of viral multiplication to observe cytopathic changes occurring in L cells infected by these two related RNA viruses. EMC and Mengovirus, which multiply at equal rates, in most respects elicit similar alterations in cell fine structure. Rearrangement and changes in nuclear material accompanied by formation of small vesicles in the centrosphere region commence at 4 to 6 hours after infection. Thereafter a progressive degeneration of the nucleus and vesiculation of the cytoplasm are observed up to 18 to 20 hours. Increased numbers of small dense granules, indistinguishable from ribonucleoprotein particles, appear in the cytoplasm between 8 and 14 hours after infection. L cells infected with Mengovirus become permeable to Erythrocin more slowly than those infected with EMC. Only in the case of Mengovirus infection are large aggregates of dense material first observed in the cytoplasm at 8 hours, followed by the appearance of crystals probably composed of Mengovirus particles, at 12 hours. Differences in the rates of cell permeability after infection with EMC and Mengovirus are discussed in relation to formation of virus crystals and plaque-type mutants.     

Ultrathin structure of normally growing and regenerating cells of Acetabularia mediterranea]

The ultrastructure of nucleus and cytoplasm in regenerating cells of Acetabularia mediterranea differs from that in normally growing ones: the nucleus of regenerating cells form numerous outgrowths; the cortical layer of nucleolus disintegrates markedly; the emission of nucleolar material in the nucleoplasm is observed. In the cytoplasm the portion of active chloroplasts increases and a great number of tubular fibrils appears. In the cytoplasm of regenerating cells the Golgi dictyosomes occur more frequently and the vesicles at their ends are larger than in that of normally growing cells. The changes of macromolecular organization of the nucleus and cytoplasm in question observed at the earlier stages of regeneration (2--12 hrs) suggest the increase of metabolic activity of cells.     

EVIDENCE FROM ELECTRON MICROGRAPHS FOR THE PASSAGE OF MATERIAL THROUGH PORES OF THE NUCLEAR MEMBRANE

1. The nurse cells of Rhodnius possess nucleoli that stain with Heidenhain's hematoxylin but give a negative Feulgen reaction. In localized positions adjacent to the nuclear membrane are seen masses of material both within the nucleus and the adjoining cytoplasm that stain with Heidenhain's hematoxylin, but, like the nucleolus, give a negative Feulgen reaction. 2. Electron micrographs of the nurse cells of Rhodnius reveal the nuclear membrane to contain pores approximately 400 A in diameter. 3. In electron micrographs the nucleolus is seen to be composed of a reticulum containing tightly packed granules. Between the centrally located nucleolus and the nuclear membrane are observed relatively small bunches of granules of the same relative size as those occurring in the nucleolus. Aggregated at certain positions adjacent to the nuclear membrane both within the nucleus and in the adjoining cytoplasm are irregularly shaped masses of granules. Certain of these masses within the nucleus are seen to be continuous with those in the cytoplasm through narrow isthmuses of material extending through pores of the nuclear membrane. Other masses of granules show evidence of preparing to enter the pores by projecting tongues of material toward and into them. In the adjacent cytoplasm pear-shaped masses of granules are seen in front of and in contact with the pores which suggests that they were fixed in the process of or just after completing passage through the pores.     

水稻淀粉胚乳细胞编程性死亡中细胞核变化特征

应用透射电子显微镜技术 ,观察了水稻 (OryzasativaL .)淀粉胚乳细胞编程性死亡过程中核的变化特征。伴随胚乳的发育进程 ,淀粉胚乳细胞核表现出衰退特征 :核变形、染色质凝缩、核膜多处被降解破坏、核基质外泄等。DNALadder显示核内大片段DNA呈严重的弥散状拖尾现象 ,而核内和胞质中在 14 0～ 180bp处有明显的条带。在核衰退的同时 ,其胞质中的粗面内质网、淀粉质体和线粒体等细胞器具有正常的代谢功能 ,细胞仍在合成并积累营养物质 ,淀粉胚乳细胞一边衰退一边行使其功能 ,直至死亡。这些结果表明 ,水稻淀粉胚乳在核衰退的同时 ,细胞仍在积极合成与积累贮藏产物 ,表现为一种特殊形式的植物细胞编程性死亡现象。此外 ,对淀粉胚乳细胞特有的核质关系、植物细胞编程性死亡过程中细胞核的变化等问题进行了讨论。     

Ultrastructure of the larval corpus allatum of Hyphantria cunea drury (Insecta,Lepidoptera)

Summary The corpora allata of the three last larval instars were studied in newly molted animals, at the beginning, middle, and end of the feeding period, and during the molt period. They were found to consist of uniform gland cells, whose ultrastructure changes in the course of the instars.In gland cells considered to be resting, the outer and inner nuclear membranes run in parallel without forming a dilated perinuclear space. Mitochondria are small, polymorphic, with an electron-dense matrix. The smooth endoplasmic reticulum (SER) appears as stacks of parallel cisternae near the nuclear envelope and in the rest of the cytoplasm, and as accumulations of twisted profiles. Occasionally, the SER takes the form of paracrystalline bodies. There are few small smooth-surfaced vesicles in the cytoplasm.In cells considered as active, a dilated perinuclear space occurs. The peripheral ends of profiles forming the SER are swollen, and numerous vesicles and vacuoles bud off from them to fill the cytoplasm. Mitochondria are large, with a more transparent matrix. The plasma membrane of gland cells located just beneath the connective tissue sheath forms numerous small invaginations.The corpora allata consist of resting cells during the molt periods. At the beginning of each instar, few active gland cells appear. In the middle of the second to last and the third to last instars, the bulk of the gland cells is active. At the end of these instars, there are both active and inactive cells. In the middle of the last instar, the gland cells are inactive or subactive, and at its end, all gland cells are completely inactive.     

雌激素受体α和β亚型在文昌鱼神经系统、哈氏窝和性腺中的定位

用兔抗人ER-α和ER-β多克隆抗体对文昌鱼神经系统、轮器哈氏窝和性腺进行免疫细胞化学的定位研究。结果揭示幼年和成年两性不同发育时期文昌鱼在这些部位分布ER-α和ER-β蛋白。ER-α定位在端脑、中脑、后脑和神经管中大多数神经细胞核,少数在胞质及其突起和神经纤维,ER-β则定位在细胞质或细胞膜上,少数在核内。ER—α免疫阳性物质主要分布在哈氏窝下层的上皮细胞核,少数在上层细胞质,β受体则在上层细胞核。在性腺,ER-α分布在卵巢中卵原细胞和小生长期卵母细胞胞质与核仁,生发泡(核)显免疫阴性,在大生长期卵母细胞核膜和核仁的免疫阳性显著增强,成熟期则在卵细胞生发泡表达,ER-β免疫阳性物质分布在卵原细胞和早期卵母细胞质以及成熟卵细胞的卵被膜检测到,生发泡显免疫阴性。在精巢,这两种ER亚型均定位在精原细胞、初级与次级精母细胞和足细胞质,精子细胞在胞核,精子显免疫阴性。另外,双染结果还揭示ER-α和ER-β在上述部位多数共存于同一细胞,少数在不同细胞表达,且在细胞定位有不同。首次发现这两种雌激素受体亚型在文昌鱼有广泛分布,它们介导雌激素对文昌鱼神经内分泌组织的调节作用。α和β受体在靶细胞定位的不同,提示两者在介导雌激素信号路线和基因转录机制可能有不同生理作用。     

Structure of Crystal Cells and Influences of Leaf Development on Crystal Cell Development and Vice Versa in Phaseolus vulgaris (Leguminosae)

The structure of cells with calcium oxalate crystals and their nelghbouring cells has been studied by light and transmission electron microscopy at different stages of bean leaf development. Plants were grown with varying calcium supply to identify a possible influence of calcium nutrition on cell structure. Crystals are formed inside the vacuole of already highly vacuolated cells of bundle sheath extensions. The membrane around the crystal vacuole is continuous with the plasmalemma. The crystal vacuole contains membraneous structures. In the fully expanded leaf the crystal becomes ensheathed by wall material. Chloroplasts of bundle sheath extension cells, with or without crystals, are smaller, with fewer membranes, and with much narrower stroma regions than those of the palisade parenchyma. There is a stage in the young leaf when only the bundle sheath extension cells without crystals have starch grains in their chloroplasts. As their number is lower in plants grown with high calcium supply this means that, in this case, less cells are competent for photosynthesis.     

HuD distribution changes in response to heat shock but not neurotrophic stimulation.

Cellular stress leads to a change in distribution of RNA-binding proteins. HuR, a member of the ELAV/Hu family of RNA-binding proteins, is nuclear in distribution and following heat shock is found in large cytoplasmic stress granules where translation is inhibited. HuD, another ELAV/Hu RNA-binding protein, stabilizes the GAP-43 mRNA in response to nerve growth factor (NGF) stimulation in PC12 cells. We were interested in determining the nuclear distribution of HuD and if neurotrophic stimulation induced changes in the distribution of HuD. In PC12 cells, we found, as expected, that HuR translocates from the nucleus to the cytoplasm in response to heat shock. In response to heat shock, HuD forms large cytoplasmic stress granules, consistent with a role for HuD in the cessation of translation. In unstimulated cells, HuD is distributed in small granules in the cytoplasm and is consistently present at low levels in the nucleus. Stimulation of PC12 cells with NGF induces neuronal differentiation including outgrowth of neurites and increased levels of GAP-43 protein, whereas HuD remains localized in small cytoplasm granules and is still present in the nucleus. These results suggest that, following neurotrophic stimulation, the lack of changes in HuD distribution are due to continued steady state of HuD nuclear shuttling in PC12 cells, or that HuD is not normally shuttled from the nucleus in response to NGF.     

CELL DIVISION IN SPIROGYRA. I. MITOSIS

Dividing cells of Spirogyra sp. were examined with both the light and electron microscopes. By preprophase many of the typical transverse wall micro-tubules disappeared while others were seen in the thickened cytoplasmic strands. Microtubules appeared in the polar cytoplasm at prophase and by prometaphase they penetrated the nucleus. They were attached to chromosomes at metaphase and early anaphase, and formed a sheath surrounding the spindle during anaphase; they were seen in the interzonal strands and cytoplasmic strands at telophase. The interphase nucleolus, containing 2 distinct zones and chromatinlike material, fragmented at prophase; at metaphase and anaphase nucleolar material coated the chromosomes, obscuring them by late anaphase. The chromosomes condensed in the nucleoplasm at prophase, moving into the nucleolus at prometaphase. The nuclear envelope was finally disrupted at anaphase during spindle elongation; at telophase membrane profiles coated the reforming nuclei. During anaphase and early telophase the interzonal region contained vacuoles, a few micro-tubules, and sometimes eliminated n ucleolar material; most small organelles, including swollen endoplasmic reticulum and tubular membranes, were concentrated in the polar cytoplasm. Quantitative and qualitative cytological observations strongly suggest movement of intact wall rnicrotubules to the spindle at preprophase and then back again at telophase.     

Hepatitis delta virus ribonucleoproteins shuttle between the nucleus and the cytoplasm

Hepatitis delta virus (HDV) infection of individuals infected with hepatitis B virus (HBV) is associated with more severe liver damage and an increased risk of fulminant disease. HDV is a single-stranded RNA virus that encodes a single protein, the delta antigen, which is expressed in two forms, small (S-HDAg) and large (L-HDAg). Here we show that although HDV ribonucleoproteins are mainly detected in the nucleus, they are also present in the cytoplasm of cells infected with HDV or transfected with HDV cDNA. Making use of an heterokaryon assay, we demonstrate that HDV ribonucleoproteins shuttle continuously between the nucleus and the cytoplasm. In the absence of HDV RNA, both forms of the delta antigen are retained in the nucleus, whereas in the absence of the delta antigen, HDV RNA is predominantly detected in the cytoplasm. Coexpression of HDV RNA and S-HDAg (which binds to the viral RNA and contains a nuclear localization signal) results in nuclear accumulation of the viral RNA. This suggests that HDV RNA mediates export of viral particles to the cytoplasm whereas the delta antigen triggers their reimport into the nucleus.     

Experimental study of intracellular ice growth in human umbilical vein endothelial cells

Study of the intracellular ice formation (IIF) and growth is essential to the mechanistic understanding of cellular damage through freezing. In the aid of high speed and high resolution cryo-imaging technology, the transient intracellular ice formation and growth processes of the attached human umbilical vein endothelial cells (HUVEC) were successfully captured during freezing. It was found that the intracellular ice nucleation site was on the cell membrane closer to the nucleus. The ice growth was directional and toward the nucleus, which covered the whole nucleus before growing into the cytoplasm. The crystal growth rate in the nucleus was much larger than that in the cytoplasm, and its morphology was influenced by the cooling rate. During the thawing process, small crystals fused into larger ones inside the nucleus. Moreover, the cumulative fraction of the HUVEC with IIF was mainly dependent on the cooling rate not the confluence of the cells attached.     

Ultrastructure of Mikrocytos mackini, the cause of Denman Island disease in oysters Crassostrea spp. and Ostrea spp. in British Columbia, Canada

An ultrastructural study was carried out on Mikrocytos mackini, the cause of Denman Island disease in Pacific oysters Crassostrea gigas in western Canada. Three forms were identified, quiescent cells (QC), vesicular cells (VC) and endosomal cells (EC). QC occurred in the vesicular connective tissue (VCT), haemocytes (hyalinocytes), adductor and heart myocytes, and extracellularly. They had a central round to ovoid nucleus, < 7 cisternae of inactive nuclear membrane-bound Golgi, few vesicles and lysosome-like bodies. VC were rarely extracellular and usually occurred in adductor and heart myocytes, in close association with host cell mitochondria. The contents of the host cell mitochondria appeared to pass through a tubular extension into the cytoplasm of the parasite. Cytoplasmic vesicles resembled the tubular structure in appearance and size. EC occurred in the VCT, in haemocytes and extracellularly. They had a dilated nuclear membrane, sometimes containing a looped membranous structure that appeared to derive from the nucleus, and pass into the cytoplasm. A well-developed anastomosing endoplasmic reticulum connected the nuclear and plasma membranes, and endosomes were present in the cytoplasm. QC and EC cells were frequently observed tightly against, or between, the nuclear membranes of the host cell. Few organelles occurred in all forms of M. mackini, especially QC. The lack of organelles found in most eukaryotic cells, including mitochondria or their equivalents, may be due to obligate parasitism and the utilization of host cell organelles reducing the need for parasite organelles. Alternatively, perhaps M. mackini is a primitive eukaryote. Although phylogenetic affinities could not be determined, it is not a haplosporidian. A developmental cycle is proposed from these findings.     

Fine structure of the giant aflagellate spermatozoon in pseudostomum quadrioculatum (leuckart) (platyhelminthes,prolecithophora)

The giant aflagellate spermatozoa of P. quadrioculatum are composed of two different parts: a thicker head piece and a more slender tail piece. In the head there exist a large elongated nucleus and an elongated mitochondrial derivative situated in a groove-like cavity of the nucleus. In mature spermatozoa the nuclear material is arranged in many small membrane bounded areas. Both structures, nucleus and mitochondrial derivative, are spirally coiled. The outer part of the membrane in the mitochondrial derivative forms many loop-like foldings. Both organelles continue to the tail in form of two small, helically coiled ribbons; the nucleus is anchored within the mitochondrial derivative by an electron-opaque process. A sheath of spirally-orientated cortical microtubules starting from the tip of the head runs to the tip of the tail under the cell membrane. In addition, a second sheath of tubules occurs in the tail region, these tubules also run parallel to each other, but in the opposite direction to the microtubules of the outer sheath.The possible relations between the structures observed and the motility of the spermatozoa are discussed; in addition, some phylogenetic comments are attempted.Abbreviations c — cerebrum - com — cortical microtubules - cop — copulatory organ - fm — foldings of the mitochondrial membrane - l — lattice - mid — mitochondrial derivative - mt — microtubules - n — nucleus - ne — nuclear envelope - ph — pharynx - pn — protonephidium - rp — ribbon-like nuclear process - te — testis - tt — testis - tt — tip of the tail - vi — vitellarium - vs — vesicula seminalis     

Mechanism of Intranuclear Crystal Formation of Herpes Simplex Virus as Revealed by the Negative Staining of Thin Sections

Structural alterations induced in HeLa cells by herpes simplex virus and the mechanism whereby the virus is formed in the nucleus in crystal arrays were studied by electron microscopy with both the usual and negatively stained sections. Aggregates of granular and filamentous material were observed in the cytoplasm of infected cells with both sections. On the other hand, no remarkable alterations in appearance of the cytoplasmic ground substance were observed with the usual sections of infected cells. However, the cytoplasmic ground substance of infected cells when negatively stained consisted of granular material which was different in appearance from the spongy material constituting the cytoplasmic matrix of uninfected cells. In the nucleus of infected cells, complexes consisting of round bodies, amorphous material, aggregates of uniform granules in rows, and viral crystals were often observed near the nuclear membrane in both types of sections. Examinations of the granular aggregates with negatively stained sections suggested that each granule represents a subunit and that the several adjoining subunits (approximately eight) constitute the requirement for formation of a single viral capsid with a core. Thus, rapid and simultaneous formation of the core and capsid within the aggregate would replace the rows of the granules with the viral crystal. The advantages of negative staining of thin sections for visualization of fine structural alterations are discussed.     

Fine structure of the spermatozoa of Chiton marginatus (mollusca: Amphineura), with special reference to nucleus maturation

The spermatozoon of Chiton marginatus is a long uniflagellate cell displaying structural features of “modified sperm.” The nucleus presents a conical shape with a long apical cylindrical extension. The chromatin is homogeneously dense. Scattered inside the condensed nucleus, a few nuclear lacunae are visible. The acrosomal complex is lacking. Some mitochondria are located in a laterofrontal structure side by side with the nucleus. The typical midpiece is absent. The cytoplasm forms a thin layer around the nucleus and the mitochondria. The proximal centriole is in a basal nuclear indent. The distal centriole serves to form the axoneme tail with the usual microtubular pattern. During nuclear maturation, the early spermatid nucleus is spherical and contains fine granular chromatin patches. The nuclear envelope shows a deposit of dense material at the base of the nucleus, forming a semicircular invagination occupied by a flocculent mass. In middle spermatid stage, the chromatin gets organized in filaments, coiled as a hank, attached over the inner surface of the basal thickening of the nuclear envelope. The nucleus starts to elongate anteroposteriorly. At the pointed apical portion of the spermatid, a group of microtubules is observed seeming to impose external pressure to the nucleus giving rise to the long apical nuclear point. The mitochondria have a basal position. Late spermatids have an elongated conical nucleus. The chromatin filaments are further condensed, and lacunae appear inside the nucleus. Some mitochondria migrate to a lateral position.     

Monitoring the disruption of nuclear envelopes in interphase cells with GFP-beta-galactosidase.

The nuclear envelope of eukaryotic cells provides a barrier separating nucleus from cytoplasm, thereby regulating the exchange of macromolecules between both compartments. However, in cells exposed to severe forms of stress this barrier may break down, resulting in the mixing of nuclear and cytoplasmic contents. We show here that the fusion protein GFP-beta-galactosidase can be used to evaluate the intactness of nuclear envelopes in HeLa cells that have been exposed to heat and oxidative stress. GFP-beta-galactosidase is restricted to the cytoplasm of interphase cells, but enters the nucleus when nuclear membranes are disrupted. For comparison, we have analyzed the barrier function of nuclear membranes with antibodies against lamin B. Treatment of fixed cells with digitonin permeabilizes the plasma membrane, but leaves nuclear envelopes intact. Consequently, after digitonin incubation antibodies to lamin B can bind their antigen only if nuclear membranes are damaged. For various heat and oxidative stress conditions, we have compared the distribution of GFP-beta-galactosidase with the accessibility of lamin B to antibodies. Our results demonstrate that nuclear envelopes are permeable to antibodies whenever GFP-beta-galactosidase enters the nucleus. GFP-beta-galactosidase is therefore a useful tool for evaluating the disintegration of the nuclear envelope and identifying cells in which a mixing of nuclear and cytoplasmic material takes place.