Changes In Cytoplasmic Organization Research Articles

Actin and myosin regulate cytoplasm stiffness in plant cells: a study using optical tweezers

Here, we produced cytoplasmic protrusions with optical tweezers in mature BY-2 suspension cultured cells to study the parameters involved in the movement of actin filaments during changes in cytoplasmic organization and to determine whether stiffness is an actin-related property of plant cytoplasm. Optical tweezers were used to create cytoplasmic protrusions resembling cytoplasmic strands. Simultaneously, the behavior of the actin cytoskeleton was imaged. After actin filament depolymerization, less force was needed to create cytoplasmic protrusions. During treatment with the myosin ATPase inhibitor 2,3-butanedione monoxime, more trapping force was needed to create and maintain cytoplasmic protrusions. Thus, the presence of actin filaments and, even more so, the deactivation of a 2,3-butanedione monoxime-sensitive factor, probably myosin, stiffens the cytoplasm. During 2,3-butanedione monoxime treatment, none of the tweezer-formed protrusions contained filamentous actin, showing that a 2,3-butanedione monoxime-sensitive factor, probably myosin, is responsible for the movement of actin filaments, and implying that myosin serves as a static cross-linker of actin filaments when its motor function is inhibited. The presence of actin filaments does not delay the collapse of cytoplasmic protrusions after tweezer release. Myosin-based reorganization of the existing actin cytoskeleton could be the basis for new cytoplasmic strand formation, and thus the production of an organized cytoarchitecture.

High Viscosity and Anisotropy Characterize the Cytoplasm of Fungal Dormant Stress-Resistant Spores

Ascospores of the fungus Talaromyces macrosporus are dormant and extremely stress resistant, whereas fungal conidia--the main airborne vehicles of distribution--are not. Here, physical parameters of the cytoplasm of these types of spores were compared. Cytoplasmic viscosity and level of anisotropy as judged by spin probe studies (electron spin resonance) were extremely high in dormant ascospores and during early germination and decreased only partly after trehalose degradation and glucose efflux. Upon prosilition (ejection of the spore), these parameters fell sharply to values characteristic of vegetative cells. These changes occurred without major volume changes that suggest dramatic changes in cytoplasmic organization. Azide reversibly inhibited prosilition as well as the decline in cytoplasmic parameters. No organelle structures were observed in etched, cryoplaned specimens of ascospores by low-temperature scanning electron microscopy (LTSEM), confirming the high cytoplasmic viscosity. However, cell structures became visible upon prosilition, indicating reduced viscosity. The viscosity of fresh conidia of different Penicillium species was lower, namely, 3.5 to 4.8 cP, than that of ascospores, near 15 cP. In addition the level of anisotropic motion was markedly lower in these cells (h(0)/h(+1) = 1.16 versus 1.4). This was confirmed by LTSEM images showing cell structures. The decline of cytoplasmic viscosity in conidia during germination was linked with a gradual increase in cell volume. These data show that mechanisms of cytoplasm conservation during germination differ markedly between ascospores and conidia.

Relative messenger RNA abundance in bovine oocytes collected in vitro or in vivo before and 20 hr after the preovulatory luteinizing hormone surge

In the cyclic cow, final maturation of the ovulatory follicle is initiated by the preovulatory luteinizing hormone (LH) surge. During the subsequent 24 hr period, the oocyte nucleus undergoes meiotic progression to metaphase II and several changes in cytoplasmic organization take place. We have previously shown that oocytes recovered at the time of the LH peak and matured in vitro are less competent to reach the blastocyst stage than their counterparts recovered 20 hr later following in vivo maturation, despite both groups undergoing IVF and culture in parallel. The objective of this study was to compare, using real-time quantitative RT-PCR, the relative abundance of various developmentally important gene transcripts in these oocytes. The groups used were mature bovine oocytes originating from: (1) 2-6 mm follicles from slaughterhouse ovaries; (2) preovulatory follicles punctured by ovum pick-up just before the LH surge (i.e., immature) and matured in vitro; or (3) preovulatory follicles punctured 20 hr later, just prior to ovulation (i.e., in vivo matured). In addition, immature oocytes from 2-6 mm follicles were examined. We examined the relative mRNA expression of five enzymes involved in protection against free oxygen radicals (mitochondrial Mn-superoxide dismutase, MnSOD, cytosolic Cu/Zn superoxide dismutase, Cu/ZnSOD, gamma-glutamyl-cysteine transferase, GCS, glutathione peroxidase, GPX, sarcosine oxidase, SOX), a transcript involved in follicular development (growth differentiation factor-9, GDF-9), transcripts involved in glucose metabolism (glucose-6-phosphate dehydrogenase, G6PDH, glucose transporter type-1 and -8, Glut-1, Glut-8) and genes involved in cell cycle events, Cyclin A and B, and poly(A) polymerase (PAP). Transcripts for all genes were detected, irrespective of oocyte origin. While differences were not significant in all cases, variations in levels of transcript abundance between the groups were related to developmental competence. In particular, transcripts for GDF-9 were expressed at significantly higher levels in oocytes recovered at the LH peak and matured in vitro than in those matured in vivo. The observations with GDF-9 are interesting as this gene is believed to be essential for normal folliculogenesis and may be important in the regulation of early follicle and oocyte growth. In conclusion, the results of this study demonstrate differences in the relative mRNA abundance of several developmentally important gene transcripts in bovine oocytes which may be related to developmental competence.

Ultrastructural characterization and Fourier analysis of fiber cell cytoplasm in the hyperbaric oxygen treated guinea pig lens opacification model

The structural characteristics of differentiated fiber cells in control and hyperbaric oxygen (HBO)-treated guinea pig lenses were examined by transmission electron microscopy (TEM). Emphasis was placed on cell damage, membrane integrity, and cytoplasmic texture. Given the faint gross opacities observed in HBO-treated lenses in previous studies, it was hypothesized that subtle but significant morphological differences due to oxidative damage exist between control and treated animals. Experimental animals received either 70 or 85 treatments with HBO (2·5 atm of 100% O 2 for 2·5 hr, 3 times per week for 5–7 months). All specimens were obtained within 24 hr of death. Freshly cut Vibratome lens sections were fixed and processed for low and high-magnification thin-section TEM analysis. Cytoplasmic texture was analyzed using Fourier and autocorrelation image processing techniques. Low-magnification analysis revealed relatively insignificant differences in general appearance between the fiber cells of the inner fetal and embryonic nuclei in control and HBO-treated guinea pigs. Both groups demonstrated cells of similar morphology with equivalent membrane complexity and homogeneous cytoplasmic texture. Evidence of any major cellular damage or extracellular space debris was not obvious. High-magnification analysis of the cytoplasm of the treated lenses exhibited a mild, yet detectable increase in texture compared with controls and was confirmed by Fourier analysis. Cytoplasmic texture increased in complexity with additional treatments. The absence of major cellular damage in the lenses of HBO-treated animals suggests a less conspicuous source of light scattering. The small changes in cytoplasmic organization observed between treated and control animals may entirely account for the increase in nuclear light scattering observed by slit lamp. The results obtained with this guinea pig/HBO model parallel many of the morphological data associated with human nuclear cataracts. The high-angle scattering observed in the lens of the HBO-treated guinea pig may represent the type of cytoplasmic reorganization that occurs with mild oxidation, effectively making it a valuable model for human lens aging.

Human Uterine Epithelial RL95-2 Cells Reorganize Their Cytoplasmic Architecture with Respect to Rho Protein and F-Actin in Response to Trophoblast Binding

Embryo implantation is initiated by interaction of trophoblast with uterine epithelium via the apical cell poles of both partners. Using spheroids of human trophoblastoid JAR cells and monolayers of human uterine epithelial RL95-2 cells to simulate this initial interaction, we previously demonstrated that formation of stable cell-to-cell bonds depends on actin cytoskeleton (F-actin) and small GTPases of the Rho family, most likely RhoA. In this study, we determined the apical as well as the basal distribution of these proteins by fluorescence confocal microscopy before and after binding of JAR spheroids. We focussed on changes in cytoplasmic organization with respect to apicobasal polarity of RL95-2 cells. Before binding of spheroids, significantly higher fluorescence signals of RhoA [37 ± 6 grey scale values (gsv)] and of F-actin (41 ± 3 gsv) were found in the basal region of RL95-2 cells as compared to the apical pole (RhoA: 24 ± 3 gsv, F-actin: 28 ± 2 gsv). After binding of JAR spheroids, this apicobasal asymmetry was inverted (RhoA: 55 ± 10 gsv apical vs. 25 ± 3 gsv basal; F-actin: 108 ± 17 gsv apical vs. 57 ± 7 gsv basal). Inactivation of Rho GTPases in RL95-2 cells by Clostridium difficile toxin A leads to a loss of their apical adhesion competence, as previously published. Here, we observed a uniform distribution of RhoA and F-actin between apical and basal region rather than an asymmetric one in toxin A-treated cells. These data suggest that activation of Rho GTPases and coordinated rearrangement of F-actin within uterine epithelial cells in response to trophoblast binding are part of a generalized structural and functional reorganization of the cytoplasm. This involves not only the immediate contact zone (apical) but also the opposite (basal) cell pole and may be a critical element of uterine epithelial reactions during transition between trophoblast adhesion and transmigration.

Microtubule organization in root cells of Medicago truncatula during development of an arbuscular mycorrhizal symbiosis with Glomus versiforme.

The colonization of plants by arbuscular mycorrhizal fungi has been shown to induce changes in cytoplasmic organization and morphology of root cells. Because of their role in a variety of cellular functions in plants, it is likely that microtubules are involved either in the signaling events leading to the establishment of the symbiosis or in changes in host cell morphology and cytoplasmic architecture. Recent studies of the arbuscular mycorrhizal symbiosis have shown that root cortical cells reorganize their microtubules upon colonization. These studies, however, have focused primarily on the cells containing hyphal coils or arbuscules and did not include descriptions of microtubule changes in adjacent cells. To probe further into the potential role of the microtubule cytoskeleton in the establishment of arbuscular mycorrhizal symbiosis, we examined the three-dimensional arrangement of microtubules in roots of the model legume Medicago truncatula colonized by the arbuscular mycorrhizal fungus Glomus versiforme by indirect immunofluorescence and confocal microscopy. Our data show extensive remodeling of the microtubule cytoskeleton from the early stages of arbuscule development until arbuscule collapse and senescence. While confirming some of the microtubule patterns shown in other mycorrhizal systems, our results also reveal that cortical cells adjacent to those containing arbuscules or adjacent to intercellular hyphae reorganize their microtubules. This indicates that the cortical cells initiate the modification of their cytoskeleton prior to entry of the fungus and is consistent with signal exchange between the symbionts prior to fungal penetration of the cells.

Aluminum toxicity studies in Vaucheria longicaulis var. macounii (Xanthophyta, Tribophyceae). I. Effects on cytoplasmic organization

Using differential interference contrast (DIC) and epifluorescence microscopy, we tested the hypothesis that exposure to environmentally significant levels of aluminum (Al) would cause rapid changes in cytoplasmic organization in vegetative filaments of the coenocytic alga, Vaucheria longicaulis Hoppaugh var. macounii Blum resulting in the loss of cytoplasmic streaming. In untreated cells, DIC microscopy revealed the presence of cortical cytoplasmic strands that were oriented longitudinally to the cell axis as well as sub-cortical cytoplasmic strands that exhibited a reticulate morphology. Organelles such as chloroplasts and mitochondria translocated throughout the cell in close association with the cortical longitudinal cytoplasmic strands. Staining with the lipophilic dye, 3,3-dihexyloloxacarbocyanine, revealed structures that appeared to be endoplasmic reticulum (ER). This organelle closely resembled, in location and appearance, the cytoplasmic strands visualized using DIC microscopy. The addition of Al (80 μM) resulted in the inhibition of cytoplasmic streaming as well as the dissipation of the putative cortical longitudinal ER within one minute. Subsequently, the DIC-visible cortical cytoplasmic strands exhibited progressive degrees of disorganization. Throughout these changes, chloroplasts and mitochondria remained visibly associated with the cortical cytoplasmic strands.

Nuclear differentiation in the filamentous caulonema of the moss Funaria hygrometrica.

Nuclei from different cell types in plants and animals show many features of differentiation; they differ in shape, volume, structure, ultrastructure and in the distribution of nuclear components. Using the filamentous caulonema of the moss Funaria hygrometrica Hedw. this study records the changes in cytoplasmic organization alongside the reorganization of the interphase nucleus, Events taking place in the meristematic cells at or near the lip of the advancing caulonemal filaments (e.g. acquisition of polarity, tip growth, nuclear and cell division, side branch initiation] are associated with haploid nuclei (1C DXA amount 0.5 pg) that are spherical or slightly oval, with no blocks of condensed chromatin, and a large central nucleolus with a large granular component. Maturation of the caulonemal cells involves wall thickening and pigmentation concomitant with suspension of elongate plastids in linear arrays along endoplasmic strands. Many cells become highly polarized with the majority of the organelles at their apical ends. These eytoplasmic changes are associated with endoreduplication of the genome to about 8C, endoreduplication occurs by amplification of the 1C genome to give nuclei with IC-SC DNA amounts. There is no evidence of differential amplification of the genome. The amplification in the copy number of ribosomal RNA genes is associated with the heterochromatinisation of the genes within the nucleolus. At the same time the nucleolus reduces in volume owing to a diminution of the granular component and all components of the nucleolus become spatial separate. There is an increased nuclear volume associated with endoreduplication and the nucleus elongates causing an increase in the surface area of the nuclear envelope. These major nuclear reorganizations are associated with a stable distribution of the 'D' polypeptide involved in pre-mRNA splicing. Scrutiny of published data suggests that similar differentiation events might be encountered commonly in other organisms. The changing nuclear morphology probably reflects the changing activity of the nucleus and the cell. It might be that nuclear reorganization changes the balance of genes or gene products and the spatial distribution of the component pans to enable the new nuclear functions. These results suggest that nuclear differentiation is a fundamental feature of cell differentiation.

Comparative ultrastructural study ofDirofilaria repens(Nematoda: Filarioidea) development in susceptible and refractory strains ofAedes aegypti

Abstract A comparative study of the development of Dirofilaria repens in resistent and susceptible strains of Aedes aegypti was performed to acquire more information about the ultrastructural events in the Malpighian tubules and hemocoel of the mosquito, and to investigate the defense mechanisms activated by the host. Larval growth in the primary cells of the Malpighian tubules of the susceptible strain, associated with increasing changes in cytoplasmic organization of the host‐cell leading to cell death, were observed by scanning and transmission electron microscopy. When the parasite reaches the L3 stage and migrates to the hemocoel, hemocytes rarely intervene and seem ineffective. In the resistent strain, it was observed that the destiny of D. repens is decided in the Malpighian tubules where the intracellular development of penetrating microfilaria is blocked by chemical and/or biochemical defense mechanisms activated by the host.

Ultrastructure and histochemistry of lipoidal droplets in vessel contact cells and adjacent parenchyma cells in cotton plants infected by Fusarium oxysporum f.sp. vasinfectum

Osmiophilic droplets developed in contact cells and adjacent vascular parenchyma cells of cotton accompanied by changes in cytoplasmic organization after vessel infection by Fusarium oxysporum f.sp. vasinfectum. In the early stages of the cellular response to vessel infection, the droplets were observed in dilations of ER cisternac, the membrane envelopes of mitochondria, and occasionally the membrane envelopes of plastids and nuclei. As the cellular response to vessel infection proceeded and cytoplasmic reorganization became apparent, the droplets were associated with vesicles or remained as individual droplets within the cytoplasm. The developmental sequence suggests that the osmiophilic substances in these droplets may be secreted through the plasmalemma, infused into apposition layers, and exuded into the infected vessels through pits. Sudan Black B staining and organic solvent extraction indicate that the osmiophilic droplets are lipoidal in nature. It is suggested that the lipoidal substances are terpenoids that could function as phytoalexins.

Serum mediated changes in cell attachment and cytoplasmic organization of B16 melanoma correlates with selective alterations in secreted proteins

B16 melanoma cells exhibited decreased adhesion to substration and the presence of cytoplasmic granules, resembling lipid inclusions, when cultured in vitro in the presence of syngenic serum from C57 BL6 mice. This constrasted with the rapid cell attachment and absence of cytoplasmic granules in cultures seeded in medium supplemented with an identical concentration of fetal bovine serum. Electrophoretic comparison of intracellular proteins revealed similar patterns in detergent-soluble and matrix-associated proteins from cells grown with bovine or mouse serum. However, a similar analysis of the conditioned media showed clear differences in methionine-rich species which migrated in the 100 kd region in cells grown with bovine serum, and as 110 kd component in cells grown with mouse serum. Our data indicate that the poor attachment to substratum and changes in cytoplasmic organization of B16 melanoma cells, is primarily associated with specific changes in secreted proteins.

Sporangiogenesis in Three Genera of the Saprolegniaceae. I. Pre-Sporangium Hyphae to Early Primary Spore Initial Stage

The changes in cytoplasmic organization up to the early stages of primary spore initial delimitation are described in sporangia of four species of Saprolegniaceae. Organelle associations characteristic of asexual and vegetative stages are present in the cytoplasm of presporangium hyphae of Achlya debaryana. Organelles are aligned parallel to the axis of hyphal growth and the apex assumes the characteristic profile of a sporangium. Dense-body vesicles possibly originate from expanded cisternae of endoplasmic reticulum at this stage. In Dictyuchus anomalus, D. pseudodictyon, and Brevilegnia minutandra, nuclei of the primary spore initial stage orient radially in the cytoplasm with nucleus-associated organelles close to the sporangium wall. Nuclei in the central region of sporangia degenerate.

Sporangiogenesis in Three Genera of the Saprolegniaceae. I. Pre-Sporangium Hyphae to Early Primary Spore Initial Stage

The changes in cytoplasmic organization up to the early stages of primary spore initial delimitation are described in sporangia of four species of Saprolegniaceae. Organelle associations characteri...

Observations on chick-embryo lens histogenesis in vivo and in vitro.

The 6-day chick-embryo lens anterior epithelium will undergo histogenesis in vitro. A 'normal' epithelial curvature in vitro is required for extensive fiber-cell elongation; a reversed curvature, or flat epithelial conformation, results in considerably reduced cell extension. With a 'normal' epithelial curvature, cell elongation in vitro progresses rapidly for about 3 days, and changes in cytoplasmic organization (ultrastructure) take place that are similar to those associated with fiber-cell differentiation in vivo. However, these cells do not achieve as full a cytoplasmic differentiation as was observed in the most differentiated cells of the normal embryonic lens. Epithelia explanted with a reversed curvature, while evidencing reduced cell elongation, had, after 3 days in vitro, differentiated (according to criteria of ultrastructure and relative proportions of soluble proteins) in a manner similar to those epithelia explanted with a 'normal' curvature and in which extensive cell elongation had been demonstrated.