Intranuclear actin microfilaments in the oocytes of the common frog

For identification and distribution of actin microfilaments in hand-isolated nuclei of R. temporaria oocytes (stage 6, according to Dumont, 1972) different methods were used: heavy meromyosin decoration, antiactin immunofluorescence with monoclonal antibodies, staining with rhodamine phalloidin, and electrophoresis in polyacrylamide gel. The nuclei of R. temporaria oocytes contain a considerable quantities of actin microfilaments which form intranuclear meshwork. Microfilaments are connected with the nucleoli, nucleolar RNP-complexes and nuclear envelope. Immunofluorescence with antiactin monoclonal antibodies reveals a strong staining of microfilaments and nucleoli. A slight staining of nucleoli is observed after the treatment of nuclei with rhodamine phalloidin. A specific role of intranuclear microfilaments in direct transport of nucleolar material from the nucleus into the oocyte cytoplasm, in stabilization of the karyosphere (the late diplotene oocyte complex of chromosomes with numerous nucleoli) is discussed in addition to its keeping in a definite region of the nucleus. A supposition is drawn on the functional significance of the connection between microfilaments and nuclear matrix. Based on our own and literature data, a conclusion is drawn, that the intranuclear filament actin may be one of the leading components in morpho-functional organization of the nucleus as the whole.     

An actin filament matrix in hand-isolated nuclei of X. laevis oocytes

The nuclear gel of Xenopus oocytes contains a meshwork of randomly oriented microfilaments which have been identified as F-actin by decoration with rabbit skeletal muscle myosin subfragment-1 (S-1). Nuclear gel preparations treated with S-1 differ in several respects from control preparations incubated in either aqueous medium alone, or medium containing BSA. Actin filaments in control preparations appear less well preserved than those in S-1 treated preparations of the nuclear gel. The nucleoli of control preparations are extremely dense, while those of S-1 treated preparations have a more open, granular appearance. Large granular aggregates, which are a prominent feature of the controls, are seen much less frequently in S-1-treated preparations of the nuclear gel. These morphological differences appear to be correlated with the binding of protein to F-actin, since nuclear gel preparations incubated in tropomyosin, which also binds to actin filaments, appear similar to those treated with S-1. Approximately 63% of the total nuclear actin exists in a globular state, while 37% is filamentous.     

Dynamics of nuclear actin bundle induction by dimethyl sulfoxide and factors affecting its development

We have reported in a previous paper that dimethyl sulfoxide (DMSO) induces the formation of huge bundles of actin filaments in the nuclei of Dictyostelium mucoroides. The present study was performed to provide electron microscope data on the induction of nuclear actin bundles, illustrating both their formation and their reversion, as well as on the effects of various factors on the induction. The large nuclear bundles of actin appeared after 20--30 min of treatment with 10% DMSO. A DMSO concentration of 5 or 10% was optimal for the induction of the bundles. The nuclear actin bundle reverted to the original morphology within 5 min after removing DMSO. Induction of nuclear actin bundles was inhibited by Mg++ and low temperatures, but not by Na+, K+, Ca++, ATP, 3'5'-cyclic adenosine monophosphate (cAMP), phosphate buffer, or cytochalasin B. Neither NaN3 nor cycloheximide totally inhibited the induction of the bundles.     

Regulation of microvillus structure: calcium-dependent solation and cross-linking of actin filaments in the microvilli of intestinal epithelial cells

The bundle of filaments within microvilli of intestinal epithelial cells contains five major proteins including actin, calmodulin, and subunits of 105-, 95-, and 70-kdaltons. It has been previously shown (Howe, C. L., M. S. Mooseker, and T. A. Graves. 1980. Brush-border calmodulin: a major component of the isolated microvillus core. J. Cell Biol. 85: 916-923) that the addition of Ca++ (> 10(-6) M) to microvillus cores causes a rapid, drastic, but at least partially reversible disruption of this actin filament bundle. High-speed centrifugation of microvillus cores treated with Ca++ indicates that several core proteins are solubilized, including 30-50% of the actin and calmodulin, along with much of the 95- and 70-kdalton subunits. Gel filtration of such Ca++ extracts in the presence and absence of Ca++ indicates that microvillar actin "solated" by Ca++ is in an oligomeric state probably complexed with the 95-kdalton subunit. Removal of Ca++ results in the reassembly of F-actin, probably still complexed with 95- kdalton subunit, as determined by gel filtration, cosedimentation, viscometry, and electron microscopy. The 95-kdalton subunit (95K) was purified from Ca++ extracts by DEAE-Sephadex chromatography and its interaction with actin characterized by viscometry, cosedimentation, and EM in the presence and absence of Ca++. In the presence, but not absence, of Ca++, 95K inhibits actin assembly (50% inhibition at 1:50- 60 95K to actin) and also reduces the viscosity of F-actin solutions. Similarly, sedimentation of actin is inhibited by 95K, but a small, presumably oligomeric actin- 95K complex formed in the presence of Ca++ is pelletable after long-term centrifugation. In the absence of Ca++, 95K cosediments with F-actin. EM of 95K-actin mixtures reveals that 95K "breaks" actin into small, filamentous fragments in the presence of Ca++. Reassembly of filaments occurs once Ca++ is removed. In the absence of Ca++, 95K has no effect on filament structure and, at relatively high ratios (1:2-6) of 95K to actin, this core protein will aggregate actin filaments into bundles.     

Actin cytoskeleton reorganization of the apoptotic nurse cells during the late developmental stages of oogenesis in Dacus oleae

In the present study, we demonstrate the actin cytoskeleton reorganization during nurse cells apoptosis of the olive fruit fly Dacus oleae. At the developmental stage 9A of oogenesis, the actin microfilaments are assembled in numerous ring canals and subcortically support all the nurse cells, as is shown by phalloidin-FITC staining. During the following stages, 9B and 10A, this structural pattern remains the same. The developmental stage 10B is characterized by actin microfilament rearrangement and formation of actin cables that are symmetrically organized around the nurse cell nuclei. At stage 11, when the dumping process begins, these actin cables seem to retain each nurse cell nucleus in the cell center, away from blocking the ring canals. The early stage 12 is characterized by an asynchronous nurse cell nuclear chromatin condensation, while at late stage 12 the actin cables become very thick, as adjacent ones overlap one another and traverse the disorganized apoptotic nurse cell nuclei that already have fragmented DNA, as is demonstrated by acridine orange staining and TUNEL assay. Finally, during stage 13, the apoptotic nuclear remnants are phagocytosed by the neighboring follicle cells. The data presented herein compared to previous reported results in Drosophila [Nezis et al., 2000: Eur J Cell Biol 79:610-620], demonstrate that actin cytoskeleton reorganization during nurse cell apoptosis is a developmentally regulated physiological mechanism, phylogenetically conserved in higher Dipteran.     

Intercellular cytoplasm transport during oogenesis of the moth midge, Tinearia alternata Say (Diptera: Psychodidae)

Polytrophic ovaries of the nematocerous dipteran, Tinearia alternata Say consists of several developmentally synchronized ovarioles each housing only one functional egg chamber with 15 nurse cells and an oocyte. At the early stages of previtellogenesis the nurse cells become polyploid and synthetically active. Their nuclei contain polytene chromosomes and prominent nucleoli. With the advance of previtellogenic growth the nurse cell cytoplasm is loaded with the growing number of ribosomes and contain perinuclear nuage material, mitochondria, electron dense bodies and aggregations of endoplasmic reticulum. All these organelles are transported into the oocyte thanks to the massive and rapid flow of the nurse cell cytoplasmic contents. Nurse cell-oocyte transport is mediated by actin cytoskeleton. Prior to the rapid cytoplasm transfer, F-actin network is associated with the nurse cell membranes while tiny bundles of microfilaments form actin baskets connected with ring canals. Nurse cells in Tinearia lack an extensive scaffold of radially oriented, F-actin bundles (cables) that would tether their nuclei in place, thus preventing ring canals from plugging. The way the nuclei are anchored to their central positions within the cells remains unclear. Towards the final stages of oogenesis nurse cells are almost devoid of cytoplasm and degenerate. Although their nuclei undergo dramatic morphological transformations, typical hallmarks of apoptotic pathway could not be clearly observed. Rapid ooplasmic streaming does not occur.     

The effects of insulin,cycloheximide and phalloidin on the content of actin and p35 in extracts prepared from the nuclear fraction of Krebs II ascites cells

The nuclear fraction isolated from Krebs II ascites cells following cell disruption by nitrogen cavitation was separated into four fractions by salt/detergent extraction: NP-40 soluble fraction, 130 mM KCl extract, DOC/Triton × 100 soluble fraction and salt/detergent treated nuclei. The protein composition of the individual fractions was studied by SDS-PAGE and the relative amounts of actin and a 35 kDa protein (p35) were measured from gel scans. There was a time-dependent shift of actin from the 130 mM KCl extract to the NP-40 soluble fraction upon storage of the nuclear fraction on ice, indicating a progressive depolymerization of microfilaments. Compared with actin there was a slower release of p35 into the NP-40 soluble fraction. The results suggest that p35 is not integrated in the microfilament network. Phalloidin, which stabilizes the microfilaments, enriched the amount of both proteins in the 130 mM KCl extracts, together with a series of other proteins in the range 50–205 kDa. The presence of phalloidin also resulted in a large increase in the actin content in both the DOC/Triton × 100 extract and the fraction containing salt/detergent treated nuclei. Incubation of cells with insulin and/or cycloheximide enriched the amount of actin in the 130 mM KCl fraction. The results show that short term incubation of cells with phalloidin, insulin or cycloheximide increases the actin content of the nuclear fraction and also affects the presence of several other proteins.     

Involvement of microtubules and 10-nm filaments in the movement and positioning of nuclei in syncytia

Previous studies (Holmes, K.V., and P.W. Choppin. J. Exp. Med. 124:501- 520; J. Cell Biol. 39:526-543) showed that infection of baby hamster kidney (BHK21-F) cells with the parainfluenza virus SV5 causes extensive cell fusion, that nuclei migrate in the syncytial cytoplasm and align in tightly-packed rows, and that microtubules are involved in nuclear movement and alignment. The role of microtubules, 10-nm filaments, and actin-containing microfilaments in this process has been investigated by immunofluorescence microscopy using specific antisera, time-lapse cinematography, and electron microscopy. During cell fusion, micro tubules and 10-nm filaments from many cells form large bundles which are localized between rows of nuclei. No organized bundles of actin fibers were detected in these areas, although actin fibers were observed in regions away from the aligned nuclei. Although colchicine disrupts microtubules and inhibits nuclear movement, cytochalasin B (CB; 20-50 microgram/ml) does not inhibit cell fusion or nuclear movement. However, CB alters the shape of the syncytium, resulting in long filamentous processes extending from a central region. When these processes from neighboring cells make contact, fusion occurs, and nuclei migrate through the channels which are formed. Electron and immunofluorescence microscopy reveal bundles of microtubules and 10-nm filaments in parallel arrays within these processes, but no bundles of microfilaments were detected. The effect of CB on the structural integrity of microfilaments at this high concentration (20 microgram/ml) was demonstrated by the disappearance of filaments interacting with heavy meromyosin. Cycloheximide (20 microgram/ml) inhibits protein synthesis but does not affect cell fusion, the formation of microtubules and 10-nm filament bundles, or nuclear migration and alignment; thus, continued protein synthesis is not required. The association of microtubules and 10-nm filaments with nuclear migration and alignment suggests that microtubules and 10-nm filaments are two components in a system which serves both cytoskeletal and force-generating functions in intracellular movement and position of nuclei.     

The actin cytoskeleton is involved in signalling protoplast embryogenesis induced by agarose embedding

The organization of actin microfilaments was studied by immunofluorescence in protoplasts isolated from sunflower hypocotyls and cultured in an agarose matrix. Removal of the cell wall completely disrupted the actin cytoskeleton, which became progressively reorganized into cortical microfilament arrays and actin cables during protoplast culture. Treatment of protoplasts with arginine-glycine-aspartic acid (Arg-Gly-Asp) motif-containing peptides, to inhibit putative cell contacts with the agarose matrix, strongly affected this repair process: microfilament elongation and cable formation were inhibited and the connectivity between the cortical network and the perinuclear basket was lost. Furthermore, embryoid formation induced by agarose embedding was reduced. Similar effects were observed with a short treatment with latrunculin B, known to disrupt actin microfilaments. These results indicate that the actin network is involved in the signalling process that leads to polarity acquisition and embryoid determination in agarose-embedded protoplasts.     

Sequential rearrangement and nuclear polymerization of actin in baculovirus-infected Spodoptera frugiperda cells.

Proper assembly of nucleocapsids of the baculovirus Autographa californica nuclear polyhedrosis virus is prevented by cytochalasin D, a drug that interferes with actin microfilament function. To investigate the involvement of microfilaments in A. californica nuclear polyhedrosis virus replication, a fluorescence microscopy study was conducted that correlated changes in distribution of microfilaments with events in the life cycle of the virus. Tetramethylrhodamine isothiocyanate-labeled phalloidin was used to label microfilaments, and monoclonal antibody was used to label p39, the major viral capsid protein. Three microfilament arrangements were found in infected cells. During uptake of virus, thick cables were formed. These were insensitive to cycloheximide, indicating that this configuration was a rearrangement of preexisting cellular actin mediated by a component of the viral inoculum. At the time of cell rounding and before viral DNA replication, ventral aggregates of actin were observed. These were sensitive to cycloheximide but not to aphidicolin, indicating that an early viral gene mediated this actin rearrangement. Ventral aggregates did not result from the rounding process itself. Uninfected cells prerounded with colchicine did not form ventral aggregates. Cells prerounded with colchicine and then infected did form aggregates. At the time of exponential production of progency virus, microfilaments were found in the nucleus surrounding the virogenic stroma. In this area (where nucleocapsid assembly is known to take place) microfilaments colocalized with p39. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western immunoblot analysis identified p39 among proteins retained on an f-actin affinity column. We postulate that microfilaments in the nucleus provide a scaffold to position capsids for proper assembly and filling with DNA.     

The viscoelastic properties of actin solutions

The viscoelastic properties in actin solutions were investigated by measuring their elastic modulus and viscous modulus using a rheometer. The polymerization/gelation process of actin solutions was accompanied by an increase of both parameters, indicating the formation of a protein network. High shear rotational motion destroyed this network which, however, would reanneal if left undisturbed. At 25 °C under low ionic strength conditions, the viscoelastic moduli of a Spudich-Watt globular (G) actin preparation increased with time, while G-actin, purified by gel filtration maintained low viscoelastic moduli. The rigidity of the filamentous (F) actin network in a solution of Spudich-Watt actin, measured by the elastic modulus, was somewhat lower than that of gel-filtration-purified actin at the same protein concentration. The crosslink density of these F-actin networks was estimated, using models from rubber elasticity theory. The calculated density was 1 crosslink/50 actin monomers for the purified actin and 1 crosslink/120 actin monomers for Spudich-Watt actin. The results are consistent with the idea that a small amount of regulatory factor(s), which could be removed by the gel filtration step, modulates the structure of an actin network.     

The central role of septa in the basidiomycete Schizophyllum commune hyphal morphogenesis

The purpose of the present research was to observe in the filamentous basidiomycete Schizophyllum commune, the connection between the nuclear division and polymerization of the contractile actin ring with subsequent formation of septa in living hyphae. The filamentous actin was visualized using Lifeact-mCherry and the nuclei with EGFP tagged histone 2B (H2B). Time-lapse fluorescence microscopy confirmed that in monokaryotic and dikaryotic hyphae, the first signs of the contractile actin ring occur at the site of the nuclear division, in one to two minutes after division. At this stage, the telophase nuclei have moved tens of micrometers from the division site. The actin ring is replaced by the septum in six minutes. The apical cells treated with filamentous actin disrupting drug latrunculin A, had swollen tips but the cells were longer than in control samples due to the absence of the actin rings. The nuclear pairing and association with clamp cell development as well as the clamp cell fusion with the subapical cell was disrupted in latrunculin-treated dikaryotic hyphae, indicating that actin filaments are involved in these processes, also regulated by the A and B mating-type genes. This suggests that the actin cytoskeleton may indirectly be a target for mating-type genes.     

Fate of microfilaments in vero cells infected with measles virus and herpes simplex virus type 1.

In herpes simplex virus type 1-infected Vero cells, reorganization of microfilaments was observed approximately 4 h postinfection. Conversion of F (filamentous) actin to G (globular) actin, as assessed by a DNase I inhibition assay, was continuous over the next 12 to 16 h, at which time a level of G actin of about twice that observed in uninfected cells was measured. Fluorescent localization of F actin, using 7-nitrobenz-2-oxa-1,3-diazole (NBD)-phallacidin, demonstrated that microfilament fibers began to diminish at about 16 to 18 h postinfection, roughly corresponding to the time that G actin levels peaked and virus-induced cytopathology was first observable. In measles virus-infected cells, no such disassembly of microfilaments occurred. Rather, there was a modest decrease in G actin levels. Fluorescent localization of F actin showed that measles virus-infected Vero cells maintained a complex microfilament network characterized by fibers which spanned the entire length of the newly formed giant cells. Disruption of microfilaments with cytochalasin B, which inhibits measles virus-specific cytopathology, was not inhibitory to measles virus production at high multiplicities of infection (MOI) but was progressively inhibitory as the MOI was lowered. The carbobenzoxy tripeptide SV-4814, which inhibits the ability of Vero cells to fuse after measles virus infection, like cytochalasin B, inhibited measles virus production at low MOI but not at high MOI. Thus, it appears that agents which affect the ability of Vero cells to fuse after measles virus infection may be inhibitory to virus production and that the actin network is essential to this process.     

Mechanics in human fibroblasts and progeria: Lamin A mutation E145K results in stiffening of nuclei

The lamina is a filamentous meshwork beneath the inner nuclear membrane that confers mechanical stability to nuclei. The E145K mutation in lamin A causes Hutchinson‐Gilford progeria syndrome (HGPS). It affects lamin filament assembly and induces profound changes in the nuclear architecture. Expression of wild‐type and E145K lamin A in Xenopus oocytes followed by atomic force microscopy (AFM) probing of isolated oocyte nuclei has shown significant changes in the mechanical properties of the lamina. Nuclei of oocytes expressing E145K lamin A are stiffer than those expressing wild‐type lamin A. Here we present mechanical measurements by AFM on dermal fibroblasts obtained from a 4‐year‐old progeria patient bearing the E145K lamin A mutation and compared it to fibroblasts obtained from 2 healthy donors of 10 and 61 years of age, respectively. The abnormal shape of nuclei expressing E145K lamin A was analyzed by fluorescence microscopy. Lamina thickness was measured using electron micrographs. Fluorescence microscopy showed alterations in the actin network of progeria cells. AFM probing of whole dermal fibroblasts did not demonstrate significant differences in the elastic moduli of nuclear and cytoplasmic cell regions. In contrast, AFM measurements of isolated nuclei showed that nuclei of progeria and old person's cells are significantly stiffer than those of the young person, indicating that the process of aging, be it natural or abnormal, increases nuclear stiffness. Our results corroborate AFM data obtained using Xenopus oocyte nuclei and prove that the presence of E145K lamin A abnormally increases nuclear stiffness.     

Actin distribution in somatic embryos and embryogenic protoplasts of white spruce (Picea glauca)

Summary Examination of unfixed immature somatic embryos of white spruce (Picea glauca) with fluorescent rhodamine-labeled phalloidin revealed an extensive network of fine actin microfilaments (MFs) in the embryonal region which were not detected in specimens fixed with formaldehyde. Transition cells linking the embryonal region and suspensor cells contained fine MFs as well as bundles of MFs. The large, highly vacuolated suspensor cells were characterized by actin MF cables only. Treatment of embryos with cytochalasin B (CB) removed the fine MFs from the embryonal region and transition cells, but many MF cables in suspensor cells were resistant. Full recovery from CB treatment was observed in most somatic embryos. Embryogenic protoplasts capable of regenerating to somatic embryos in culture were released from only the embryonal region of somatic embryos. Both uninucleate and multinucleate embryogenic protoplasts retained the extensive network of fine actin MFs. In contrast, protoplasts derived from vacuolated suspensor cells and vacuolated free-floating cells contained thick MF bundles and were not embryogenic. Distinct MF cages enclosed nuclei in multinucleate protoplasts and may be responsible for preventing nuclear fusion. Microspectrophotometric analyses showed that the DNA contents of embryonal cells in the embryo and embryogenic protoplasts were similar and characteristic of rapidly dividing cell populations. However, transition and suspensor cells which released nonembryogenic protoplasts appeared to be arrested in G₁, and suspensor cells showed signs of DNA degradation.     

Fractionation of the detergent-resistant filamentous network of Ehrlich ascites tumour cells

Previous investigations of the filamentous network in eukaryotic cells have been based on observations by electron and fluorescence microscopy. In order to examined, in more detail, the interconnection of the various components of th filamentous network, we have treated Ehrlich ascites tumour cells with Triton X-100 in the presence of Mg++, disassembled the detergent-resistant, residual cell structure with Tris-EDTA and subjected the postnuclear supernatant to sucrose density gradient equilibrium centrifugation. Using this technique we are able to demonstrate 1) the association of the major part of intermediate-sized filament protein (vimentin) with unfolded ribosomal subunits, 2) the nearly identical sedimentation behavior of the boundary lamina and actin, and a minor part of the intermediate-sized filament protein respectively, and 3) the association of a Ca++-dependent protease specific for vimentin intermediate-sized filament protein with the Triton X-100 resistant, residual cell structure. Furthermore, we are able to confirm, by labelling intact Ehrlich ascites tumour cells with [3H] concanavalin A and recovering radioactivity in the lighter sucrose gradient fractions, that the detergent-resistant boundary lamina is derived from the plasma membrane. The presence of coated vesicles in Triton X-100-treated cells as well as of coated pits in the derived membrane point at the same origin of the boundary lamina. The results of the fractionation study are correlated with structures observed by electron microscopy of ultrathin sections of the intact filamentous network.     

Pulsation of nuclei in insect oocytes is reversibly inhibited by cytochalasin B

Oocyte nuclei of the dipteran insect Heteropeza pygmaea display swift pulsating movements during in vitro follicle formation in the ovaries. Low doses of cytochalasin B (CB) completely inhibit the nuclear movements within a few minutes and cause the nuclei to assume spherical shapes. If the drug is removed, nuclear pulsation is resumed within 5–10 min. Phalloidin and colchicine do not affect the nuclear movements. Actin is shown by indirect immunofluorescence microscopy to be present in considerable amounts all over the cytoplasm of the oocytes. It is concluded that microfilaments are responsible for pulsation of the oocyte nuclei, whereas microtubules are not involved.