Reorganization of microfilaments and microtubules by thermal stress in two-cell bovine embryos

Two-cell bovine embryos become arrested in development when exposed to a physiologically relevant heat shock. One of the major ultrastructural modifications caused by heat shock is translocation of organelles toward the center of the blastomere. The objective of the present study was to determine if heat- shock-induced movement of organelles is a result of cytoskeletal rearrangement. Two-cell bovine embryos were cultured at 38.5 degrees C (homeothermic temperature of the cow), 41.0 degrees C (physiologically relevant heat shock), or 43.0 degrees C (severe heat shock) for 6 h in the presence of either vehicle, latrunculin B (a microfilament depolymerizer), rhizoxin (a microtubule depolymerizer), or paclitaxel (a microtubule stabilizer). Heat shock caused a rearrangement of actin-containing filaments as detected by staining with phalloidin. Moreover, latrunculin B reduced the heat-shock-induced movement of organelles at 41.0 degrees C but not at 43.0 degrees C. In contrast, movement of organelles caused by heat shock was inhibited by rhizoxin at both temperatures. Furthermore, rhizoxin, but not latrunculin B, reduced the swelling of mitochondria caused by heat shock. Paclitaxel, while causing major changes in ultrastructure, did not prevent the movement of organelles or mitochondrial swelling. It is concluded that heat shock disrupts microtubule and microfilaments in the two-cell bovine embryo and that these changes are responsible for movement of organelles away from the periphery. In addition, intact microtubules are a requirement for heat-shock-induced swelling of mitochondria. Differences in response to rhizoxin and paclitaxel are interpreted to mean that deformation of microtubules can occur through a mechanism independent of microtubule depolymerization.     

Heat shock induction of intranuclear actin rods in cultured mammalian cells

Incubation of cultured cells of mouse C3H-2K fibroblastic cell line and other mammalian cell lines at 42.0-43.0 degrees C for 30 min or longer caused disintegration of normal actin structures including stress fibers, and induced formation of intranuclear actin paracrystal-like structures, called actin rods. When cells exposed to the elevated temperatures were shifted back to 37 degrees C, normal actin structures were regained. Pretreatment of cells at moderately high temperatures such as 38.5 degrees C inhibited formation of the actin rods upon subsequent exposure to 42.0 degrees C. Neither microtubules nor intermediate filaments were disrupted by the heat treatment. Several heat shock proteins were found to be synthesized under the conditions where actin rods were induced. However, there is no causal relationship between two cellular events, the induction of intranuclear actin rods and the synthesis of heat shock proteins.     

Cell-type-specific disruption and recovery of the cytoskeleton in Arabidopsis thaliana epidermal root cells upon heat shock stress

Summary. The cytoskeleton in plant cells plays an important role in controlling cell shape and mediating intracellular signalling. However, almost nothing is known about the reactions of cytoskeletal elements to heat stress, which represents one of the major environmental challenges for plants. Here we show that living epidermal root cells of Arabidopsis thaliana could cope with short-term heat shock stress showing disruption and subsequent recovery of microtubules and actin microfilaments in a time-dependent manner. Time-lapse imaging revealed a very dynamic behavior of both cytoskeletal elements including transient depolymerization and disassembly upon heat shock (40–41 °C) followed by full recovery at room temperature (20 °C) within 1–3 h. Reaction of microtubules, but not actin filaments, to heat shock was dependent on cell type and developmental stage. On the other hand, recovery of actin filaments, but not microtubules, from heat shock stress was dependent on the same parameters. The relevance of this adaptive cytoskeletal behavior to intracellular signalling is discussed. Correspondence and reprints: Institute of Cellular and Molecular Botany, University of Bonn, Kirschallee 1, 53115 Bonn, Federal Republic of Germany.     

Role of microfilaments and microtubules in the invasion of INT-407 cells by Campylobacter jejuni

The internalization mechanisms triggered by Campylobacter jejuni were studied by invasion assays conducted with different inhibitors that act on the cytoskeleton structure of eukaryotic cells. The depolymerization of microfilaments by cytochalasin-D and that of microtubules by colchicines and nocodazole inhibited the uptake of C. jejuni into INT-407 cells in a dose-dependent manner. The inhibitory effect of microfilament depolymerization on C. jejuni internalization was more pronounced than that of microtubule depolymerization. By immunofluorescence microscopic observations, it was demonstrated that both microfilaments and microtubules were localized in INT-407 cells after C. jejuni infection. These data suggest that the internalization mechanism triggered by C. jejuni is associated with the combined effect of microfilaments and microtubules of host cells.     

Cell wall and cytoskeleton reorganization as the response to hyperosmotic shock in Saccharomyces cerevisiae

Transfer of exponentially growing cells of the yeast Saccharomyces cerevisiae to hyperosmotic growth medium containing 0.7-1 M KCl, 1 M mannitol, and/or 1 M glycerol caused cessation of yeast growth for about 2 h; thereafter, growth resumed at almost the original rate. During this time, formation of fluorescent patches on the inner surface of cell walls stained with Primulin or Calcofluor white was observed. The fluorescent patches also formed in solutions of KCl or when synthesis of the cell wall was blocked with cycloheximide and/or 2-deoxyglucose. The patches gradually disappeared as the cells resumed growth, and the new buds had smooth cell walls. Electron microscopy of freeze-etched replicas of osmotically stressed cells revealed deep plasma membrane invaginations filled from the periplasmic side with an amorphous cell wall material that appeared to correspond to the fluorescent patches on the cell surface. The rate of incorporation of D-[U-14C]glucose from the growth medium into the individual cell wall polysaccharides during osmotic shock followed the growth kinetics. No differences in cell wall composition between osmotically stressed yeast and control cells were found. Hyperosmotic shock caused changes in cytoskeletal elements, as demonstrated by the disappearance of microtubules and actin microfilaments. After 2-3 h in hyperosmotic medium, both microtubules and microfilaments regenerated to their original polarized forms and the actin patches resumed their positions at the apices of growing buds. The response of S. cerevisiae strains with mutations in the osmosensing pathway genes hog1 and pbs2 to hyperosmotic shock was similar to that of the wild-type strain. We conclude that, besides causing a temporary disassembling of the cytoskeleton, hyperosmotic shock induces a change in the organization of the cell wall, apparently resulting from the displacement of periplasmic and cell wall matrix material into invaginations of the plasma membrane created by the plasmolysis.     

Evidence for reversible, non-microtubule and non-microfilament-dependent nuclear translocation of hsp90 after heat shock in human fibroblasts.

A monoclonal antibody (29A) directed against rat liver heat shock protein M(r) 90,000 (hsp90) was produced. By Western immunoblotting of cytosols prepared from several different tissues and species, 29A was shown to specifically recognize only one band with M(r) approximately 90,000. Localization of hsp90 in human gingival fibroblasts was studied using the 29A antibody by indirect mono- and double-staining immunofluorescence and confocal laser scanning microscopy. The distribution was compared to that of the glucocorticoid receptor (GR) and various cytoskeletal structures. Cells were analyzed in interphase and mitosis under basal culture conditions, after heat shock and after microtubule and microfilament depolymerization, sometimes combined with heat shock. A major part of hsp90 immunoreactivity was diffusely distributed throughout the interphase cytoplasm, but a weak nuclear staining with non-stained nucleoli was also present, however, only detectable after methanol and not after formaldehyde/Triton X-100 fixation. Heat shock induced a time-dependent translocation of hsp90 from the cytoplasm to the cell nucleus reaching a plateau after 15 h. This compartment shift was reversible and also occurred in the absence of intact microtubules or intact microfilaments.     

Osmotic regulation of the heat shock response in H4IIE rat hepatoma cells.

The influence of cell hydration on the heat shock response was investigated in H4IIE hepatoma cells at the levels of HSP70 expression, MAP kinase activation, induction of c-jun and the MAP kinase phosphatase MKP-1, heat resistance, and development of tolerance/sensitization to arsenite after a priming heat treatment. Induction of HSP70, MKP-1, and c-jun by heat was delayed, but more pronounced or sustained, under hyperosmotic conditions compared with normo- and hypo-osmotically exposed cells. Anisosmolarity per se was ineffective to induce HSP70; some expression of the mRNAs for MKP-1 and c-jun in response to hyperosmolarity was found, but was small compared with the response to heat. Heat-induced activation of JNK-1 was increased under hyperosmotic conditions and more sustained than the JNK-activity induced by hyperosmolarity at 37 degrees C. A prominent Erk-2 activation was found immediately after heat shock under hypo- and normo-osmotic conditions, but Erk-2 activation was weak in hyperosmolarity-exposed cells. Despite anisosmotic alterations of the heat shock response at the molecular level, the heat resistance of H4IIE cells toward heat shock was not affected by ambient osmolarity. However, an osmolarity-dependent sensitization to arsenite was induced by a priming heat shock. The osmodependence of the H4IIE cell response to heat differs from that recently found in primary rat hepatocytes. The data are discussed in terms of cellular adaption mechanisms and their physiological relevance.     

Host cell actin polymerization is required for cellular retention of Trypanosoma cruzi and early association with endosomal/lysosomal compartments

One of the hallmarks of Trypanosoma cruzi invasion of non-professional phagocytes is facilitation of the process by host cell actin depolymerization. Host cell entry by invasive T. cruzi trypomastigotes is accomplished by exploiting a cellular wound repair process involving Ca(2+)-regulated lysosome exocytosis (i.e. lysosome-dependent) or by engaging a recently recognized lysosome-independent pathway. It was originally postulated that cortical actin microfilaments present a barrier to lysosome-plasma membrane fusion and that transient actin depolymerization enhances T. cruzi entry by increasing access to the plasma membrane for lysosome fusion. Here we demonstrate that cytochalasin D treatment of host cells inhibits early lysosome association with invading T. cruzi trypomastigotes by uncoupling the cell penetration step from lysosome recruitment and/or fusion. These findings provide the first indication that lysosome-dependent T. cruzi entry is initiated by plasma membrane invagination similar to that observed for lysosome-independent entry. Furthermore, prolonged disruption of host cell actin microfilaments results in significant loss of internalized parasites from infected host cells. Thus, the ability of internalized trypomastigotes to remain cell-associated and to fuse with host cell lysosomes is critically dependent upon host cell actin reassembly, revealing an unanticipated role for cellular actin remodelling in the T. cruzi invasion process.     

Internalization and recycling of insulin receptors in hepatoma cells. Absence of regulation by receptor occupancy.

Insulin receptors of Fao hepatoma cells were labelled with a 125I-labelled photoreactive insulin analogue or by surface iodination catalysed by lactoperoxidase. Cells were then incubated at 37 degrees C, and the cellular localization of the labelled receptors was assessed by limited exposure of intact cells to trypsin. The results show that: (1) photolabelled insulin-receptor complexes are internalized and recycled in Fao hepatoma cells; (2) the dynamics of photolabelled insulin receptors (internalization and recycling) is similar before and after down-regulation; (3) the unoccupied receptors labelled by surface iodination are internalized and recycled similarly to covalent insulin-receptor complexes; (4) insulin does not induce internalization of surface-iodinated insulin receptors. We conclude that internalization and recycling of insulin receptors are independent of receptor occupancy by insulin in Fao hepatoma cells.     

Ultrastructural Identification of Caveolae and Immunocytochemical as Well as Biochemical Detection of Caveolin in Chondrocytes

Using fluorescence immunocytochemistry, transmission electron microscopy and Western blotting, we have shown that caveolae and caveolin are abundant on chondrocytes of different cartilaginous structures of newborn and adult rat knee joints. Caveolin was detected in chondrocytes of the outer layer of articular cartilage, in the fibrocartilage of the menisci, and in fibrocartilage-like cells at tendon and ligament insertions. Electron microscopical studies revealed caveolae-like invaginations along the plasmalemmal membrane of articular chondrocytes and fibrocartilage cells. Immunoblot analysis demonstrated caveolin in detergent-insoluble and soluble complexes isolated from cultured rat chondrocytes.     

Agonist-dependent trafficking of alpha2-adrenoceptor subtypes: dependence on receptor subtype and employed agonist

Many G protein-coupled receptors (GPCRs) are internalized from the plasma membrane after agonist exposure. Previously, marked agonist-induced internalization of human alpha2A- and alpha2B-adrenergic receptors (AR) was observed in transfected neuronal rat pheochromocytoma (PC12) cells; alpha2A- and alpha2B-AR were internalized into partly distinct intracellular vesicles (Olli-L?hdesm?ki et al., J. Neurosci. 19, 9281-9288, 1999). In this paper, the extent of alpha2-AR internalization was quantitated in human embryonic kidney (HEK-293) and PC12 cells by combined application of cell surface biotinylation and ELISA methods, which allow measurement of protein trafficking in intact, differentiated and undifferentiated cells. Significant subtype-specific (but not cell type-dependent) trafficking of human alpha2-AR was observed by quantitation and immunocytochemistry. Agonist-induced sequestration of alpha2B-AR was markedly reduced after blocking the formation of clathrin-coated vesicles by hyperosmotic sucrose pretreatment. The sequestration of alpha2A-AR was partly inhibited after sucrose pretreatment but could be further reduced after inhibiting the formation of both clathrin-coated and caveolin vesicles by combined pretreatment with hyperosmotic sucrose and filipin. Differences were also observed in the recycling of alpha2A- and alpha2B-AR. The extent of maximal agonist-induced sequestration in PC12 cells was not directly dependent on relative agonist efficacy.     

The dynamic state of heat shock proteins in chicken embryo fibroblasts

Subcellular fractionation and immunofluorescence microscopy have been used to study the intracellular distributions of the major heat shock proteins, hsp 89, hsp 70, and hsp 24, in chicken embryo fibroblasts stressed by heat shock, allowed to recover and then restressed. Hsp 89 was localized primarily to the cytoplasm except during the restress when a portion of this protein concentrated in the nuclear region. Under all conditions, hsp 89 was readily extracted from cells by detergent. During stress and restress, significant amounts of hsp 70 moved to the nucleus and became resistant to detergent extraction. Some of this hsp 70 was released from the insoluble form in an ATP-dependent reaction. Hsp 24 was confined to the cytoplasm and, during restress, aggregated to detergent-insoluble perinuclear phase-dense granules. These granules dissociated during recovery and hsp 24 could be solubilized by detergent. The nuclear hsps reappeared in the cytoplasm in cells allowed to recover at normal temperatures. Sodium arsenite also induces hsps and their distributions were similar to that observed after a heat shock, except for hsp 89, which remained cytoplasmic. We also examined by immunofluorescence the cytoskeletal systems of chicken embryo fibroblasts subjected to heat shock and found no gross morphological changes in cytoplasmic microfilaments or microtubules. However, the intermediate filament network was very sensitive and collapsed around the nucleus very shortly after a heat shock. The normal intermediate filament morphology reformed when cells were allowed to recover from the stress. Inclusion of actinomycin D during the heat shock--a condition that prevents synthesis of the hsps--did not affect the intermediate filament collapse, but recovery of the normal morphology did not occur. We suggest that an hsp(s) may aid in the formation of the intermediate filament network after stress.     

A role for caveolin and the urokinase receptor in integrin-mediated adhesion and signaling

The assembly of signaling molecules surrounding the integrin family of adhesion receptors remains poorly understood. Recently, the membrane protein caveolin was found in complexes with beta1 integrins. Caveolin binds cholesterol and several signaling molecules potentially linked to integrin function, e.g., Src family kinases, although caveolin has not been directly implicated in integrin-dependent adhesion. Here we report that depletion of caveolin by antisense methodology in kidney 293 cells disrupts the association of Src kinases with beta1 integrins resulting in loss of focal adhesion sites, ligand-induced focal adhesion kinase (FAK) phosphorylation, and adhesion. The nonintegrin urokinase receptor (uPAR) associates with and stabilizes beta1 integrin/caveolin complexes. Depletion of caveolin in uPAR-expressing 293 cells also disrupts uPAR/integrin complexes and uPAR-dependent adhesion. Further, beta1 integrin/caveolin complexes could be disassociated by uPAR-binding peptides in both uPAR-transfected 293 cells and human vascular smooth muscle cells. Disruption of complexes by peptides in intact smooth muscle cells blocks the association of Src family kinases with beta1 integrins and markedly impairs their migration on fibronectin. We conclude that ligand-induced signaling necessary for normal beta1 integrin function requires caveolin and is regulated by uPAR. Caveolin and uPAR may operate within adhesion sites to organize kinase-rich lipid domains in proximity to integrins, promoting efficient signal transduction.     

Cytoskeletal regulation of Caco-2 intestinal monolayer paracellular permeability

An abnormal increase in intestinal paracellular permeability may be an important pathogenic factor in various intestinal diseases. The intracellular factors and processes that regulate and cause alteration of intestinal paracellular permeability are not well understood. The purpose of this study was to examine some of the intracellular processes involved in cytoskeletal regulation of intestinal epithelial paracellular permeability using the filter-grown Caco-2 intestinal epithelial monolayers. Cytochalasin-b and colchicine were used to disrupt the cytoskeletal elements, actin microfilaments, and microtubules. Cytochalasin-b (5 m?g/ml) and colchicine (2 × 10^?5M) at the doses used caused marked depolymerization and disruption of actin microfilaments and microtubules, respectively. Cytochalasin-b-induced disruption of actin microfilaments resulted in perturbation of tight junctions and desmosomes and an increase in Caco-2 monolayer paracellular permeability. The cytochalasin-b-induced disruption of actin microfilaments and subsequent changes in intercellular junctional complexes and paracellular permeability were not affected by inhibitors of protein synthesis (actinomycin-D or cycloheximide) or microtubule function (colchicine), but were inhibited by metabolic energy inhibitors (2,4-dinitrophenol or sodium azide). The cytochalasin-b-induced disturbance in Caco-2 actin microfilaments and intercellular junctional complexes and increase in paracellular permeability were rapidly reversed. The paracellular pathway “re-tightening” following cytochalasin-b removal was not affected by actinomycin-D, cycloheximide, or colchicine, but was inhibited by 2,4-dinitrophenol and sodium azide. The colchicine-induced disruption of microtubules did not have significant effect on actin microfilaments, intercellular junctions, or paracellular permeability. These findings suggest that cytochalasin-b-induced increase in Caco-2 monolayer paracellular permeability was due to actin microfilament mediated perturbation of intercellular junctional complexes. The re-tightening of paracellular pathways (following removal of cytochalasin-b) resulted from energy-mediated re-assembly of pre-existing actin microfilaments and intercellular junctional complexes. This re-closure process did not require protein synthesis or microtubule-mediated shuttling process. © 1995 Wiley-Liss, Inc.     

Role of cytoskeletal elements in the recruitment of Cx43-GFP and Cx26-YFP into gap junctions

Cytoskeletal elements may be important in connexin transport to the cell surface, cell surface gap junction plaque formation and/or gap junction internalization. In this study, fluorescence recovery after photobleaching was used to examine the role of microfilaments and microtubules in the recruitment and coalescence of green fluorescent protein-tagged Cx43 (Cx43-GFP) or yellow fluorescent tagged-Cx26 (Cx26-YFP) into gap junctions in NRK cells. In untreated cells, both Cx26-YFP and Cx43-GFP were recruited into gap junctions within photobleached areas of cell-cell contact within 2 hrs. However, disruption of microfilaments with cytochalasin B inhibited the recruitment and assembly of both Cx26-YFP and Cx43-GFP into gap junctions within photobleached areas. Surprisingly, disruption of microtubules with nocodazole inhibited the recruitment of Cx43-GFP into gap junctions but had limited effect on the transport and clustering of Cx26-YFP into gap junctions within the photobleached regions of cell-cell contact. These results suggest that the recruitment of Cx43-GFP and Cx26-YFP to the cell surface or their lateral clustering into gap junctions plaques is dependent in part on the presence of intact actin microfilaments while Cx43-GFP was more dependent on intact microtubules than Cx26-YFP.     

Role of Cytoskeletal Elements in the Recruitment of Cx43-GFP and Cx26-YFP into Gap Junctions

Cytoskeletal elements may be important in connexin transport to the cell surface, cell surface gap junction plaque formation and/or gap junction internalization. In this study, fluorescence recovery after photobleaching was used to examine the role of microfilaments and microtubules in the recruitment and coalescence of green fluorescent protein-tagged Cx43 (Cx43-GFP) or yellow fluorescent tagged-Cx26 (Cx26-YFP) into gap junctions in NRK cells. In untreated cells, both Cx26-YFP and Cx43-GFP were recruited into gap junctions within photobleached areas of cell-cell contact within 2 hrs. However, disruption of microfilaments with cytochalasin B inhibited the recruitment and assembly of both Cx26-YFP and Cx43-GFP into gap junctions within photobleached areas. Surprisingly, disruption of microtubules with nocodazole inhibited the recruitment of Cx43-GFP into gap junctions but had limited effect on the transport and clustering of Cx26-YFP into gapjunctions within the photobleached regions of cell-cell contact. These results suggest that the recruitment of Cx43-GFP and Cx26-YFP to the cell surface or their lateral clustering into gap junctions plaques is dependent in part on the presence of intact actin microfilaments while Cx43-GFP was more dependent on intact microtubules than Cx26-YFP.     

Role of Cytoskeletal Elements in the Recruitment of Cx43-GFP and Cx26-YFP into Gap Junctions

Cytoskeletal elements may be important in connexin transport to the cell surface, cell surface gap junction plaque formation and/or gap junction internalization. In this study, fluorescence recovery after photobleaching was used to examine the role of microfilaments and microtubules in the recruitment and coalescence of green fluorescent protein-tagged Cx43 (Cx43-GFP) or yellow fluorescent tagged-Cx26 (Cx26-YFP) into gap junctions in NRK cells. In untreated cells, both Cx26-YFP and Cx43-GFP were recruited into gap junctions within photobleached areas of cell-cell contact within 2 hrs. However, disruption of microfilaments with cytochalasin B inhibited the recruitment and assembly of both Cx26-YFP and Cx43-GFP into gap junctions within photobleached areas. Surprisingly, disruption of microtubules with nocodazole inhibited the recruitment of Cx43-GFP into gap junctions but had limited effect on the transport and clustering of Cx26-YFP into gapjunctions within the photobleached regions of cell-cell contact. These results suggest that the recruitment of Cx43-GFP and Cx26-YFP to the cell surface or their lateral clustering into gap junctions plaques is dependent in part on the presence of intact actin microfilaments while Cx43-GFP was more dependent on intact microtubules than Cx26-YFP.     

Stabilizing microtubules with taxol increases microfilament stability during freezing of rye root tips

We have used double fluorescence labelling to investigate the effect of freezing on microtubules and microfilaments in root-tip cells of rye (Secale cereale L. cv Rymin). Freezing to -5°C (which does not kill these cells) caused partial depolymerization of both, but microfilaments were more resistant than microtubules. When microtubules were stabilized against freeze-induced depolymerization by pre-treating seedlings with taxol, microfilaments exhibited enhanced stability as well. Almost all the frozen cells containing taxol-stabilized microtubules also contained microfilaments. When seedlings were treated with the microtubule-destabilizing drug APM prior to freezing, microfilaments became more susceptible to freeze-induced depolymerization than in controls. These data suggest a physical interaction between microtubules and microfilaments in these cells.     

Depolymerization of actin microfilaments in the I--Z--I brushes of transverse striated muscles by using DNAse I

To release Z-discs of skeletal muscles myofibrils from actin microfilaments, I--Z--I-brushes (complexes of Z-discs and thin filaments) were treated with DNAse I-both in suspension and on electron microscopical grids. It was shown that such a treatment resulted in depolymerization of actin filaments. The preparations obtained were heterogeneous and contained I--Z--I-brushes with shorter actin filaments and single Z-discs. The structure of Z-discs released from actin filament remained intact. Therefore these preparations may be used in studies on regulation of actin microfilaments assembly.