Trypanosoma cruzi infection affects actin mRNA regulation in heart muscle cells

We have previously described alterations in the cytoskeletal organization of heart muscle cells (HMC) infected with Trypanosoma cruzi in vitro. Our aim was to investigate whether these changes also affect the regulation of the actin mRNAs during HMC differentiation. Northern blot analysis revealed that alpha-cardiac actin mRNA levels increased during cell differentiation while beta-actin mRNA levels declined. Nonmuscle cells displayed beta-actin mRNA signal localized at the cell periphery, while alpha-cardiac actin mRNA had a perinuclear distribution in myocytes. Trypanosoma cruzi-infected cells showed 50% reduction in alpha-cardiac actin mRNA expression after 72 h of infection. In contrast, beta-actin mRNA levels increased approximately 79% after 48 h of infection. In addition, in situ beta-actin mRNA was delocalized from the periphery into the perinuclear region. These observations support the hypothesis that Trypanosoma cruzi affects actin mRNA regulation and localization through its effect on the cytoskeleton of heart muscle cells.     

The changes in structural organization of actin in the sea urchin egg cortex in response to hydrostatic pressure

We have used hydrostatic pressure to study the structural organization of actin in the sea urchin egg cortex and the role of cortical actin in early development. Pressurization of Arbacia punctulata eggs to 6,000 psi at the first cleavage division caused the regression of the cleavage furrow and the disappearance of actin filament bundles from the microvilli. Within 30 s to 1 min of decompression these bundles reformed and furrowing resumed. Pressurization of dividing eggs to 7,500 psi caused both the regression of the cleavage furrow and the complete loss of microvilli from the egg surface. Following release from this higher pressure, the eggs underwent extensive, uncoordinated surface contractions, but failed to cleave. The eggs gradually regained their spherical shape and cleaved directly into four cells at the second cleavage division. Microvilli reformed on the egg surface over a period of time corresponding to that required for the recovery of normal egg shape and stability. During the initial stages of their regrowth the microvilli contained a network of actin filaments that began to transform into bundles when the microvilli had reached approximately 2/3 of their final length. These results demonstrate that moderate levels of hydrostatic pressure cause the reversible disruption of cortical actin organization, and suggest that this network of actin stabilizes the egg surface and participates in the formation of the contractile ring during cytokinesis. The results also demonstrate that actin filament bundles are not required for the regrowth of microvilli after their removal by pressurization. Preliminary experiments demonstrate that F-actin is not depolymerized in vitro by pressures up to 10,000 psi and suggest that pressure may act indirectly in vivo, either by changing the intracellular ionic environment or by altering the interaction of actin binding proteins with actin.     

Changes in distribution of actin mRNA in different polysome fractions following stimulation of MPC-11 cells

Individual mRNA species have been shown to differ both with respect to localization in the cell, and in their distribution upon stimulation of cells with different signals. In this study we have examined the distribution of actin mRNA in the free, cytoskeletal-bound, and membrane-bound RNA fractions, both in starved cells, and in response to stimulation by feeding. These results were then compared with mRNAs for glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and histone H4. The results we obtained showed that actin mRNA was located in the free RNA fraction in starved cells, while upon stimulation it was located both in the free, and in the cytoskeletal fraction; no redistribution of GAPDH mRNA occurred between the three RNA fractions, while H4 mRNA showed a different localization upon stimulation. Incubation with the drugs actinomycin-D and cycloheximide showed that an altered localization of actin mRNA from free in starved cells to free and cytoskeletal mRNA fractions following stimulation, was dependent on RNA synthesis, and not on protein synthesis.     

Intracellular localization of messenger RNAs for cytoskeletal proteins

We have analyzed intracellular distributions of mRNAs for the cytoskeletal proteins actin, vimentin, and tubulin by in situ hybridization. Although polyadenylated RNA was homogeneously distributed throughout the cell, actin mRNA demonstrated a nonhomogeneous distribution in 95% of randomly selected chicken embryonic myoblasts and fibroblasts, as detected by isotopic and nonisotopic techniques. Actin mRNA concentrations were highest at cell extremities, generally in lamellipodia, where grain densities were up to 16-fold higher than in areas near the nucleus. Vimentin mRNA, unlike actin mRNA, was distributed near the nucleus. Tubulin mRNA appeared most concentrated in the peripheral cytoplasm. These results demonstrate that cytoplasmic mRNAs are localized in specific, nonrandom cellular patterns and that localized concentrations of specific proteins may result from corresponding localization of their respective mRNAs. Hence, actin mRNA distribution may result in increased concentration of actin filaments in lamellipodia of motile cells.     

2'-Deoxy-GTP in the microtubule cytoskeleton of neuronal cells cultured with nerve growth factor.

Tubulin, widely recognized as a GTP/GDP-binding protein, has been isolated in its polymerized state from rat PC12 cells and embryonic chick dorsal root ganglion neurons by Triton X-100 detergent extraction of the cytoskeletal fraction. Perchloric acid extraction and deproteinization of this fraction permitted subsequent analysis of nucleotide identity and content by high performance liquid chromatography. PC12 cells grown in the absence of nerve growth factor (NGF) contained ADP, ATP, GDP, and GTP at levels consistent with the actin and tubulin content of the cytoskeletal fraction. Microtubules from PC12 cells cultured in the presence of NGF contain an additional nucleotide that we have identified as dGTP. Analysis of whole cell nucleotide extracts from PC12 cells grown in the absence or presence of NGF revealed no evidence for the presence of dGTP at 4 and 14 days, respectively. We have determined that embryonic chick dorsal root ganglion neurons also contain this deoxyribonucleotide, and we found virtually no ADP or ATP in the extracted dorsal root ganglion cytoskeletal fraction. On the basis of metabolic labeling studies with [14C] guanine, we have inferred that the presence of dGTP in NGF-treated PC12 cells probably arises either from binding to the nonexchangeable nucleotide site of tubulin undergoing dynamic assembly/disassembly or from binding to the exchangeable site of tubulin subsequently incorporated into highly stabilized microtubules.     

Tropomyosin co-localizes with actin microfilaments and microtubules within supporting cells of the inner ear

Summary The distribution of tropomyosin, actin and tubulin in the supporting cells of the organ of Corti was studied by immunofluorescent localization of antibodies to these proteins. Tropomyosin colocalizes with actin and tubulin in the regions of the tunnel pillar and Deiters cells where actin microfilaments and microtubules had previously been observed ultrastructurally. Despite the implications of the presence of antiparallel actin filaments in the supporting cells, the presence of tropomyosin and the absence of myosin suggest that the role of tropomyosin may be to confer rigidity to the actin filaments. Thus the primary function of the cytoskeletal proteins in the supporting cells may be structural.     

Heavy Water and Hydrostatic Pressure Effects on Tetrahymena pyriformis1

SYNOPSIS. Heat-synchronized cultures of Tetrahymena pyriformis strain GL subjected to pulses of high hydrostatic pressure (10,000 psi for 2 min) had increasing division delays during the 1st 40 min after the last heat shock (40 min after heat treatment). Pressure treatment during the subsequent 10-min interval disrupted cell synchrony. Comparable pressures applied to the cells at later stages, before the 1st synchronous division, caused negligible division delay. Continuous exposure to 10% (v/v) heavy water hardly affected division; higher concentrations delayed or blocked division. Ten-min pulses with heavy water (40%, 50%, 70%) resulted in increasing division delays depending on the stage of the cell cycle during which the heavy water was applied. Amelioration of the division-delaying effects of pressure was observed in cells treated simultaneously with pressure (3,000 psi for 30 min), and 30% D₂O. The results are consistent with the hypothesis that some of the pressure and D₂O effects could be attributed to changes in the sol-gel state of the cytoplasm.     

Adenine and guanine nucleotide content of Triton-extracted cytoskeletal fractions of nonmuscle cells.

Determination of the adenine and guanine nucleotides in Triton X-100-extracted cytoskeletal fractions was utilized to estimate the actin and tubulin content of the assembled cytoskeletons in nonmuscle cells. Results with stable cell lines (i.e., rat pheochromocytoma PC12 and neuroblastoma NB41A3) and with primary cultures (i.e., human foreskin fibroblasts and chick embryonic dorsal root ganglion neurons) exhibited levels of cytoskeletal fraction ADP and GDP consistent with their assembly-induced nucleoside-5'-triphosphatase activities only previously analyzed in vitro. Likewise, estimates of actin and tubulin content fall in the range of values obtained by other experimental approaches. In contrast, analysis of whole cell nucleotides showed high [ATP]/[ADP] and [GTP]/[GDP] ratios, suggesting there is little, if any, contamination of the cytoskeletal nucleotide pool by other cellular nucleotides.     

Control of tubulin and actin gene expression in Tetrahymena pyriformis during the cell cycle

Poly(A)-containing mRNA was isolated from division synchronized populations of the ciliated protozoan, Tetrahymena pyriformis. The level of tubulin and actin mRNA at specific cell cycle stages was analyzed by hybridization to tubulin and actin cDNA probes and by gel analysis of their in vitro translation products. The pattern of fluctuation of tubulin mRNA levels was similar to that observed for the in vivo tubulin synthesis previously reported [1]. This suggests that as the cells progress through the cell cycle, tubulin synthesis is controlled at the mRNA level. There was little fluctuation of actin synthesis or actin mRNA levels during the cell cycle, which may be indicative of a different regulatory mechanism for actin than for tubulin.     

Fragmentation of the Golgi apparatus: an early apoptotic event independent of the cytoskeleton

The Golgi apparatus undergoes irreversible fragmentation during apoptosis, in part as a result of caspase-mediated cleavage of several Golgi-associated proteins. However, Golgi structure and orientation is also regulated by the cytoskeleton and cytoskeletal changes have been implicated in inducing apoptosis. Consequently, we have analyzed the role of actin filaments and microtubules in apoptotic Golgi fragmentation. We demonstrate that in Fas receptor-activated cells, fragmentation of the Golgi apparatus was an early event that coincided with release of cytochrome c from mitochondria. Significantly, Golgi fragmentation preceded major changes in the organization of both the actin cytoskeleton and microtubules. In staurosporine-treated cells, actin filament organization was rapidly disrupted; however, the Golgi apparatus maintained its juxtanuclear localization and underwent complete fragmentation only at later times. Attempts to stabilize actin filaments with jasplakinolide prior to treatment with staurosporine did not prevent Golgi fragmentation. Finally, in response to Fas receptor activation or staurosporine treatment the levels of beta-actin or alpha-tubulin remained unaltered, whereas several Golgi proteins, p115 and golgin-160, underwent caspase-mediated cleavage. Our data demonstrate that breakdown of the Golgi apparatus is an early event during apoptosis that occurs independently of major changes to the actin and tubulin cytoskeleton.     

Tubulin, actin, and tau protein interactions and the study of their macromolecular assemblies

The intracellular polymerization of cytoskeletal proteins into their supramolecular assemblies raises many questions regarding the regulatory patterns that control this process. Binding experiments using the ELISA solid phase system, together with protein assembly assays and electron microscopical studies provided clues on the protein-protein associations in the polymerization of tubulin and actin networks. In vitro reconstitution experiments of these cytoskeletal filaments using purified tau, tubulin, and actin proteins were carried out. Tau protein association with tubulin immobilized in a solid phase support system was inhibited by actin monomer, and a higher inhibition was attained in the presence of preassembled actin filaments. Conversely, tubulin and assembled microtubules strongly inhibited tau interaction with actin in the solid phase system. Actin filaments decreased the extent of in vitro tau-induced tubulin assembly. Studies on the morphological aspects of microtubules and actin filaments coexisting in vitro, revealed the association between both cytoskeletal filaments, and in some cases, the presence of fine filamentous structures bridging these polymers. Immunogold studies showed the association of tau along polymerized microtubules and actin filaments, even though a preferential localization of labeled tau with microtubules was revealed. The studies provide further evidence for the involvement of tau protein in modulating the interactions of microtubules and actin polymers in the organization of the cytsokeletal network.     

Effect of Collagen Gel Configuration on the Cytoskeleton in Cultured Rat Hepatocytes

The cytoskeleton is important in the maintenance of cellular morphology and differentiated function in a number of cell types, including hepatocytes. In this study, adult rat hepatocytes sandwiched between two layers of collagen gel were compared to cells cultured on a single collagen gel for differences in the organization and expression of the cytoskeletal proteins actin and tubulin. Hepatocytes cultured between two layers of hydrated rat tail tendon collagen (sandwich gel) morphologically resembled cells in intact liver for several weeks. Actin filaments (F-actin) in these hepatocytes were concentrated under the plasma membrane in regions of cell-cell contact. In contrast, hepatocytes cultured on a single collagen gel were flattened and motile and had F-actin containing stress fibers. This was accompanied by a severalfold increase in actin mRNA. Microtubules formed an interwoven network in hepatocytes cultured in a sandwich gel, but in single gel cultures they formed long parallel arrays extending out to the cell periphery. Tubulin mRNA was severalfold greater in hepatocytes cultured on a single gel. Fibronectin and laminin staining were greater in single gel cultures, and these proteins were concentrated in fibrils radiating from the cell periphery. Overlaying a second collagen gel onto hepatocytes that had been cultured on a single gel (double gel rescue) reversed cell spreading and reduced stress fibers. Double gel rescue also resulted in a decrease in actin and tubulin mRNA to levels present in sandwich gel cultures and freshly isolated hepatocytes. These results show that the configuration of the external matrix has a dynamic effect on cytoskeletal proteins in cultured rat hepatocytes.     

Altered expression and distribution of the cytoskeletal-associated p35 protein in NIH 3T3 cells transformed with the Harvey sarcoma virus v-ras oncogene

1. Cytoskeletal events associated with retroviral oncogene (v-ras)-mediated transformation were studied in NIH 3T3 fibroblasts and their v-ras-transfected counterparts (3T3/H-1 cells). 2. Abnormal microfilament networks seen in 3T3/H-1 cells reflected significant decreases (approximately 90%) in two cytoskeletal-associated proteins (tropomyosin-1, p35). Neither actin content nor actin mRNA levels were altered, however, v-ras transfectants. 3. p35 mRNA activity in both NIH 3T3 and 3T3/H-1 cells was similar although differential compartmentalization of p35 to the detergent-resistant cytoskeletal fraction was evident only in normal fibroblasts. 4. Proper cytoskeletal organization may be a factor in the regulation of p35 mRNA translation in situ or influence the stability of p35 independent of translational rate.     

CANNABINOID ENANTIOMER ACTION ON THE CYTOARCHITECTURE

The negative and positive enantiomers of 7-hydroxy- Δ⁶-tetrahydrocannabinol-dimethylheptyl (designated HU-210 and HU-211 respectively) differentially affect undifferentiated and differentiating cultured pheochromocytoma cells (PC-12 cells). In general, cell viability and cell proliferation were suppressed to a much greater extent with HU-210 than with HU-211 in differentiating cells. The effects of these synthetic cannabinoids on the cytoskeleton of PC-12 cells were examined by epifluorescence and confocal microscopy. In both undifferentiated and differentiating PC-12 cells, HU-211 has little effect on the cytoarchitecture whereas HU-210 disrupts the distribution of microtubules and microfilaments. Vacuoles (2–4 μm) were evident in the cytoplasm of HU-210-treated cells but not in the cytoplasm of HU-211-treated cells or in vehicle controls. Tubulin and actin mRNA levels were reduced to 5 and 40 %, respectively (relative to untreated controls) in 10 μmHU-210-treated cells whereas the same concentration of HU-211 reduced tubulin and actin mRNA levels to 90 and 95 %, respectively. A comparison of the effects of the paired enantiomers and Δ¹-THC on the cellular parameters studied reveals that in differentiating cells the action of Δ¹-THC is intermediate between that of HU-210 and HU-211. This study demonstrates that compared to HU-210 and Δ¹-THC the positive enantiomer HU-211 has little cellular activity.     

In vitro regulation of granulosa cell differentiation. Involvement of cytoskeletal protein expression

The link between the biochemical and morphological differentiation of granulosa cells was studied by investigating the organization and the expression of cytoskeletal proteins which determine cell shape and contacts. In cells treated with follicle-stimulating hormone (FSH), in a serum- and growth factor-free medium, or with other compounds which elevate cellular cAMP levels, the synthesis of the adherens junction proteins, vinculin, alpha-actinin, and actin was reduced significantly when compared to unstimulated cells (7-fold for vinculin, 5-fold for alpha-actinin, and 3-fold for actin). The in vitro translatability of the mRNAs coding for these proteins and the level of actin mRNA determined by RNA blot hybridization were generally reduced in differentiating cells. The synthesis and the organization of vimentin and tubulin was unaffected during this process, whereas the organization of actin and vinculin was dramatically affected, with FSH-treated cells displaying a diffuse pattern of actin and vinculin, with very little vinculin in adhesion plaques. Gonadotropin-releasing hormone agonist and the phorbol ester 12-O-tetradecanoylphorbol-13-acetate which are known to antagonize the cAMP-mediated biochemical differentiation of granulosa cells by reducing cAMP levels or by activating protein kinase C and phospholipid turnover, blocked to a large extent the FSH-induced effect on the adherens junction proteins. Epidermal growth factor, which blocked the FSH-induced cAMP increase, but not the FSH-induced progesterone production, failed to block the synthesis of vinculin, alpha-actinin, and actin. Cytochalasin B could induce steroidogenesis and similar changes in the synthesis of these cytoskeletal proteins, whereas fibronectin, which causes cell spreading, blocked in part the FSH-induced effect on the expression of cytoskeletal proteins. The modulation of cytoskeletal proteins may therefore be an essential feature of programmed differentiation events leading to the final phenotype of granulosa cells.     

Actin and Myosin Synthesis During Differentiation of Neuroblastoma Cells

Abstract: The levels and rates of synthesis of actin and myosin in murine neuroblastoma S20 cells have been examined during neurite formation in the absence of serum. Two forms of actin (β, -γ) have been identified in these cells; they have isoelectric points of about 5.43 and 5.45, respectively, which differ from that of murine skeletal muscle actin (5.41). Although the neuroblastoma actins have the same molecular weight as skeletal muscle actin, they can be clearly distinguished from this protein by peptide maps. Actin comprises about 7% of the total protein in these cells and neither the level nor the proportion of the two isoelectric forms change during neurite extension (β: γ:60: 40). Myosin ATPase activity is also similar in morphologically differentiated and undifferentiated cells. The rates of synthesis of actin and myosin relative to total protein synthesis undergo small changes during differentiation. Actin synthesis increases about 30% in the 1st day of induction and falls off to the undifferentiated level by day 4. Myosin synthesis increases by about 30% within 2 days of induction and remains at that level for at least 2 days. In contrast, no change in the relative synthesis of tubulin occurs over 4 days of neurite induction. Thus, neurite extension by these cells is not accompanied by large changes in either the level or rates of synthesis of actin and/or myosin.     

Changes in cytoskeletal protein synthesis following axon injury and during axon regeneration

Injury to the axons of facial motoneurons stimulates increases in the synthesis of actin, tubulins, and GAP-43, and decreases in the synthesis of neurofilament proteins: mRNA levels change correspondingly. In contrast to this robust response of peripheral neurons to axotomy, injured central nervous system neurons show either an attenuated response that is subsequently aborted (rubrospinal neurons) or overall decreases in cytoskeletal protein mRNA expression (corticospinal and retinal ganglion neurons). There is evidence that these changes in synthesis are regulated by a variety of factors, including loss of endoneurially or target-derived trophic factors, positive signals arising from the site of injury, changes in the intraaxonal turnover of proteins, and substitution of target-derived trophic support by factors produced by glial cells. It is concluded that there is, as yet, no coherent explanation for the upregulation or downregulation of any of the cytoskeletal proteins following axotomy or during regeneration. In considering the relevance of these changes in cytoskeletal protein synthesis to regeneration, it is emphasized that they are unlikely to be involved in the initial outgrowth of the injured axons, both because transit times between cell body and injury site are too long, and because sprouting can occur in isolated axons. Injuryinduced acceleration of the axonal transport of tubulin and actin in the proximal axon is likely to be more important in providing the cytoskeletal protein required for initial axonal outgrowth. Subsequently, the increased synthesis and transport velocity for actin and tubulin increase the delivery of these proteins to support the increased volume of the maturing regenerating axons. Reduction in neurofilament synthesis and changes in neurofilament phosphorylation may permit the increased transport velocity of the other cytoskeletal proteins. There is little direct evidence that alterations in cytoskeletal protein synthesis are necessary for successful regeneration, nor are they sufficient in the absence of a supportive environment. Nevertheless, the correlation that exists between a robust cell body response and successful regeneration suggests that an understanding of the regulation of cytoskeletal protein synthesis following axon injury must be a part of any successful strategy to improve the regenerative capacity of the central nervous system.     

Effects of the piezo-tolerance of cultured deep-sea eel cells on survival rates, cell proliferation, and cytoskeletal structures

We investigated the pressure tolerance of deep-sea eel (Simenchelys parasiticus; habitat depth, 366–2,630 m) cells, conger eel (Conger myriaster) cells, and mouse 3T3-L1 cells. Although there were no living mouse 3T3-L1 and conger eel cells after 130 MPa (0.1 MPa = 1 bar) hydrostatic pressurization for 20 min, all deep-sea eel cells remained alive after being subjected to pressures up to 150 MPa for 20 min. Pressurization at 40 MPa for 20 min induced disruption of actin and tubulin filaments with profound cell-shape changes in the mouse and conger eel cells. In the deep-sea eel cells, microtubules and some actin filaments were disrupted after being subjected to hydrostatic pressure of 100 MPa and greater for 20 min. Conger eel cells were sensitive to pressure and did not grow at 10 MPa. Mouse 3T3-L1 cells grew faster under pressure of 5 MPa than at atmospheric pressure and stopped growing at 18 MPa. Deep-sea eel cells were capable of growth in pressures up to 25 MPa and stopped growing at 30 MPa. Deep-sea eel cells required 4 h at 20 MPa to finish the M phase, which was approximately fourfold the time required under atmospheric conditions.     

Distinct effects of calcium- and cyclic AMP-enhancing factors on cytoskeletal synthesis and assembly in mouse osteoblastic cells.

Previous studies have indicated that the effects of parathyroid hormone (PTH) on osteoblastic function involve alteration of cytoskeletal assembly. We have reported that after a transitory cell retraction, PTH induces respreading with stimulation of actin, vimentin and tubulins synthesis in mouse bone cells and that this effect is not mediated by cAMP. In order to further elucidate the role of intracellular cAMP and calcium on PTH action on bone cell shape and cytoskeleton we have compared the effects of calcium- and cAMP-enhancing factors on actin, tubulin and vimentin synthesis in relation with mouse bone cell morphology, DNA synthesis and alkaline phosphatase activity as a marker of differentiation. Confluent mouse osteoblastic cells were treated with 0.1 mM isobutylmethylxanthine (IBMX) for 24 h. This treatment caused an increase in the levels of cytoskeletal subunits associated with an elevation of cAMP. Under these conditions, PTH (20 nM) and forskolin (0.1 microM) produced persistent cytoplasmic retraction. PTH and forskolin treatment in presence of IBMX (24 h) induced inhibitory effects on actin and tubulin synthesis evaluated by [35S]methionine incorporation into cytoskeletal proteins identified on two-dimensional gel electrophoresis. Under these culture conditions PTH and forskolin also caused disassembly of microfilament and microtubules as shown by the marked reduction in Triton X soluble-actin and alpha- and beta-tubulins. In contrast, incubation of mouse bone cells with 1 microM calcium ionophore A23187 (24 h) resulted in increased monomeric and polymeric forms of actin and tubulin while not affecting intracellular cAMP. Alkaline phosphatase activity was increased in all conditions while DNA synthesis evaluated by [3H]thymidine incorporation into DNA was stimulated by PTH combined with forskolin and inhibited by the calcium ionophore. These data indicate that persistent elevation of cAMP levels induced by PTH and forskolin with IBMX cause cell retraction with actin and tubulin disassembly whereas rising cell calcium induces cytoskeletal protein assembly and synthesis in mouse osteoblasts. The results point to a distinct involvement of calcium and cAMP in both cytoskeletal assembly and DNA synthesis in mouse bone cells.