Cytochalasins as Modulators of Stem Cell Differentiation.

Regenerative medicine aims to identify new research strategies for the repair and restoration of tissues damaged by pathological or accidental events. Mesenchymal stem cells (MSCs) play a key role in regenerative medicine approaches due to their specific properties, such as the high rate of proliferation, the ability to differentiate into several cell lineages, the immunomodulatory potential, and their easy isolation with minimal ethical issues. One of the main goals of regenerative medicine is to modulate, both in vitro and in vivo, the differentiation potential of MSCs to improve their use in the repair of damaged tissues. Over the years, much evidence has been collected about the ability of cytochalasins, a large family of 60 metabolites isolated mainly from fungi, to modulate multiple properties of stem cells (SCs), such as proliferation, migration, and differentiation, by altering the organization of the cyto- and the nucleo-skeleton. In this review, we discussed the ability of two different cytochalasins, cytochalasins D and B, to influence specific SC differentiation programs modulated by several agents (chemical or physical) or intra- and extra-cellular factors, with particular attention to human MSCs (hMSCs).

One new cytochalasin metabolite isolated from a mangrove-derived fungus Daldinia eschscholtzii HJ001.

One new cytochalasin metabolite [11]-cytochalasa-5(6),13-diene-1,21-dione-7,18-dihydroxy-16,18-dimethyl-10-phenyl-(7S*,13E,16S*,18R*) (1), together with three known compounds (2-4) were obtained from the EtOAc extract of the endophytic fungus Daldinia eschscholtzii HJ001 isolated from the mangrove Brguiera sexangula var. rhynchopetala collected in the South China Sea. Their structures were elucidated by the detailed analysis of comprehensive spectroscopic data. Compounds 1 and 2 were evaluated for their antibacterial and cytotoxic activities.

Improving the productivity of 19,20-epoxy-cytochalasin Q in Xylaria sp. sof11 with culture condition optimization.

19,20-Epoxy-cytochalasin Q (B5A) is a cytochalasin with a wide range of biological activities, which can be produced by Xylaria sp. sof11, a strain isolated from the seafloor of the northern South China Sea. Since the low titer of B5A has greatly limited its further studies, we have systematically conducted the fermentative optimization for B5A production in this article. The effects of major medium components, including the carbon and organic nitrogen sources, as well as of the concentration of sea salt, were respectively investigated through single-factor experiments. As a result, sucrose and fish meal were determined to be the key factors affecting the production of B5A. Then three important variables, sucrose, fish meal, and filling volume, were screened out by the Plackett-Burman (PB) design. The optimal level of these variables was further confirmed by response surface analysis. The final formulated medium was set as 35.2 g/L sucrose and 18.0 g/L fish meal, with filling volume of 34.6 mL, which could afford 440.3 mg/L production of B5A, approximately 4.4-fold higher than that in the original medium. The significantly improved productivity of B5A will facilitate the subsequent mechanistic and clinical studies of B5A.

Effect of Culture Conditions on Metabolite Production of Xylaria sp.

Seeking a strategy for triggering the cryptic natural product biosynthesis to yield novel compounds in the plant-associated fungus Xylaria sp., the effect of culture conditions on metabolite production was investigated. A shift in the production of five known cytochalasin-type analogues 1-5 to six new α-pyrone derivatives, xylapyrones A-F (compounds 6-11), from a solid to a liquid medium was observed. These compounds were identified by analysis of 1D and 2D NMR and HRMS data. Compounds 1-3 showed moderate cytotoxicity against HepG2 and Caski cancer cell lines with IC50 values ranging from 25 to 63 µM and compounds 4-11 were found to be inactive, with IC50 values>100 µM.

Nuclear receptor nur77 promotes cerebral cell apoptosis and induces early brain injury after experimental subarachnoid hemorrhage in rats.

Nur77 is a potent proapoptotic member of the nuclear receptor superfamily that is expressed predominantly in brain tissue. It has been demonstrated that Nur77 mediates apoptosis in multiple organs. Nur77-mediated early brain injury (EBI) involves a conformational change in BCL-2 and triggers cytochrome C (cytoC) release resulting in cellular apoptosis. This study investigates whether Nur77 can promote cerebral cell apoptosis after experimentally induced subarachnoid hemorrhage (SAH) in rats. Sprague Dawley rats were randomly assigned to three groups: 1) untreated group, 2) treatment control group, and 3) SAH group. The experimental SAH group was divided into four subgroups, corresponding to 12 hr, 24 hr, 48 hr, and 72 hr after experimentally induced SAH. It remains unclear whether Nur77 can play an important role during EBI after SAH as a proapoptotic protein in cerebral cells. Cytosporone B (Csn-B) was used to demonstrate that Nur77 could be enriched and used to aggravate EBI after SAH. Rats treated with Csn-B were given an intraperitoneal injection (13 mg/kg) 30 min after experimentally induced SAH. We found that Nur77 promotes cerebral cell apoptosis by mediating EBI and triggering a conformational change in BCL-2, resulting in cytoC release. Nur77 activity, along with cerebral cell apoptosis, peaked at 24 hr after SAH onset. After induction of SAH, an injection of Csn-B, an agonist for Nur77, enhanced the expression and function of Nur77. In summary, we have demonstrated the proapoptotic effect of Nur77 within cerebral cells, an effect that can be further exacerbated with Csn-B stimulation.

Signaling by intracellular Ca2+ and H+ in larval mosquito (Aedes aegypti) midgut epithelium in response to serosal serotonin and lumen pH.

The midgut of larval mosquitoes (Aedes aegypti) mediates a cycle of alkali secretion in the anterior segment (AMG) followed by partial reacidification in the posterior segment (PMG); both processes are serotonin-dependent. Here we report that intracellular Ca(2+)(Ca(i)(2+)) as indicated by Fura-2 fluorescence, is elevated in both tissues in response to serotonin, but the time courses differ characteristically in the two gut segments, and Ca(2+)-free solution abolishes the serotonin response in AMG, but not in PMG, whereas Thapsigargin, an inhibitor of endoplasmic Ca(2+) transport, abolished responsiveness to 5-HT in PMG. These results suggest the origins for the Ca(2+) signal differ between the two tissues. Quantitative real-time RT-PCR revealed expression of 5 putative 5-HT receptor types in AMG, including 5-HT(2)-like receptors which would be expected to initiate a Ca(2+) signal. None of these receptors were highly expressed in PMG. Cyclic AMP (cAMP) is a secretagogue for both tissues, but H89, an inhibitor of Protein Kinase A (PKA), is also a secretagogue, suggesting that the stimulatory effect of cAMP involves a non-PKA pathway. Cytochalasins B and D block the effect of 5-HT in AMG, suggesting a vesicle-fusion mechanism of activation of the basal V-ATPase in this tissue. Finally, in PMG, elevation of luminal pH increases (Ca(i)(2+)) and decreases intracellular pH as measured by BCECF fluorescence. These responses suggest that the rate of acid secretion by PMG might be responsive to local demand for luminal reacidification as well as to serosal serotonin.

Exclusion of actin-binding protein p57/coronin-1 from bacteria-containing phagosomes in macrophages infected with Legionella.

Legionella pneumophila, the causative agent of Legionnaires' disease, is a human pathogen that multiplies within alveolar macrophages. L. pneumophila establishes specialized phagosomes in which it evades the host defense through largely unknown mechanisms. Here we analyzed the role of an actin-binding protein, p57/coronin-1, a member of the coronin protein family, during Legionella infection. On fluorescence microscopy, p57/coronin-1 and F-actin were found to be co-localized at the sites on the plasma membrane where L. pneumophila adhered to U937 human macrophage-like cells. The localization of p57/coronin-1 at the sites of bacterial adherence was inhibited by treatment with cytochalasin D (an inhibitor of actin polymerization), suggesting that p57/coronin-1 is involved in the actin-dependent uptake of L. pneumophila into U937 cells. In addition, we showed that p57/coronin-1 was excluded from phagosomes containing live L. pneumophila throughout the infection, whereas transient accumulation of p57/coronin-1 was observed on phagosomes containing Texas-Red-labeled opsonized zymosan (TROpZ) or heat-killed L. pneumophila at an early stage of phagocytosis. The exclusion of p57/coronin-1 from phagosomes containing live another Legionella species Legionella gratiana at an early stage of infection was also observed. Taken together, these results suggest that the endocytic pathways of live Legionella species are distinct from general phagocytic pathways, which lead to lysosomal degradation.

Localization of RHO-4 indicates differential regulation of conidial versus vegetative septation in the filamentous fungus Neurospora crassa.

Rho-4 mutants of the filamentous fungus Neurospora crassa lack septa and asexual spores (conidia) and grow slowly. In this report, localization of green fluorescent protein-tagged RHO-4 is used to elucidate the differences in factors controlling RHO-4 localization during vegetative growth versus asexual development. RHO-4 forms a ring at incipient vegetative septation sites that constricts with the formation of the septum toward the septal pore; RHO-4 persists around the septal pore after septum completion. During the formation of conidia, RHO-4 localizes to the primary septum but subsequently is relocalized to the cytoplasm after the placement of the secondary septum. Cytoplasmic localization and inactivation of RHO-4 are mediated by a direct physical interaction with RDI-1, a RHO guanosine nucleotide dissociation inhibitor. Inappropriate activation of the cyclic AMP-dependent protein kinase A pathway during vegetative growth causes mislocalization of RHO-4 away from septa to the cytoplasm, a process which was dependent upon RDI-1. An adenylate cyclase cr-1 mutant partially suppresses the aconidial defect of rho-4 mutants but only rarely suppresses the vegetative septation defect, indicating that conidial septation is negatively regulated by CR-1. These data highlight the differences in the regulation of septation during conidiation versus vegetative septation in filamentous fungi.

Uptake and intracellular fate of Francisella tularensis in human macrophages.

Francisella tularensis is an intracellular pathogen that survives and multiplies within host mononuclear phagocytes. We have shown that uptake of the bacterium by human macrophages occurs by a novel process, "looping phagocytosis," in which the bacterium is engulfed in a spacious, asymmetric, pseudopod loop. Whereas looping phagocytosis is resistant to treatment of the F. tularensis with formalin, proteases, or heat, the process is abolished by oxidation of the bacterial carbohydrates with periodate, suggesting a role for preformed surface carbohydrate molecules in triggering looping phagocytosis. Following uptake, F. tularensis initially resides in a spacious vacuole at the periphery of the cell, but this vacuole rapidly shrinks in size. The nascent F. tularensis vacuole transiently acquires early endosomal markers, but subsequently exhibits an arrested maturation, manifest by only limited amounts of lysosome-associated membrane glycoproteins (consistent with limited interaction with late endosomes), nonfusion with lysosomes, and minimal acidification. In ultrastructural studies, we have observed that the F. tularensis phagosome displays a novel feature in that many of the phagosomes acquire an electron dense fibrillar coat. This fibrillar coat forms blebs and vesicles, and with time, is seen to be fragmented and disrupted. With increasing time after infection, increasing numbers of the F. tularensis are found free in the macrophage cytoplasm, such that by 14 h after infection, less than 15% of the bacteria are surrounded by any discernible phagosomal membrane. Further research is needed to determine the mechanisms underlying looping phagocytosis, and the maturational arrest, fibrillar coat formation, and disruption of the phagosome.

Conversion of serine to aspartate imitates phosphorylation-induced changes in the structure and function of microtubule-associated protein tau.

Microtubule-associated protein tau is a neuronal phosphoprotein that promotes microtubule assembly in vitro and has been shown to play a role in the development of axonal morphology. Tau can be phosphorylated in vitro by several kinases, some of which cause a change in the conformation and activities of tau. Here we report the consequences of converting two of the protein kinase A phosphorylation sites (positions 156 and 327), first to alanine to eliminate phosphorylation, and second to aspartate, to mimic phosphorylation. We show that a serine to aspartate mutation at position 327 results in a conformational change similar to that caused by phosphorylation of this residue. This mutation does not affect the activities of tau in microtubule assembly as compared with wild-type tau. However, an additional mutation at position 156 to aspartate drastically decreases the microtubule nucleation activity of tau but does not affect the activity of tau to promote microtubule growth. All constructs are similarly bound to microtubules and promote process formation when expressed in cytochalasin-treated PC12 cells. We conclude that serine to aspartate mutations provide a useful system for analyzing the effect of individual phosphorylation sites on the conformation and function of tau in vitro and in cells. The results provide evidence that microtubule growth and nucleation can be differentially affected by phosphorylation of individual residues in a region amino-terminally flanking the microtubule binding domain of tau.

Effects of multidrug resistance-related ATP-binding-cassette transporter proteins on the cytoskeletal activity of cytochalasins.

Cytochalasins are microfilament-active mould metabolites, widely utilized to study the involvement of the actin cytoskeleton in cellular processes as well as in genotoxicity and cell kinetic research. In this study we have investigated whether multidrug-resistance phenotypes, caused by overexpression of the ATP-binding-cassette transporter proteins P-glycoprotein (P-gp) or multidrug-resistance-associated protein (MRP), influence the microfilament-depolymerizing effect of cytochalasins. Using four well-characterized multidrug-resistance cell models, we have shown that both the microfilament-disrupting (phalloidine staining) and the cytotoxic (MTT-assay) activity of cytochalasins are reduced in parallel with increased P-gp expression and restorable by P-gp-modulating agents. This also applied to the cytochalasin D-mediated induction of polykaryons (microscopic evaluation) which arise as a consequence of impaired cytokinesis but unaffected karyokinesis. The reduced cellular activity of cytochalasins in P-gp-positive cell lines was correlated with decreased intracellular accumulation ([3H]cytochalasin B accumulation) which was also restorable by P-gp modulators. Moreover, the dose-dependent inhibition of P-gp photoaffinity labeling ([3H]-azidopine) suggested cytochalasins as P-gp-binding agents. In contrast, MRP overexpression had no effect on either cytochalasin microfilament activity or cytotoxicity. In conclusion, data indicate that the microfilament-destructive effects of cytochalasins are impaired due to a reduction of the intracellular cytochalasin accumulation by P-gp but not by MRP. Results are discussed with regard to P-gp as a resistance factor when cytochalasins are utilized to study microfilament dynamics, cell cycle kinetics or chromosomal damage. Moreover, the polykaryon-inducing activity of cytochalasin D is suggested as a specific indicator for a P-gp-mediated multidrug-resistance phenotype and the reversing potency of chemosensitizers.

The complex of actin and deoxyribonuclease I as a model system to study the interactions of nucleotides, cations and cytochalasin D with monomeric actin.

The stoichiometric actin--DNase-I complex was used to study the actin--nucleotide and actin--divalent-cation interactions and its ATPase activity in the presence of MgCl2 and cytochalasin D. Treatment of actin--DNase-I complex with 1 mM EDTA results in almost complete restoration of its otherwise inhibited DNase I activity, although the complex does not dissociate, as verified by size-exclusion chromatography. This effect is due to a loss of actin-bound nucleotide but is prevented by the presence of 0.1-0.5 mM ATP, ADP and certain ATP analogues. In this case no increase in DNase I activity occurs, even in the presence of EDTA. At high salt concentrations and in the presence of Mg2+ ('physiological conditions') the association rate constants for ATP, ADP and epsilon ATP (1,N6-ethenoadenosine 5'-triphosphate) and the dissociation rate constant for epsilon ATP were determined. Both the on and off rates were found to be reduced by a factor of about 10 when compared to uncomplexed actin. Thus the binding constant of epsilon ATP to actin is almost unaltered after complexing to DNase I (2.16 x 10(8) M-1). Titrating the increase in DNase I activity of the actin--DNase I complex against nucleotide concentration in the presence of EDTA, the association constant of ATP to the cation-free form of actin--DNase I complex was found to be 5 x 10(3) M-1, which is many orders of magnitude lower than in the presence of divalent metal ions. The binding constant of Ca2+ to the high-affinity metal-binding site of actin was found not to be altered when complexed to DNase I, although the rate of Ca2+ release decreases by a factor of 8 after actin binding to DNase I. The rate of denaturation of nucleotide-free and metal-ion-free actin--DNase I complex was found to be reduced by a factor of about 15. The ATPase activity of the complex is stimulated by addition of Mg2+ and even more effectively by cytochalasin D, proving that this drug is able to interact with monomeric actin.

Stimulation of a calcium-dependent actin nucleation activity by phorbol 12-myristate 13-acetate in rabbit macrophage cytoskeletons.

Cytoskeletons of detergent-extracted quiescent macrophages have nucleation sites that increase the rate of pyrene-labeled actin assembly in vitro. Cytochalasin D, which inhibits actin assembly at the fast-exchanging ends of filaments (barbed with respect to heavy meromyosin decorated filaments), only partially inhibits the increased assembly rate, demonstrating that pyrene-actin monomers add to both ends of filaments present in the cytoskeletons. Cytoskeletons prepared from macrophages treated with phorbol 12-myristate 13-acetate for 20-30 s before permeabilization, markedly stimulated (300% of control) the rate of actin assembly, and this increment was completely cytochalasin-sensitive, indicating that exposure to phorbol leads to formation of free barbed ends. Nucleation activity required more than 5 nM free calcium only in the assay and was maximal in the presence of 200 nM calcium. Concentrations of calcium of at least 30 nM dissociate the nucleation activity from the cytoskeleton, and it is recovered fully active in the calcium wash.

Polyphosphoinositide micelles and polyphosphoinositide-containing vesicles dissociate endogenous gelsolin-actin complexes and promote actin assembly from the fast-growing end of actin filaments blocked by gelsolin.

The Ca2+-activated actin-binding protein gelsolin regulates actin filament length by severing preformed filaments and by binding actin monomers, stabilizing nuclei for their assembly into filaments. Gelsolin binds to phosphatidylinositol 4,5-bisphosphate (PIP2), with consequent inhibition of its filament severing activity and dissociation of EGTA-resistant complexes made with rabbit macrophage or human plasma gelsolin and rabbit muscle actin. This study provides evidence for an interaction of gelsolin with phosphatidylinositol monophosphate (PIP) as well as PIP2 and further describes their effects on gelsolin's function. Both phosphoinositides completely dissociate EGTA-insensitive rabbit macrophage cytoplasmic gelsolin-actin complexes and inhibit gelsolin's severing activity. The magnitude of inhibition depends strongly on the physical state of the phosphoinositides, being maximal in preparations that contain small micelles of either purified PIP or PIP2. Aggregation of PIP or PIP2 micelles by divalent cations or insufficient sonication or their incorporation into vesicles containing other phospholipids decreases but does not eliminate the inhibitory properties of the polyphosphoinositides. The presence of gelsolin partly inhibits the divalent cation-induced aggregation of PIP2 micelles. PIP2 in combination with EGTA inactivates gelsolin molecules that block the fast-growing end of actin filaments, thereby accelerating actin polymerization. Regulation of gelsolin by the intracellular messengers Ca2+ and polyphosphoinositides allows for the formation of several different gelsolin-actin intermediates with distinct functional properties that may be involved in changes in the state of cytoplasmic actin following cell stimulation.

Actin polymerization. The mechanism of action of cytochalasin D.

Fluorescence changes using actin covalently labeled with N-(1-pyrenyl)iodoacetamide have been used to determine the effect of cytochalasin D on actin polymerization. A mechanism for the effect of cytochalasin D on actin polymerization is presented, which explains the experimental observation of a cytochalasin D-induced increase in the initial rate of polymerization and a decrease in the final extent of the reaction. Central to this mechanism is the Mg2+-dependent formation of cytochalasin D-induced dimers. The dimers serve as nuclei to enhance the polymerization rate. Binding of Mg2+ to a low affinity site on the dimer induces a conformational change which can be observed as a rapid fluorescence increase. A subsequent time-dependent fluorescence decrease observed prior to polymerization appears to represent ATP hydrolysis resulting in dissociation of the dimer and release of actin monomers containing ADP. We postulate that a slow rate of exchange of ATP for bound ADP relative to hydrolysis results in the accumulation of monomers containing ADP. As these monomers have a high critical concentration, the final extent of polymerization is reduced dramatically. The Mg2+ dependence of the final extent of polymerization in the presence of cytochalasin D is also explained in the context of this mechanism.

Cytoplasmic concentrations of inorganic phosphate affect the critical concentration for assembly of actin in the presence of cytochalasin D or ADP.

Skeletal muscle actin labelled with pyrene was used to measure the critical concentration (Cc) for assembly in conditions designed to approximate the ionic environment in the cytoplasm. Under these conditions (0.1 M-KCl, 2 mM-MgCl2, 1.1 mM-ATP, 0.1 mM-CaCl2, 0.5 mM-ethyleneglycol-bis(beta-aminoethylether)N,N'-tetraacetic acid, 0.25 mM-2-mercaptoethanol, 20 mM-imidazol X HCl, pH 7.0), the steady-state Cc value was estimated to be 0.07 microM (3.0 micrograms/ml), and, consistent with previous observations, the Cc increased to 0.20 microM (8.7 micrograms/ml) in the presence of 10(-6) M-cytochalasin D, and to 1.10 microM (47 micrograms/ml) after conversion of ATP to ADP using hexokinase and glucose. Addition of inorganic phosphate (Pi) at concentrations up to 20 mM caused only a slight decrease in the steady-state Cc, but at 2 mM-Pi (a reasonable estimate of cytoplasmic concentrations) the increase in Cc due to cytochalasin D was abolished, and at higher Pi concentrations there was even a slight decrease. Increasing Pi concentrations also progressively reduced the steady-state Cc for ADP-actin close to that for ATP-actin. These results are consistent with an increased affinity of ADP-actin for the polymer in the presence of Pi. To determine whether these effects of Pi were simply mass action effects on hydrolysis of bound ATP by polymerized actin, the stoichiometry of ATP hydrolysis during actin assembly was estimated and found to be at unity within the limits of experimental error and to be unaffected by Pi up to 20 mM. In addition, actin depolymerized by removal of ATP using glucose and hexokinase rapidly reassembled after addition of 20 mM-Pi. These results are interpreted by a mechanism involving the formation of ADP-Pi-actin species and are discussed in relation to the phenomenon of treadmilling and the theory of dynamic instability, and the potential for their occurrence in cells.

Interaction of cytochalasin D with actin filaments in the presence of ADP and ATP.

Cytochalasin D strongly inhibits the faster components in the reactions of actin filament depolymerization and elongation in the presence of 10 mM Tris-Cl-, pH 7.8, 0.2 mM dithiothreitol, 1 mM MgCl2, 0.1 mM CaCl2, and 0.2 mM ATP or ADP. Assuming an exclusive and total capping of the barbed end by the drug, the kinetic parameters derived at saturation by cytochalasin D refer to the pointed end and are 10-15-fold lower than at the barbed end. In ATP, the critical concentration increases with cytochalasin D up to 12-fold its value when both ends are free; as a result of the lowering of the free energy of nucleation by cytochalasin D, short oligomers of F-actin exist just above and below the critical concentration. Cytochalasin D interacts strongly with the barbed ends independently of the ADP-G-actin concentration (K = 0.5 nM-1). In contrast, the affinity of cytochalasin D decreases cooperatively with increasing ATP-G-actin concentration. These data are equally well accounted for by two different models: either cytochalasin D binds very poorly to ATP-capped filament ends whose proportion increases with actin concentration, or cytochalasin D binds equally well to ATP-ends and ADP-ends and also binds to actin dimers in ATP but not in ADP. A linear actin concentration dependence of the rate of growth was found at the pointed end, consistent with the virtual absence of an ATP cap at that end.

Microtubule-disrupting drugs increase the frequency of conversion of a rat mammary epithelial stem cell line to elongated, myoepithelial-like cells in culture.

Rat mammary (Rama) 25 cuboidal epithelial stem cells convert at a low frequency to elongated, Thy-1-positive, myoepithelial-like cells in culture; one such cell line is termed Rama 29. Addition of increasing concentrations of the microtubule-disrupting drug colchicine to sparse cultures of Rama 25 dramatically increases the percentage of colonies containing elongated cells and the percentage of Thy-1-positive cells when the drug is removed. Similar results on the formation of elongated cell colonies are obtained with other microtubule disruptors, such as vinblastine, vincristine, demecolcine, and nocodazole. The inactive analogues of colchicine beta- and delta-lumicolchicine and the microfilamental-disruptors cytochalasin B and D are without effect on the formation of elongated cell colonies and Thy-1-positive cells. For a given concentration of colchicine the percentage of elongated cell colonies and Thy-1-positive cells increases the longer the cells are exposed to the drug (range 8-96 hr) and the longer the drug-treated cultures are subsequently grown in drug-free medium. Colchicine fails to display this morphological change on Rama 29 elongated cells and on Rama 600 epithelial cells from a rat mammary metastasizing tumor. Immunofluorescent localization of antisera to tubulin confirms that colchicine disrupts the microtubules in all three cell lines at similar concentrations (0.1 to 1 microM) to those required to increase the percentage of elongated cell colonies in Rama 25. The DNA synthesis inhibitor cytosine arabinoside fails to inhibit this conversion process, and time-lapse cinematographic studies confirm that the conversion of a cuboidal to an elongated cell can take place without cell division. However, cell division may sometimes be required for subsequent stabilization events. Treatment of Rama 25 cells with colchicine under the same conditions also increases the abundance of elongated cell (Rama 29)-associated polypeptides, and elongated cell clones isolated after such treatment show an overall pattern of protein synthesis very similar to that of Rama 29.

Evidence for regulation of actin synthesis in cytochalasin D-treated HEp-2 cells.

In HEp-2 cells treated with 0.2 or 2.0 microM cytochalasin D (CD), the relative rate of actin synthesis increased for about 12 h and then reached a plateau; this increase was suppressed by actinomycin D (AD). When CD was washed from cells which had been treated for 20 h, the elevated rate of actin synthesis declined to the control value within ca 4 h, as the actin-containing cytoskeletal components rearranged by CD recovered their normal morphology. Subsequently, actin synthesis was depressed below control values for a prolonged period; during recovery from 2 h treatment with CD, this depression was of much shorter duration. Re-addition of CD to cells after a 3 h recovery period again induced the cytoskeletal alterations characteristic of CD treatment but did not reverse the prior decline in the rate of actin synthesis. In HEp-2 cells treated with cycloheximide during exposure to CD for 20 h, the relative rate of actin synthesis measured after removal of cycloheximide was twofold higher than with CD alone and such cells exhibited a twofold slower decline in the rate of actin synthesis during recovery from CD in the continued presence of cycloheximide. These effects of cycloheximide, which resemble observations on "super-induction", suggest that actin synthesis in CD-treated and recovering HEp-2 cells may be regulated by a repressor protein. The possibility that the proposed repressor protein is actin and that actin may thus be a feedback inhibitor of its own synthesis is discussed.