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Autophagy is a central intracellular catabolic mechanism that mediates the degradation of cytoplasmic proteins and organelles, and regulation of autophagy is essential for homeostasis. HMGB1 is an important sepsis mediator when secreted and also functions as an inducer of autophagy by binding to Beclin 1. In this study, we studied the effect of inflachromene (ICM), a novel HMGB1 secretion inhibitor, on autophagy. ICM inhibited autophagy by inhibiting nucleocytoplasmic translocation of HMGB1 and by increasing Beclin 1 ubiquitylation for degradation by enhancing the interaction between Beclin 1 and E3 ubiquitin ligase RNF216. These data suggest that ICM could be used as a potential autophagy suppressor., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

Nonsense-mutation-containing messenger ribonucleoprotein particles (mRNPs) transit through cytoplasmic foci called P-bodies before undergoing nonsense-mediated mRNA decay (NMD), a cytoplasmic mRNA surveillance mechanism. This study shows that the cytoskeleton modulates transport of nonsense-mutation-containing mRNPs to and from P-bodies. Impairing the integrity of cytoskeleton causes inhibition of NMD. The cytoskeleton thus plays a crucial role in NMD. Interestingly, disruption of actin filaments results in both inhibition of NMD and activation of premature termination codon (PTC) readthrough, while disruption of microtubules causes only NMD inhibition. Activation of PTC readthrough occurs concomitantly with the appearance of cytoplasmic foci containing UPF proteins and mRNAs with nonsense mutations but lacking the P-body marker DCP1a. These findings demonstrate that in human cells, PTC readthrough occurs in novel 'readthrough bodies' and requires the presence of UPF proteins., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2017. Published by The Company of Biologists Ltd.)

The transcription factor p53 plays a crucial role in coordinating the cellular response to various stresses. Therefore, p53 protein levels and activity need to be kept under tight control. We report here that diacylglycerol kinase ζ (DGKζ) binds to p53 and modulates its function both in the cytoplasm and nucleus. DGKζ, a member of the DGK family that metabolizes a lipid second messenger diacylglycerol, localizes primarily to the nucleus in various cell types. Recently, reports have described that excitotoxic stress induces DGKζ nucleocytoplasmic translocation in hippocampal neurons. In the study reported here we found that cytoplasmic DGKζ attenuates p53-mediated cytotoxicity against doxorubicin-induced DNA damage by facilitating cytoplasmic anchoring and degradation of p53 through a ubiquitin-proteasome system. Concomitantly, decreased levels of nuclear DGKζ engender downregulation of p53 transcriptional activity. Consistent with these in vitro cellular experiments, DGKζ-deficient brain exhibits high levels of p53 protein after kainate-induced seizures and even under normal conditions. These findings provide novel insights into the regulation of p53 function and suggest that DGKζ serves as a sentinel to control p53 function both during normal homeostasis and in stress responses.

Here, we describe a phosphorylation-based reverse myristoyl switch for mammalian ZNRF2, and show that this E3 ubiquitin ligase and its sister protein ZNRF1 regulate the Na(+)/K(+) pump (Na(+)/K(+)ATPase). N-myristoylation localizes ZNRF1 and ZNRF2 to intracellular membranes and enhances their activity. However, when ZNRF2 is phosphorylated in response to agonists including insulin and growth factors, it binds to 14-3-3 and is released into the cytosol. On membranes, ZNRF1 and ZNRF2 interact with the Na(+)/K(+)ATPase α1 subunit via their UBZ domains, while their RING domains interact with E2 proteins, predominantly Ubc13 that, together with Uev1a, mediates formation of Lys63-ubiquitin linkages. ZNRF1 and ZNRF2 can ubiquitylate the cytoplasmic loop encompassing the nucleotide-binding and phosphorylation regions of the Na(+)/K(+)ATPase α1 subunit. Ouabain, a Na(+)/K(+)ATPase inhibitor and therapeutic cardiac glycoside, decreases ZNRF1 protein levels, whereas knockdown of ZNRF2 inhibits the ouabain-induced decrease of cell surface and total Na(+)/K(+)ATPase α1 levels. Thus, ZNRF1 and ZNRF2 are new players in regulation of the ubiquitous Na(+)/K(+)ATPase that is tuned to changing demands in many physiological contexts.

The yeast cyclin-C-Cdk8p kinase complex represses the transcription of a subset of genes involved in the stress response. To relieve this repression, cyclin C is destroyed in cells exposed to H(2)O(2) by the 26S proteasome. This report identifies Not4p as the ubiquitin ligase mediating H(2)O(2)-induced cyclin C destruction. Not4p is required for H(2)O(2)-induced cyclin C destruction in vivo and polyubiquitylates cyclin C in vitro by utilizing Lys48, a ubiquitin linkage associated with directing substrates to the 26S proteasome. Before its degradation, cyclin C, but not Cdk8p, translocates from the nucleus to the cytoplasm. This translocation requires both the cell-wall-integrity MAPK module and phospholipase C, and these signaling pathways are also required for cyclin C destruction. In addition, blocking cytoplasmic translocation slows the mRNA induction kinetics of two stress response genes repressed by cyclin C. Finally, a cyclin C derivative restricted to the cytoplasm is still subject to Not4p-dependent destruction, indicating that the degradation signal does not occur in the nucleus. These results identify a stress-induced proteolytic pathway regulating cyclin C that requires nuclear to cytoplasmic relocalization and Not4p-mediated ubiquitylation.

In response to environmental stress, cytoplasmic mRNAs aggregate to form stress granules (SGs). SGs have mainly been studied indirectly using protein markers, but the real-time behavior of endogenous mRNAs in SGs remains uncertain. Here, we visualized endogenous cytoplasmic poly(A)(+) mRNAs in living mammalian cells using a linear antisense 2'-O-methyl RNA probe. In arsenite-stressed cells, endogenous mRNAs aggregated in granules that colocalized with SGs marked by TIA-1-GFP. Moreover, analysis of mRNA dynamics using fluorescence recovery after photobleaching showed that approximately one-third of the endogenous mRNAs in SGs was immobile, another one-third was diffusive, and the remaining one-third was in equilibrium between binding to and dissociating from SGs, with a time constant of approximately 300 seconds. These dynamic characteristics of mRNAs were independent of the duration of stress and microtubule integrity. Similar characteristics were also observed from fos mRNA labeled with an antisense 2'-O-methyl RNA probe. Our results revealed the behavior of endogenous mRNAs, and indicated that SGs act as dynamic harbors of untranslated poly(A)(+) mRNAs.

Distinct opioid receptor agonists have been proved to induce differential patterns of ERK activation, but the underlying mechanisms remain unclear. Here, we report that Ser363 in the δ-opioid receptor (δOR) determines the different abilities of the δOR agonists DPDPE and TIPP to activate ERK by G-protein- or β-arrestin-dependent pathways. Although both DPDPE and TIPP activated ERK1/2, they showed different temporal, spatial and desensitization patterns of ERK activation. We show that that DPDPE employed G protein as the primary mediator to activate the ERK cascade in an Src-dependent manner, whereas TIPP mainly adopted a β-arrestin1/2-mediated pathway. Moreover, we found that DPDPE gained the capacity to adopt the β-arrestin1/2-mediated pathway upon Ser363 mutation, accompanied by the same pattern of ERK activation as that induced by TIPP. Additionally, we found that TIPP- but not DPDPE-activated ERK could phosphorylate G-protein-coupled receptor kinase-2 and β-arrestin1. However, such functional differences of ERK disappeared with the mutation of Ser363. Therefore, the present study reveals a crucial role for Ser363 in agonist-specific regulation of ERK activation patterns and functions.

Water is the dominant ingredient of cells and its dynamics are crucial to life. We and others have suggested a physical picture of the cell as a soft, fluid-infiltrated sponge, surrounded by a water-permeable barrier. To understand water movements in an animal cell, we imposed an external, inhomogeneous osmotic stress on cultured cancer cells. This forced water through the membrane on one side, and out on the other. Inside the cell, it created a gradient in hydration, that we visualized by tracking cellular responses using natural organelles and artificially introduced quantum dots. The dynamics of these markers at short times were the same for normal and metabolically poisoned cells, indicating that the cellular responses are primarily physical rather than chemical. Our finding of an internal gradient in hydration is inconsistent with a continuum model for cytoplasm, but consistent with the sponge model, and implies that the effective pore size of the sponge is small enough to retard water flow significantly on time scales ( approximately 10-100 seconds) relevant to cell physiology. We interpret these data in terms of a theoretical framework that combines mechanics and hydraulics in a multiphase poroelastic description of the cytoplasm and explains the experimentally observed dynamics quantitatively in terms of a few coarse-grained parameters that are based on microscopically measurable structural, hydraulic and mechanical properties. Our fluid-filled sponge model could provide a unified framework to understand a number of disparate observations in cell morphology and motility.

Nucleoporins and the promyelocytic leukemia protein (PML) represent structural entities of nuclear pore complexes and PML nuclear bodies, respectively. In addition, these proteins might function in a common biological mechanism, because at least two different nucleoporins, Nup98 and Nup214, as well as PML, can become aberrantly expressed as oncogenic fusion proteins in acute myeloid leukemia (AML) cells. Here we show that PML and nucleoporins become directed to common cytoplasmic compartments during the mitosis-to-G1 transition of the cell cycle. These protein assemblies, which we have termed CyPNs (cytoplasmic assemblies of PML and nucleoporins), move on the microtubular network and become stably connected to the nuclear membrane once contact with the nucleus has been made. The ability of PML to target CyPNs depends on its nuclear localization signal, and loss of PML causes an increase in cytoplasmic-bound versus nuclear-membrane-bound nucleoporins. CyPNs are also targeted by the acute promyelocytic leukemia (APL) fusion protein PML-RARalpha and can be readily detected within the APL cell line NB4. These results provide insight into a dynamic pool of cytoplasmic nucleoporins that form a complex with the tumor suppressor protein PML during the G1 phase of the cell cycle.

Homeostasis in cells maintains their survival and functions. The plasticity at neurons and synapses may destabilize their signal encoding. The rapid recovery of cellular homeostasis is needed to secure the precise and reliable encoding of neural signals necessary for well-organized behaviors. We report a homeostatic process that is rapidly established through Ca(2+)-induced coordination of functional plasticity among subcellular compartments. An elevation of cytoplasmic Ca(2+) levels raises the threshold potentials and refractory periods of somatic spikes, and strengthens the signal transmission at glutamatergic and GABAergic synapses, in which synaptic potentiation shortens refractory periods and lowers threshold potentials. Ca(2+) signals also induce an inverse change of membrane excitability at the soma versus the axon. The integrative effect of Ca(2+)-induced plasticity among the subcellular compartments is homeostatic in nature, because it stabilizes neuronal activities and improves spike timing precision. Our study of neuronal homeostasis that is fulfilled by rapidly coordinating subcellular compartments to improve neuronal encoding sheds light on exploring homeostatic mechanisms in other cell types.

Programmed cell death (apoptosis) is important in tissue maintenance. Hallmarks of apoptosis include caspase activation, DNA fragmentation and an overall reduction in cell volume. Whether this apoptotic volume decrease (AVD) is a mere response to initiators of apoptosis or whether it is functionally significant is not clear. In this study, we sought to answer this question using human malignant glioma cells as a model system. In vivo, high grade gliomas demonstrate an increased percentage of apoptotic cells as well as upregulation of death ligand receptors. By dynamically monitoring cell volume, we show that the induction of apoptosis, via activation of either the intrinsic or extrinsic pathways with staurosporine or TRAIL, respectively, resulted in a rapid AVD in D54-MG human glioma cells. This decrease in cell volume could be prevented by inhibiting the efflux of Cl(-) through channels. Such suppression of AVD also reduced the activation of caspases 3, 8 and 9 and suppressed DNA fragmentation. Importantly, experimental manipulations that reduce the cell volume to 70% of the original volume for periods of at least 3 hours were sufficient to initiate apoptosis even in the absence of death ligands. Hence, this data suggests that cell condensation is both necessary and sufficient for the induction of apoptosis.

Using confocal laser scanning microscopy we investigated the Ca(2+) distribution in single corticotropin releasing factor- and urocortin-stimulated human skin cells. The models tested included melanoma cells, neonatal melanocytes and keratinocytes, and immortalized HaCaT keratinocytes. The changes in intracellular Ca(2+) signal intensities observed after stimulation of different cell types with corticotropin releasing factor and urocortin showed that: (1) the increase of intracellular Ca(2+) concentration was caused by a Ca(2+) influx (inhibition by EGTA); (2) this Ca(2+) influx took place through voltage-activated Ca(2+) ion channels (inhibition by d-cis-diltiazem, verapamil) and (3) cyclic nucleotide-gated ion channels were not involved in this process (no effect of Mg(2+)). The effects were also observed at very low peptide concentrations (10(-13) M) with no apparent linear correlation between peptide dosage and increase of fluorescence intensity, which implied co-expression of different corticotropin releasing factor receptor forms in the same cell. Immortalized (HaCaT) keratinocytes exhibited the strongest differential increases of a Ca(2+) fluorescence after peptide-stimulation. Corticotropin releasing factor induced Ca(2+) flux into the cytoplasm, while urocortin Ca(2+) flux into the nucleus with a remarkable oscillatory effect. The latter indicated the presence of an intracellular urocortin-induced signal transduction pathway that is unique to keratinocytes.

In A7r5 vascular smooth muscle cells, the two expressed inositol 1,4,5-trisphosphate receptor (IP(3)R) isoforms were differentially localized. IP(3)R1 was predominantly localized in the perinuclear region, whereas IP(3)R3 was homogeneously distributed over the cytoplasm. Prolonged stimulation (1-5 hours) of cells with 3 microM arginine-vasopressin induced a redistribution of IP(3)R1 from the perinuclear region to the entire cytoplasm, whereas the localization of IP(3)R3 appeared to be unaffected. The redistribution process occurred independently of IP(3)R downregulation. No structural changes of the endoplasmic reticulum were observed, but SERCA-type Ca(2+) pumps redistributed similarly to IP(3)R1. The change in IP(3)R1 localization induced by arginine-vasopressin could be blocked by the simultaneous addition of nocodazole or taxol and depended on Ca(2+) release from intracellular stores since Ca(2+)-mobilizing agents such as thapsigargin and cyclopiazonic acid could induce the redistribution. Furthermore, various protein kinase C inhibitors could inhibit the redistribution of IP(3)R1, whereas the protein kinase C activator 1-oleoyl-2-acetyl-sn-glycerol induced the redistribution. Activation of protein kinase C also induced an outgrowth of the microtubules from the perinuclear region into the cytoplasm, similar to what was seen for the redistribution of IP(3)R1. Finally, blocking vesicular transport at the level of the intermediate compartment inhibited the redistribution. Taken together, these findings suggest a role for protein kinase C and microtubuli in the redistribution of IP(3)R1, which probably occurs via a mechanism of vesicular trafficking.

Oocytes of the marine bivalve Mactra chinensis are spawned and arrested at the germinal vesicle stage (first meiotic prophase) until fertilization, without undergoing a process called oocyte maturation. As is the case of other animals, a fertilized oocyte of the bivalve displays increases in intracellular free Ca(2+). We have clarified here the spatiotemporal patterns and sources of the intracellular Ca(2+) changes at fertilization. Shortly after insemination, increased Ca(2+) simultaneously appeared at the whole cortical region of the oocyte and spread inwardly to the center, attaining the maximal Ca(2+) levels throughout the oocyte, including the cytoplasm and nucleus. The initial maximal Ca(2+) peak was followed by a submaximal plateau phase of cytoplasmic and nuclear Ca(2+) elevations, which persisted for several minutes. The nuclear envelope began to break down shortly before the termination of the plateau phase. These sperm-induced Ca(2+) changes were inhibited by suppression of the influx of external Ca(2+) from seawater but not by disturbance of the release of internal Ca(2+) from inositol 1,4,5-trisphosphate [Ins(1,4,5)P(3)]-sensitive stores, suggesting that the increased Ca(2+) is from an external source. In contrast to the situation observed at fertilization, an oocyte artificially stimulated with serotonin (5-hydroxytryptamine, 5-HT) displayed repetitive Ca(2+) transients, each of which started from one cortical region and propagated across the oocyte as a Ca(2+) wave. The 5-HT-induced Ca(2+) transients persisted even in the absence of external Ca(2+). Experiments with caged Ins(1,4,5)P(3) revealed that Ca(2+) release from Ins(1,4,5)P(3)-sensitive stores is another pathway that is sufficient to trigger meiosis reinitiation from the first prophase. These results demonstrate that Mactra oocytes can potentially use two different Ca(2+)-mobilizing pathways: Ca(2+) influx producing a centripetal Ca(2+) wave from the whole cortex and Ca(2+) release from Ins(1,4,5)P(3)-sensitive stores producing a point-source propagating Ca(2+) wave. However, it seems likely that the Ca(2+) influx pathway is predominantly activated at fertilization.

Telomere clustering, the defining feature of the bouquet, is an almost universal feature of meiotic prophase, yet its mechanism remains unknown. The microtubule-depolymerizing agent colchicine was found to inhibit bouquet formation. Telomeres in colchicine-treated cells remained scattered in the nuclear periphery, whereas untreated cells exhibited a prominent telomere cluster. Colchicine administered after the bouquet had formed did not affect telomere dispersal. The effect of colchicine on bouquet formation appeared to be separable from its effect on cytoplasmic microtubules; amiprophos methyl, a highly effective plant microtubule-depolymerizing drug, did not affect telomere clustering. Inhibition of bouquet formation was limited to colchicine and the related drug podophyllotoxin out of the variety of microtubule-depolymerizing drugs tested, suggesting that the target involved in bouquet formation has a structural specificity.

Macropinocytosis is an endocytic process that occurs through non-clathrin coated vesicles larger than 0.2 microm in diameter. Although macropinocytic vesicles are readily visualized in cultured cells by the introduction of fluorescent, water-soluble dyes into the culture medium, protein markers associated with this type of vesicles have not yet been well defined. Here, we report that human spectrin SH3 domain binding protein 1, or Hssh3bp1, associates with macropinosomes in NIH 3T3 fibroblasts. Hssh3bp1 macropinosomes are heterogeneous in morphology and size, do not endocytose transferrin and are resistant to brefeldin A treatment. Cytochalasin D, and wortmannin block endocytosis of fluorescent dyes into the Hssh3bp1 macropinosomes and dramatically affect their morphology. Overexpression of Hssh3bp1-green fluorescent protein abolished fusion of vesicles resulting in a decreased endocytosis of fluorescence dyes, thus suggesting a potential regulatory role of Hssh3bp1 in macropinocytosis. In the macropinosomes of NIH 3T3 cells, Hssh3bp1 associates with a 200-kDa protein that crossreacts with a monoclonal antibody to the erythroid alpha-spectrin SH3 domain. Thus macropinosomes in cells may contain a spectrin-like protein.

Developing bristles in Drosophila pupae contain 7-11 bundles of crosslinked actin filaments and a large population of microtubules. During bristle growth the rate of cell elongation increases with bristle length. Thin section EM shows that bundle size is correlated with the amount of cytoplasm at all points along the bristle. Thus, as the bristle elongates and tapers, fewer actin filaments are used. To ensure penetration of inhibitors we isolated thoraces and cultured them in vitro; bristles elongate at rates identical to bristles growing in situ. Interestingly, inhibitors of actin filament assembly (cytochalasin D and latrunculin A) dramatically curtailed bristle elongation while a filament stabilizer (jasplakinolide) accelerated elongation. In contrast, inhibitors of microtubule dynamics (nocodazole, vinblastine, colchicine and taxol) did not affect bristle elongation. Surprisingly, the bristle microtubules are stable and do not turn over. Furthermore, the density of microtubules decreases as the bristle elongates. These two facts coupled with calculations and kinetics of elongation and the fact that the microtubules are short indicate that the microtubules are assembled early in development and then transported distally as the bristle grows. We conclude that actin assembly is crucial for bristle cell elongation and that microtubules must furnish other functions such as to provide bulk to the bristle cytoplasm as well as playing a role in vesicle transport.

The hypothesis that the glucose transporter GLUT2 can function as a protein mediating transcriptional glucose signaling was addressed. To divert the putative interacting proteins from a glucose signaling pathway, two intracytoplasmic domains of GLUT2, the C terminus and the large loop located between transmembrane domains 6 and 7, were transfected into mhAT3F hepatoma cells. Glucose-induced accumulation of two hepatic gene mRNAs (GLUT2 and L-pyruvate kinase) was specifically inhibited in cells transfected with the GLUT2 loop and not with the GLUT2 C terminus. The dual effects of glucose were dissociated in cells expressing the GLUT2 loop; in fact a normal glucose metabolism into glycogen occurred concomitantly with the inhibition of the glucose-induced transcription. This inhibition by the GLUT2 loop could be due to competitive binding of a protein that normally interacts with endogenous GLUT2. In addition, the GLUT2 loop, tagged with green fluorescent protein (GFP), was located within the nucleus, whereas the GFP and GFP-GLUT2 C-terminal proteins remained in the cytoplasm. In living cells, a fraction (50%) of the expressed GFP-GLUT2 loop translocated rapidly from the cytoplasm to the nucleus in response to high glucose concentration and conversely in the absence of glucose. We conclude that, via protein interactions with its large loop, GLUT2 may transduce a glucose signal from the plasma membrane to the nucleus.

In mesophyll cells of the aquatic angiosperm Vallisneria gigantea, bundles of microfilaments (MFs) serve as tracks for the rotational streaming of the cytoplasm, which occurs along the two longer side walls and the two shorter end walls. The stationary organization of these bundles has been shown to depend on the association of the bundles with the plasma membrane at the end walls. To identify the sites of such association, the effects of cytochalasin B (CB) on the configuration of the bundles of MFs were examined. In the case of the side walls, MFs were completely disrupted after treatment with CB at 100 micrograms/ml for 24 hours. By contrast, in the case of the end walls, a number of partially disrupted MFs remained even after 48 hours of treatment. After removal of CB, a completely normal arrangement of bundles of MFs was once again evident within 24 hours after a rather complicated process of reassembly. When reassembly had been completed, the direction of cytoplasmic streaming was reversed only in a small fraction of the treated cells, suggesting that bundles of MFs are anchored and stabilized at the end walls of each cell and that the polarity of reorganized bundles and, therefore, the direction of the cytoplasmic streaming is determined in a manner that depends on the original polarity of MFs that remained in spite of the disruptive action of CB. By contrast, the direction of reinitiated cytoplasmic streaming was reversed in 50% of cells in which the bundles of MFs had been completely disrupted by exogenously applied trypsin prior treatment with CB.(ABSTRACT TRUNCATED AT 250 WORDS)

Maternally inherited cytoplasmic bacteria have occasionally been observed in embryos and adults of different strains of several Drosophila species. While there is a considerable body of data on the relationship between bacteria and embryo viability, little is known about the behavior of these bacteria during the early development of Drosophila. In eggs laid by infected Drosophila melanogaster females we showed that cytoplasmic bacteria were initially concentrated in a thin cortical layer and scattered in the yolk region. During the following syncytial blastoderm mitoses the bacteria mainly accumulated towards the poles of the mitotic spindles, suggesting that astral microtubules play a role in localizing bacteria. This is supported by the observation that treatment of the infected embryos with the microtubule-disrupting drug colchicine led to the partial dissociation of the bacteria from the spindle poles, whereas cytochalasin treatment left almost all the bacterial clusters intact. Moreover, bacteria were not found near the polar bodies and yolk nuclei, which were without astral microtubules. In mitosis-defective embryos, with centrosomes dissociated from the nuclei, the bacteria were concentrated in association with the isolated astral microtubules, and in cold-treated embryos, in which microtubules regrew from isolated centrosomes after recovering, the bacteria clustered around the newly formed asters. These observations, also supported by electron microscope analysis, indicate a close relationship between cytoplasmic bacteria and astral microtubules, and suggest that the latter were able to build discrete cytoplasmic domains ensuring the proper distribution of cytoplasmic components during the blastoderm mitoses, despite the lack of cell membranes.

In Xenopus eggs, the transient increase in intracellular free calcium ([Ca2+]i), or Ca2+ transient, which occurs 1-3 min after egg activation, is likely to be partly responsible for the release of the cell cycle blockade. In the present study, we have used microinjection of BAPTA or EGTA, two potent chelators of Ca2+, to buffer [Ca2+]i at various steps during Xenopus egg activation and evaluate the impact on some of the associated events. Microinjection of either one of the Ca2+ chelators into unactivated eggs prevented egg activation without, however, lowering [Ca2+]i, suggesting that only physiological [Ca2+]i changes, but not [Ca2+]i levels, were affected by the Ca2+ buffer. When BAPTA was microinjected around the time of occurrence of the Ca2+ transient, the egg activation-associated increase in intracellular pH (pHi) was clearly delayed. That delay was not due to a general slowing down of the cell cycle, since under the same conditions of microinjection of BAPTA the kinetics of MPF (a universal M-phase promoting factor) inactivation were unaffected. These results represent the first indication that the Ca2+ transient participates in determining the time of initiation of the pHi increase during Xenopus egg activation. The present results also demonstrate that the egg activation-associated pHi changes (a slight, transient decrease in pHi followed by a permanent increase in pHi) proceed as a wave propagating from the site of triggering of egg activation. Experiments of local microinjection of BAPTA support the view that the pH wave is a consequence of the Ca2+ wave, which it follows closely.

The possible involvement of osmotically generated hydrostatic pressure in driving actin-rich extensions of the cell surface was examined using cultures of chick neurons. Estimation of the excess internal osmotic pressure of chick neural tissue by vapor pressure deficit osmometry, and of the excess internal hydrostatic pressure in cultured chick neurons using a calibrated pressure pipette, gave upper limits of 10 mosM and 0.1 atmosphere (1 atmosphere = 101325 Pa), respectively. Increases in the osmolality of the medium surrounding cultured neurons by addition of sucrose, mannitol or polyethylene glycol by amounts that should eliminate any internal pressure not only failed to arrest the growth of filopodia but caused them to increase in length up to twofold in 3-5 min. Lamellipodia remained unchanged following hyperosmotic shifts of 20 mosM, but higher levels caused a small decrease in area. Reduction of osmolality by the addition of water to the culture fluid down to 50% of its normal value failed to show any detectable change in either filopodial length or lamellipodia area. These observations argue against an osmotic mechanism for growth cone extension and show that the growth of filopodia, in particular, is unlikely to be driven by osmotically generated hydrostatic pressure. In contrast to the short-term effects on growth cone morphology, the slower elongation of the neuritic cylinder showed a consistent osmotic response. Growth rates were reduced following addition of osmolytes and increased in rate (as much as sixfold) following addition of water to the culture medium.(ABSTRACT TRUNCATED AT 250 WORDS)

The contractile tentacles of the suctorian Discophrya collini contain a central microtubular axoneme as well as filamentous structures in the cortical epiplasm and in a fibrous collar around the axoneme at the tentacle base. The nature and possible roles of these components has been investigated by the use of reactivatable glycerinated cells. In these a mean tentacle contraction to 70% of the original length could be achieved by a 5-min treatment with a reaction mixture containing ATP, calcium and magnesium ions, the same treatment giving retraction to 30% in living cells. Both the microtubules of the axoneme and the filaments of the fibrous collar and epiplasm were present in the glycerinated cells, suggesting that these components consist of large water-insoluble molecules. The addition of heavy meromyosin to whole glycerinated cells resulted in the appearance of 36-50-nm spaced "tails" or filaments attached to the epiplasmic fibres and the aggregation of 3-6-nm filaments and electron-dense material in the region of the fibrous collar. Neither of these 2 features was apparent after treatment with ATP. It is suggested that actin-like filaments are localized in the region of the fibrous collar and in the epiplasm, and that these are involved in tentacle retraction; whilst the microtubules of the axoneme are concerned with feeding, and play only a cytoskeletal role in the contractile mechanism.

Under appropriate conditions macrophage cytosolic extracts can form a three-dimensional gel network of cross-linked actin filaments. These cytoplasmic gels are mainly composed of actin, filamin, alpha-actinin, and two new proteins of about 70,000 and 55,000 Mr (70 and 55 K). The behaviour of 70 K protein was found to be remarkably affected by Ca2+. Ca2+ treatment of isolated cytoplasmic gels led to the selective solubilization of the 70 K protein along with a 17 K polypeptide. Half-maximal recovery in the supernatant fraction was obtained from about 0.15 microM free Ca2+. The cytoplasmic gel constituents solubilized in high ionic strength buffer were able to re-assemble into an insoluble actin network when returned to near physiological ionic conditions. However, the inclusion of micromolar Ca2+ prevented the re-association of 70 K protein with actin in these complexes. As compared to the 70 K protein, alpha-actinin was fully resistant to any variations in Ca2+ concentrations. On the other hand, purified 70 K protein displayed the property of assembling actin filaments into bundles at low Ca2+ concentrations (less than 0.15 microM). However, the bundling activity decreased progressively at higher Ca2+, as detected by co-sedimentation and electron microscopy of the 70 K protein-actin mixtures. Half-maximal inhibition was observed at about 0.3 microM free Ca2+. Re-assembly of actin filaments into bundles occurred after chelation of Ca2+ by EGTA, indicating that the inhibitory effect of Ca2+ was reversible. Severing of actin filaments by 70 K protein was not observed in any of the solution conditions used. The Ca2+-dependent inhibition of the ability of 70 K protein to interact with actin networks resulted in a marked distortion of the overall organization of actin filaments, as revealed by thin-section electron microscopy of cytoplasmic gels formed in the presence and absence of Ca2+. Large zones of oriented bundles of filaments were replaced by a looser mesh. When the actin gel constituents were re-assembled in the presence of Ca2+ and exogenous gelsolin, it was also observed that the filament bundles (essentially generated by alpha-actinin) collapsed into dense aggregates. Furthermore, gelsolin did not significantly affect the ability of actin to re-combine with other proteins. The data presented here and previously led us to suspect that the Ca2+ control of the functional state of 70 K protein might be one of the prime factors in the triggering of rapid assembly and disassembly of microfilaments within macrophages.

A variety of trigger procedures were applied to analyse the exocytotic capability of different Paramecium tetraurelia strains. 7,S K 40I, kin 24I, and 9 (18 degrees C) are capable of exocytosis (permissive strains), in contrast to nd 6, nd 7, nd 9 (27 degrees C), tam 38 and ftb A, although all procedures used enhance [Ca2+]i in the cytoplasm of all strains tested and although strains nd 6, nd 7 and nd 9 (27 degrees C) contain a full set of morphologically normal trichocysts attended to the cell membrane. The results show that only those strains are permissive which were shown previously to contain a rosette of membrane-integrated particles and a Ca2+-ATPase activity in the cell membrane over the trichocyst attachment (exocytosis) sites. The results from trigger experiments with permissive and non-permissive strains would be compatible with a dual function of rosette particles as Ca2+ pumps and Ca2+ channels. Nevertheless, the latter aspect remains uncertain since we show that experiments along these lines published by others (introducing a Ca2+ ionophore from the outside) involve a solvent-induced artifact (pseudoexocytosis: matrix stretching in the absence of membrane fusion). In all strains, except for tam 38 and ftb A (which have abnormal trichocysts incapable of being attached to the cell membrane), the isolated trichocyst matrix can be transferred from the contracted to the expanded state in vitro with certain trigger procedures. Our data clearly show that an increase of [Ca2+]i in the cytoplasm is not sufficient for exocytosis to occur and that non-permissiveness is somehow due to an inability to perform membrane fusion. It remains open whether the lack of rosettes and Ca2+-ATPase activity at trichocyst attachment sites are primary cause of non-permissiveness.

Normal 3T3 cells were exposed to vinblastine and cytochalasin B in an attempt to correlate the morphological changes of the cell surface as seen in the scanning electron microscope with ultrastructural changes of the cytoskeletal elements as seen in critical-point-dried cells in the transmission electron microscope. Special attention was given to the changes in the cytoplasmic domains distinguished in a previous paper. Cytochalasin B primarily affects the ultrastructure of the cytocortical domain by inducing the formation of condensation foci on the cytoplasmic material. Vinblastine not only induces the depolymerization of microtubules and the perinuclear concentration of intermediate filaments, but it also causes the disappearance of stress fibres from the cortical cytoplasm and the widening of the cytocortex at the expense of the endoplasmic domain. These results support the hypothesis that the differentiation in ultrastructural domains is dependent on the spreading of the cells and their adhesion to substrate.

Attached, spread, chick heart fibroblasts were induced to shorten by treatment with the ionophore, A23187. The shortening resulted from the retraction of the leading lamellae and other major cell processes. The response was dependent on external calcium with a threshold close to, and a maximal effect at, physiological concentrations. The shortening was also induced in Colcemid-treated cells and therefore did not involve a depolymerization of microtubules. Indirect evidence indicates that the shortening was preceded by an increase in tension in the spread cell. The response is consistent with the effect of an increase in the cytoplasmic concentration of free calcium on a calcium-sensitive actomyosin system in the spread fibroblast. Although the retraction of non-spreading processes mimicked the intermittent retraction of similar trailing processes during normal movement of fibroblasts, the response to the ionophore differed in that the leading lamellae were also induced to retract. This difference implies that a general increase in the cytoplasmic concentration of free calcium alone cannot account for the intermittent shortening that occurs in normal movement.