Molecular dynamics simulations of the enzyme catechol-O-methyltransferase: methodological issues.

Results from extensive 70 ns all-atom molecular dynamics simulations of catechol-O-methyltransferase (COMT) enzyme are reported. The simulations were performed with explicit TIP3P water and Mg2+ ions. Four different crystal structures of COMT, with and without different ligands, were used. These simulations are among the most extensive of their kind and as such served as a stability test for such simulations. On the methodological side we found that the initial energy minimization procedure may be a crucial step: particular hydrogen bonds may break, and this can initiate an irreversible loss of protein structure that becomes observable in longer time scales of the order of tens of nanoseconds. This has important implications for both molecular dynamics and quantum mechanics-molecular mechanics simulations.

Actin dynamics in Amoeba proteus motility.

We studied the distribution of the endogenous Arp2/3 complex in Amoeba proteus and visualised the ratio of filamentous (F-actin) to total actin in living cells. The presented results show that in the highly motile Amoeba proteus, Arp2/3 complex-dependent actin polymerisation is involved in the formation of the branching network of the contractile layer, adhesive structures, and perinuclear cytoskeleton. The aggregation of the Arp2/3 complex in the cortical network, with the exception of the uroid and advancing fronts, and the spatial orientation of microfilaments at the leading edge suggest that actin polymerisation in this area is not sufficient to provide the driving force for membrane displacement. The examined proteins were enriched in the pinocytotic pseudopodia and the perinuclear cytoskeleton in pinocytotic amoebae. In migrating amoebae, the course of changes in F-actin concentration corresponded with the distribution of tension in the cell cortex. The maximum level of F-actin in migrating amoebae was observed in the middle-posterior region and in the front of retracting pseudopodia. Arp2/3 complex-dependent actin polymerisation did not seem to influence F-actin concentration. The strongly condensed state of the microfilament system could be attributed to strong isometric contraction of the cortical layer accompanied by its retraction from distal cell regions. Isotonic contraction was limited to the uroid.

How adhesion, migration, and cytoplasmic calcium transients influence interleukin-1beta mRNA stabilization in human monocytes.

We investigated the mechanisms by which primary human monocyte migration and the production of important cytokines are co-regulated. Motile monocytes underwent cyclic morphologic and adhesive changes that were associated with intracellular free calcium changes; in such cells, cytokine transcripts were unstable and translationally repressed. Agents that activate monocytes, including lipopolysacharrides (LPS), cytomegalovirus (CMV), and tumor necrosis factor (TNFalpha), have been shown to de-repress translation and these agents stabilize adhesion-induced transcripts for IL-lbeta and IL-8 and markedly diminish cell migration in the presence of autologous serum. LPS suppressed Rho A activity and either this agent or C3 transferase elevated intracellular free calcium, stabilized transcripts, and, in tandem, inhibited cell migration by preventing tail retraction, a prerequisite for cell translocation. These results, therefore, suggest that monocyte activating agents inhibit the RhoA pathway and continuously elevate intracellular calcium leading to a concomitant decrease in monocyte migration and stabilization of cytokine transcripts prior to translation.

Induction of cortical oscillations in spreading cells by depolymerization of microtubules.

Actomyosin-based cortical contractility is a common feature of eukaryotic cells but the capability to produce rhythmic contractions is found in only a few types such as cardiomyocytes. Mechanisms responsible for the acquisition of this capability remain largely unknown. Rhythmic contractility can be induced in non-muscle cells by microtubule depolymerization. Spreading epithelial cells and fibroblasts in which microtubules were depolymerized with nocodazole or colcemid underwent rhythmic oscillations of the body that lasted for several hours before the cells acquired a stable, flattened shape. By contrast, control cells spread and flattened into discoid shapes in a smooth and regular manner. Quantitative analysis of the oscillations showed that they have a period of about 50 seconds. The kinase inhibitors, HA 1077 and H7, and the more specific rho-kinase inhibitor, Y 27632, caused the oscillations to immediately cease and the cells to become flat. Transient increases in cytoplasmic calcium preceded the contractile phase of the oscillations. Wrinkle formation by cells plated on elastic substrata indicated that the contractility of colcemid-treated cells increased in comparison to controls but was drastically decreased after HA 1077 addition. These data suggest that an intact microtubular system normally prevents pulsations by moderating excessive rho-mediated actin myosin contractility. Possible mechanistic interactions between rho-mediated and calcium activated contractile pathways that could produce morphological oscillations are discussed.

Reversible changes in size of cell nuclei isolated from Amoeba proteus: role of the cytoskeleton.

Micrurgically isolated interphasal nuclei of Amoeba proteus, which preserve F-actin cytoskeletal shells on their surface, shrink after perfusion with imidazole buffer without ATP, and expand to about 200% of their cross-sectional area upon addition of pyrophosphate. These changes in size may be reproduced several times with the same nucleus. The shrunken nuclei are insensitive to the osmotic effects of sugars and distilled water, whereas the expanded ones react only to the distilled water, showing further swelling. The shrinking-expansion cycles are partially inhibited by cytochalasins. They are attributed to the state of actomyosin complex in the perinuclear cytoskeleton, which is supposed to be in the rigor state in the imidazole buffer without ATP, and to dissociate in the presence of pyrophosphate. Inflow of external medium to the nuclei during dissociation of the myosin from the perinuclear F-actin may be due to colloidal osmosis depending on other macromolecular components of the karyoplasm.

ADHESION-DEPENDENT F-ACTIN PATTERN IN AMOEBA PROTEUS AS A COMMON FEATURE OF AMOEBAE AND THE METAZOAN MOTILE CELLS

Adhesion and movement of Amoeba proteus are both dependent on the appropriate arrangement of the F-actin cytoskeleton and on the presence of the cell nucleus. In this study the F-actin organization was examined by routine FITC-phalloidin staining and confocal laser microscopy in intact amoebae and in their nucleated and anucleated fragments, at different levels of cell adherence to the substratum. In the adhering and migrating intact cells and nucleated cell fragments dot-like aggregates of F-actin are scattered over the ventral side at sites close to the substratum. In the case of de-adhesion of nucleated specimens this pattern disappears and F-actin is accumulated in the cell centre and/or dispersed in the cytoplasm. The same actin distribution, without ventral dots, is found in the anucleated fragments which usually fail to attach to the substratum. Re-adhesion of anucleated fragments, induced by a modified substratum or spontaneous, is accompanied by restoration of actin dots at the lower cell side. It is concluded that: (1) adhering specimens of A. proteus display the same dot-like actin pattern on the ventral cell side, as many metazoan motile cells; (2) organization or disorganization of this pattern may occur independently of the presence of the cell nucleus, under the control of cell adhesion to the substratum. Copyright 1998 Academic Press Limited

Differences in the motility of Amoeba proteus isolated fragments are determined by F-actin arrangement and cell nucleus presence.

Isolated fragments produced by bisection of Amoeba proteus differ by their position in the original cell and by the presence or absence of the cell nucleus. Immediately after the operation, both types of anterior fragments preserve the former motory polarity, and do not interrupt locomotion. In the same time, all posterior fragments stop, round up and fail to react stimuli. In the second phase of experiment, these anterior fragments, which had no nucleus ceased to move, whereas the nucleated posterior ones resumed locomotion. It was demonstrated, that the behaviour of a fragment is primarily determined by the peripheral F-actin distribution, which is different depending on the origin of the fragment either from the anterior or from the posterior cell region. Later, the "inherited" F-actin distribution may be stabilized or reorganized in the presence of the nucleus, or desorganized in its absence.

Phosphatidylserine synthesis in phorbol ester treated glioma C6 cells.

Phosphatidylserine (PS) synthesis was studied in glioma C6 cells with [14C]serine and in the presence or absence of the phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA). It was found that incubation of the cells with 10 nM or 100 nM TPA for 1 h inhibited PS formation by 30% and 60%, respectively. Long-term (18 h) treatment of the cells with 100 nM TPA diminished PS formation and further addition of TPA to down-regulated cells did not affect PS synthesis. The data show that the changes in PS synthesis can be associated with alterations in morphology of cell and the actin cytoskeleton organization. The role of protein kinase C in this process is discussed.

Is actin involved in the nuclear division in Amoeba proteus?

During semi-open mitosis of Amoeba proteus the nuclear envelope is not dispersed and nucleus divides by fission. The presence of actin layer close to nuclear envelope was demonstrated in interphase and telophase nuclei of that amoeba stained with rhodamine labelled phalloidin. In telophase, an accumulation of actin arises in the space between the future daughter nuclei. It appears to be comparable with the contractile ring of dividing cells. This suggests that actin associated with the nuclear envelope of Amoeba proteus may be involved in final separation of the daughter nuclei, forming a constriction ring at the middle of dividing nucleus.

Voltage-dependent L-type calcium channels in the development and plasticity of mouse barrel cortex.

Entry of calcium ions into the neuron is a triggering signal for initiation of several processes which may lead to modification of synaptic connectivity. The developmental changes of voltage-dependent L-type calcium channel (VDLCC) were studied using [3H]PN 200 110 nifedipine displaceable binding in the barrel cortex of mice, a model structure for studying cortical plasticity. In vitro binding autoradiography was used to examine quantitatively the pattern of [3H]PN 200 110 binding to brains of animals aged from 3 to 70 days. The binding values in the somatosensory cortex rose two-fold in the period examined, reaching a plateau in the 4th postnatal week. The laminar pattern of binding changed during development, with the locus of heaviest labeling shifting from layer IV to II/III in the third postnatal week and thin bands of labeling developing in layers IV and VI. A very faint barrel-like pattern of labeling in the barrel field was observed. Neither this pattern nor the binding values were altered by unilateral neonatal removal of all vibrissal follicles. Saturation studies of binding to crude synaptosomal fractions of cerebral cortex of mice aged 3, 15, 28 and 70 days revealed the presence of a single binding site, with Bmax increasing from 48.7 +/- 5.1 fmol/mg protein at postnatal day 3 to 191.7 +/- 9.6 fmol/mg protein at day 70. No developmental changes in KD values were found. No correlation was found between the critical period for cytoarchitectonic plasticity of the barrels and the time when high values of VDLCC binding were observed.