Localization of calcium and microfilament changes in mechanically stressed cells.

We combined fluorescence labeling, digital image processing, and micromanipulation to investigate the intracellular events induced by inflicting a mechanical stress on rat basophilic leukemia cells. Our findings were as follows: 1. Most cells displayed a localized calcium rise in response to micropipet aspiration. This represented an average threefold increase as compared to resting level, and it was observed during the first 10 s following aspiration. A slow return to initial level occurred within about 3 min. Further, this calcium rise involved a mobilization of intracellular stores, since it was not prevented by adding a calcium chelator into the extracellular medium. 2. All micropipet-aspirated cells displayed a local accumulation of microfilaments, with a preferential localization in the cell protrusions or near the pipet tips. 3. No absolute correlation was found between the localization of calcium rise and cytoskeletal accumulation. 4. Cell deformability was decreased when intracellular calcium was maintained at a constant (high or low) level with ionomycin and/or EGTA. It is concluded that cells have a general ability to respond to mechanical stimulation by a coordinated set of events. More parameters must be studied before the mechanisms of cell shape regulation are fully understood.     

Localization of calcium changes in stimulated rat mast cells.

We studied intracellular free, bound, and sequestered calcium in rat mast cells after various stimulations. The use of a fluorescent probe combined with digitized imaging on individual living cells demonstrated transient increases of free Ca2+ in the micromolar range. The use of histochemical techniques (K pyroantimonate and anhydrous fixation), together with X-ray microanalysis, energy electron-loss spectroscopy, and electron spectroscopic imaging, revealed large amounts of stored calcium within the cells (in the millimolar range). Chelation experiments and stimulations enabled us to identify at least two pools of bound calcium which exhibited different dynamic behaviors. Stimulation in the presence of EGTA did not modify calcium from granules, granule membranes, and heterochromatin, whereas it decreased calcium from other cell compartments. Stimulation triggered variations in the amount of bound calcium but they did not parallel free calcium movements. Hence, whereas free calcium is implicated in exocytosis, bound calcium may be involved in altogether different cell functions.     

Induced monoterpene and lignin production in mechanically stressed and fungal elicited cultured Cupressus lusitanica cells

Cultured Cupressus lusitanica cells induced by various stresses are thought to produce different complexes of defense chemicals to optimize defense. To compare the induced products of two stimulations, we investigated the emission of monoterpenes, biosynthesis of β-thujaplicin, and accumulation of lignin in mechanically stressed and fungal elicited cultured C. lusitanica cells. Both mechanical stress and fungal elicitor caused emission of qualitatively similar monoterpene blends indicating de novo biosynthesis of these compounds after stimulation, while mechanical stress alone is sufficient to induce fungal elicitor-related monoterpene emission. Sabinene and limonene were the dominant compounds over the time course in both volatile blends. Although the emitted volatile blends were qualitatively similar, the time course and the relative ratios of the constituents of the volatile blends differed with the type of stimulation. While fungal elicited cells produced significant amounts of β-thujaplicin over the 5-day time course, no β-thujaplicin was observed in the mechanically stressed cells. The production of β-thujaplicin was the main dissimilarity of the induced products of these two treatments, suggesting that synthesis of β-thujaplicin is not a general response to all types of stresses, but is a specific response and serves as a strong toxic compound against already invaded fungus. Significantly higher amounts of lignin accumulations were observed in the fungal elicited and mechanically stressed cells on the 5th day after induction. Based on these results, we suggest the composition of induced products was dependent on the method of stimulation.     

Effects of microfilament disrupters on microfilament distribution and morphology in maize root cells

Maize root tip cells were examined for the distribution of actin microfilaments in various cell types and to determine the effects of microfilament disrupters. Fluorescence microscopy on fixed, stabilized, squashed cells using the F-actin specific probe, rhodamine-labelled phalloidin, allowed for a three-dimensional visualization of actin microfilaments. Microfilaments were observed as long, meandering structures in root cap cells and meristematic cells, while those in immature vascular parenchyma were abundant in the thin band of cytoplasm and were long and less curved. By modifying standard electron microscopic fixation procedures, microfilaments in plant cells could be easily detected in all cell types. Treatment with cytochalasin B, cytochalasin D and lead acetate, compounds that interfere with microfilament related processes, re-organized the microfilaments into abnormal crossed and highly condensed masses. All the treatments affected not only the microfilaments but also the accumulation of secretory vesicles. The vivid demonstration of the effects of all of these microfilament disrupters on the number and size of Golgi vesicles indicates that these vesicles may depend on microfilaments for intracellular movement.     

Effects of microfilament disrupters on microfilament distribution and morphology in maize root cells

Summary Maize root tip cells were examined for the distribution of actin microfilaments in various cell types and to determine the effects of microfilament disrupters. Fluorescence microscopy on fixed, stabilized, squashed cells using the F-actin specific probe, rhodamine-labelled phalloidin, allowed for a three-dimensional visualization of actin microfilaments. Microfilaments were observed as long, meandering structures in root cap cells and meristematic cells, while those in immature vascular parenchyma were abundant in the thin band of cytoplasm and were long and less curved. By modifying standard electron microscopic fixation procedures, microfilaments in plant cells could be easily detected in all cell types. Treatment with cytochalasin B, cytochalasin D and lead acetate, compounds that interfere with microfilament related processes, re-organized the microfilaments into abnormal crossed and highly condensed masses. All the treatments affected not only the microfilaments but also the accumulation of secretory vesicles. The vivid demonstration of the effects of all of these microfilament disrupters on the number and size of Golgi vesicles indicates that these vesicles may depend on microfilaments for intracellular movement.     

Cooperative kinking at distant sites in mechanically stressed DNA

In cells, DNA is routinely subjected to significant levels of bending and twisting. In some cases, such as under physiological levels of supercoiling, DNA can be so highly strained, that it transitions into non-canonical structural conformations that are capable of relieving mechanical stress within the template. DNA minicircles offer a robust model system to study stress-induced DNA structures. Using DNA minicircles on the order of 100 bp in size, we have been able to control the bending and torsional stresses within a looped DNA construct. Through a combination of cryo-EM image reconstructions, Bal31 sensitivity assays and Brownian dynamics simulations, we have been able to analyze the effects of biologically relevant underwinding-induced kinks in DNA on the overall shape of DNA minicircles. Our results indicate that strongly underwound DNA minicircles, which mimic the physical behavior of small regulatory DNA loops, minimize their free energy by undergoing sequential, cooperative kinking at two sites that are located about 180° apart along the periphery of the minicircle. This novel form of structural cooperativity in DNA demonstrates that bending strain can localize hyperflexible kinks within the DNA template, which in turn reduces the energetic cost to tightly loop DNA.     

Thyrotropin induces changes in the morphology and the organization of microfilament structures in cultured thyroid cells

Thyrotropin (TSH) induces morphological changes in cultures of normal rat thyroid cell lines and in primary bovine thyroid cells. It also induces a specific reorganization of the microfilaments of the thyroid cells. Both effects are fully reversible and are mimicked by 8-bromo-cAMP. These results indicate that the trophic response of TSH involves changes in the organization of the actin-containing filaments, probably mediated through cAMP, followed by changes in cell shape.     

Localization of calcium ions in wounded characean internodal cells

Ca²⁺ was localized in chemically injured internodal cells of the characean alga Nitella flexilis (L.) Ag. using alizarin red and antimonate precipitation. The presence of Ca²⁺ in the antimonate precipitates was verified by X-ray analysis and EGTA chelation. Callose-containing amorphous wound walls were induced by 0·1 m m chlortetracycline (CTC) and cellulosic fibrillar wound walls were induced by 50 m m CaCl₂. Numerous precipitates were found in the amorphous wound walls and in the adjacent cytoplasm. Precipitates were mainly localized in single membrane-bound cisternae, probably of the endoplasmic reticulum, which accumulate at the wound and become a component of the amorphous wound wall via membrane fusion. In fibrillar wound walls, which do not contain membranous residues, precipitate density was significantly lower and similar to that in the secondary cell wall.
The data suggest that the high Ca²⁺ content of amorphous wound walls is due to incorporation of cytoplasmic Ca²⁺ stores. The possible function of amorphous wound walls in maintaining cellular Ca²⁺ homeostasis is discussed.     

Localization of intracellular calcium in root meristematic cells and root cap cells of maize

The localization of Ca²⁺ in cells of the periblem and dermatogen in the root meristem and the columella and peripheral cells of the root cap of maize was examined by the precipitation method of potassium pyroantimonate and EGTA-treatment. In periblem and dermatogen cells, Ca²⁺ was found to be localized in the nucleoplasm and granular zone of the nucleolus. Ca²⁺ was also found in most cell organelles: in the matrix in mitochondria, on the thylakoid membrane in proplastids, in the vacuoles and on the plasma membranes. Ca²⁺ was also distributed throughout the cytoplasmic ground matrix. Much Ca²⁺ was present in the cell wall soon after its formation during the cell division. Ca²⁺ was also conspicuous in the vesicles of Golgi in the dermatogen cells. In columella and peripheral cells, there was less Ca²⁺ in the organelles and cytoplasmic ground matrix, but Ca²⁺ was present in Golgi vesicles in the peripheral cells. Electron microscopic and X-ray microanalysis showed that Ca²⁺ was also present in the mucilaginous layer, the outermost cell wall of the peripheral cells.     

Mitochondrial calcium of intact and mechanically damaged bone and cartilage cells studied with K-pyroantimonate

Summary Cellular cation was localized with K-pyroantimonate osmium fixation in whole fetal mouse metatarsal bones and in deliberately mechanically damaged specimens. X-ray microprobe analysis of ultrathin sections showed a positive correlation between the concentration of Ca (and Sb) and the amount of electron-dense precipitate.In non-damaged osteoblasts and growth-plate chondrocytes dense precipitate had accumulated along the plasmalemma and the mitochondrial membranes, whereas damaged cells showed the precipitate on round granules in the mitochondrial matrix but not on membranes. Intermediate stages between these two patterns were also found.In a non-calcifying tissue such as liver no membrane-bound precipitate was found in intact cells. However, damaged liver cells showed precipitate-containing mitochondrial granules similar to those in damaged bone cells, but only after incubation of the damaged tissue for l h in a Ca-containing balanced salt solution.Freezing of fresh whole bones in liquid N₂ before fixation in K-pyroantimonate osmium did not change the precipitate pattern in the damaged cells, but in intact cells it produced a random distribution of precipitate unrelated to membranes.The results are compared with those obtained in other studies on the subcellular localization of calcium and in biochemical studies on membrane versus matrix loading in calcium-accumulating isolated mitochondria.     

Formative capacities of mechanically stressed networks: developmental and evolutionary implications.

We present a biomechanical model of morphogenesis highlighting the extensive formative capacities of stressed networks with a very simple initial geometry. They consist of a restricted number of kinematically independent elements exerting a pressure to each other and increasing thus the local curvatures. The pressure is applied as a series of periodic impulses and is opposed by a constant quasi-elastic resistance force. Single elements can be also regarded as the half wave-lengths of the undulations determined by the mechanical properties of a given body. All of the model parameters are assumed to be evenly spread throughout a body (no prepatterns are implied). On the other hand, the model parameters can be associated with genetic factors. Thus, our model relates to as yet unsolved problem of genetic regulation of shape formation. We classify the modeled shapes according to their symmetry orders and compare them with the ancient Echinodermata and with Arthropods. Possible evolutionary and developmental implications are discussed.     

Nucleotide changes in oxidatively stressed lymphocytes

Similar to HIV-1-induced suppression of thymus-derived lymphocytes (T cells), oxidatively stressed T cells show inhibited DNA synthesis and proliferation. The influence of oxidative stress on nucleotide pools was explored using ³H-uridine addition to OKT3-stimulated peripheral blood lymphocytes. The cells were preincubated and stimulated in the presence of 1 mM buthionine sulfoximine to inhibit GSH synthesis. This treatment gave rise to a significant reduction in dUDP and TTP biosynthesis following 18–32 hours stimulation, indicating possible impairment of ribonucleotide reductase activity.     

Mitochondrial calcium of intact and mechanically damaged bone and cartilage cells studied with K-pyroantimonate.

Cellular cation was localized with K-pyroantimonate osmium fixation in whole fetal mouse metatarsal bones and in deliberately mechanically damaged specimens. X-ray microprobe analysis of ultrathin sections showed a positive correlation between the concentration of Ca (and Sb) and the amount of electron-dense precipitate. In non-damaged osteoblasts and growth-plate chondrocytes dense precipitate had accumulated along the plasmalemma and the mitochondrial membranes, whereas damaged cells showed the precipitate on round granules in the mitochondrial matrix but not on membranes. Intermediate stages between these two patterns were also found. In a non-calcifying tissue such as liver no membrane-bound precipitate was found in intact cells. However, damaged liver cells showed precipitate-containing mitochondrial granules similar to those in damaged bone cells, but only after incubation of the damaged tissue for 1 h in a Ca-containing balanced salt solution. Freezing of fresh whole bones in liquid N2 before fixation in K-pyroantimonate osmium did not change the precipitate pattern in the damaged cells, but in intact cells it produced a random distribution of precipitate unrelated to membranes. The results are compared with those obtained in other studies on the subcellular localization of calcium and in biochemical studies on membrane versus matrix loading in calcium-accumulating isolated mitochondria.     

Spontaneous oscillation and mechanically induced calcium waves in chondrocytes

The characteristics of spontaneous calcium (Ca(2+)) oscillation and mechanically induced Ca(2+) waves in articular chondrocytes were studied. In some, but not all, chondrocytes in sliced cartilage and primary cultures, we observed spontaneous oscillation of intracellular Ca(2+) that never spread to adjacent cells. In contrast, a mechanical stimulus to a single cell by touching with a glass rod induced an increase of intracellular Ca(2+) that spread to neighboring cells in a wave-like manner, even though there was no physical contact between the cells. This indicated the release of some paracrine factor from the mechanically stimulated cells. Application of ultrasonic vibration also induced an oscillation of intracellular Ca(2+). The application of a uridine 5'-triphosphate (UTP), UTP, induced a transient increase in intracellular Ca(2+) and the release of adenosine 5'-triphosphate (ATP) in cultured chondrocytes. A P2 receptor antagonist (suramin) and blockers of Cl(-) channels, niflumic acid and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), reduced the UTP-induced ATP release. The results indicated that Cl(-) channels were involved in the extracellular release of ATP following mechanical or P2Y receptor stimulation. Thus, ATP stimulation of P2Y receptors elicits an increase in intracellular Ca(2+), triggering further release of ATP from adjacent cells, thereby expanding the Ca(2+) wave in chondrocytes.     

Localization and heterogeneity of agonist-induced changes in cytosolic calcium concentration in single bovine adrenal chromaffin cells from video imaging of fura-2.

Temporal and spatial changes in the concentration of cytosolic free calcium ([Ca2+]i) in response to a variety of secretagogues have been examined in adrenal chromaffin cells using digital video imaging of fura-2-loaded cells. Depolarization of the cells with high K+ or challenge with nicotine resulted in a rapid and transient elevation of [Ca2+]i beneath the plasma membrane consistent with Ca2+ entry through channels. This was followed by a late phase in which [Ca2+]i rose within the cell interior. Agonists that act through mobilization of inositol phosphates produced an elevation in [Ca2+]i that was most marked in an internal region of the cell presumed to be the site of IP3-sensitive stores. When the same cells were challenged with nicotine or high K+, to trigger Ca2+ entry through voltage-dependent channels, the rise in [Ca2+]i was most prominent in the same localized region of the cells. These results suggest that Ca2+ entry through voltage-dependent channels results in release of Ca2+ from internal stores and that the bulk of the measured rise in [Ca2+]i is not close to the exocytotic sites on the plasma membrane. Analysis of the time courses of changes in [Ca2+]i in response to bradykinin, angiotensin II and muscarinic agonists showed that these agonists produced highly heterogeneous responses in the cell population. This heterogeneity was most marked with muscarinic agonists which in some cells elicited oscillatory changes in [Ca2+]i. Such heterogeneous changes in [Ca2+]i were relatively ineffective in eliciting catecholamine secretion from chromaffin cells. A single large Ca2+ transient, with a component of the rise in [Ca2+]i occurring beneath the plasma membrane, may be the most potent signal for secretion.     

Localization of calcium in nerve fibers

Using the desheathed nerve preparation, a pyroantimonate precipitation method was used to examine the distribution of electron-dense particles seen in various organelles of the nerve fibers following exposure of nerve to various levels of Ca2+ in vitro. The presence of Ca2+ in the electron-dense particles was indicated by their extraction with EGTA and by the use of energy-dispersive X-ray microanalysis. In normal Ringer or in a Ca2+ -free medium, electron-dense particles were seen associated with the outer membrane of the mitochondria, with the smooth endoplasmic reticulum (SER), along the axolemma and yet others scattered throughout the axoplasm. When nerves were incubated in media containing higher than normal concentrations of 20-60 mM Ca2+, an increase in the number of such electron-dense particles was seen in the axoplasm and within the mitochondrial matrix. Nerves loaded with a high concentration of 60mM Ca2+ could be depleted of these particles after transfer to a Ca2+ -free or low Ca2+ Ringer medium. The sequestration of Ca2+ in axonal organelles is discussed with respect to Ca2+-regulatory mechanisms in the axon needed to maintain a low level of Ca2+ which is optimal for the support of axoplasmic transport.     

Immunofluorescent and ultrastructural studies of polygonal microfilament networks in respreading non-muscle cells

Respreading gerbil fibroma cells (CCL146) have been found to display cytoplasmic actin-based polygonal fiber networks 10 h after replating (stage III of respreading according to Vasiliev & Gelfand, [1]). The networks have been analyzed by immunofluorescence and electron microscopy. The foci, sites of actin, α-actinin and filamin distribution, are condensed meshworks of microfilaments attached to the inner surface of the plasma membrane. The interconnecting fibers, sites of uniform actin distribution and complementary periodicities of α-actinin and myosin, are bundles of parallel microfilaments with periodic dense bodies. Heavy meromyosin (HMM) labelling of the microfilaments in the foci and interconnecting bundles confirm that they contain actin. In addition, approx. 70% of the microfilaments associated with an individual focus have a uniform polarity relative to it (arrowheads pointing away) suggesting that they have their origin there. Our results support earlier conclusions [2] that polygonal networks are structural intermediates responsible for organizing contractile proteins of the cortical microfilament layer into stress fibers.