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PubMed2002

Unique ability of integrin alpha(v)beta 3 to support tumor cell arrest under dynamic flow conditions.

Shear-resistant arrest of circulating tumor cells is required for metastasis from the blood stream. Arrest during blood flow can be supported by tumor cell interaction with attached, activated platelets. This is mediated by tumor cell integrin alpha(v)beta3 and cross-linking plasma protein ligands. To analyze the mechanism of tumor cell ligand interactions under dynamic flow conditions, we used real-time video microscopy and tested human melanoma cell binding to fibrinogen, von Willebrand Factor, or fibronectin matrices in a buffer perfusion system. When perfused at venous flow, melanoma cells arrested abruptly and began to spread immediately. This was uniquely mediated by integrin alpha(v)beta3 on all tested ligands, and required alpha(v)beta3 activation and actin polymerization. Under static conditions, alpha(v)beta3 cooperated with alpha(v)beta1 and alpha5beta1 in supporting melanoma cell adhesion to fibronectin. But even when activated, beta1 integrins did not contribute to melanoma cell arrest during flow. Soluble ligand served as a cross-linker between attached and circulating tumor cells and enhanced melanoma cell arrest. Cohesion of activated melanoma cells was restricted to the matrix surface and did not occur in suspension. We conclude that the presence of alpha(v)beta3 in a functionally activated state provides a unique advantage for circulating tumor cells by promoting tumor cell arrest in the presence of flow-dependent shear forces.

Actins↗
PubMed2004

Fibroblast growth factor-2 and remodeled type I collagen control membrane protrusion in human vascular smooth muscle cells: biphasic activation of Rac1.

Plasma membrane protrusion is fundamental to cell motility, but its regulation by the extracellular environment is not well elucidated. We have quantified lamellipodial protrusion dynamics in human vascular smooth muscle cells exposed to fibroblast growth factor 2 (FGF-2) and type I collagen, two distinct ligands presented to vascular cells during arterial remodeling. Video microscopy revealed that FGF-2 stimulated a modest increase in lamellipodial protrusion rate that peaked within 15 min. This response was associated with immediate but transient activation of Rac1 and was inhibited in cells infected with retrovirus containing cDNA encoding dominant-negative Rac1. A 1-h exposure to FGF-2 also set up a second phase of more striking lamellipodial protrusion evident at 24-36 h. This delayed response was most pronounced when cells were on type 1 collagen and was associated with FGF-2-induced expression of collagenase-1 that localized to the edge of protruding lamellipodia. Moreover, late membrane protrusion was inhibited when cells were on collagenase-resistant type I collagen, implicating degraded collagen as a mediator. For cells on collagen, the immediate activation of Rac1 by FGF-2 was followed by a sustained wave of Rac1 activation that was inhibited when cleavage of the collagen triple helix was prevented and also by blockade of alpha(v)beta(3) integrin. We conclude that lamellipodial protrusion in smooth muscle cells can be regulated by waves of Rac1 activation, corresponding to the sequential presentation of FGF-2 and remodeled collagen. The findings thus reveal a previously unrecognized level of coordination among extracellular input that enables cells to maintain protrusive activity over prolonged periods.

Cell Adhesion↗
PubMed2005

Three- and four-repeat tau regulate the dynamic instability of two distinct microtubule subpopulations in qualitatively different manners. Implications for neurodegeneration.

The microtubule-associated protein tau is implicated in the pathogenesis of many neurodegenerative diseases, including fronto-temporal dementia and parkinsonism linked to chromosome 17 (FTDP-17), in which both RNA splicing and amino acid substitution mutations in tau cause dominantly inherited early onset dementia. RNA-splicing FTDP-17 mutations alter the wild-type approximately 50:50 3-repeat (3R) to 4-repeat (4R) tau isoform ratio, usually resulting in an excess of 4R tau. To examine further how splicing mutations might cause dysfunction by misregulation of microtubule dynamics, we used video microscopy to determine the in vitro behavior of individual microtubules stabilized by varying amounts of human 4R and 3R tau. At low tau:tubulin ratios (1:55 and 1:45), all 3R isoforms reduced microtubule growth rates relative to the no-tau control, whereas all 4R isoforms increased them; however, at a high tau:tubulin ratio (1:20), both 4R and 3R tau increased the growth rates. Further analysis revealed two distinct subpopulations of growing microtubules in the absence of tau. Increasing concentrations of both 4R and 3R tau resulted in an increase in the size of the faster growing subpopulation of microtubules; however, 4R tau caused a redistribution to the faster growing subpopulation at lower tau:tubulin ratios than 3R tau. This modulation of discrete growth rate subpopulations by tau suggests that tau causes a conformational shift in the microtubule resulting in altered dynamics. Quantitative and qualitative differences observed between 4R and 3R tau are consistent with a "microtubule misregulation" model in which abnormal tau isoform expression results in the inability to properly regulate microtubule dynamics, leading to neuronal death and dementia.

Alternative Splicing↗
PubMed2006

A specific isoform of nonmuscle myosin II-C is required for cytokinesis in a tumor cell line.

Nonmuscle myosin IIs play an essential role during cytokinesis. Here, we explore the function of an alternatively spliced isoform of nonmuscle myosin heavy chain (NMHC) II-C, called NMHC II-C1, in the A549 human lung tumor cell line during cytokinesis. NMHC II-C1 contains an insert of 8 amino acids in the head region of NMHC II-C. First, we show that there is a marked increase in both the mRNA encoding NMHC II-C1 and protein in tumor cell lines compared with nontumor cell lines derived from the same tissue. Quantification of the amount of myosin II isoforms in the A549 cells shows that the amounts of NMHC II-A and II-C1 protein are about equal and substantially greater than NMHC II-B. Using specific siRNAs to decrease NMHC II-C1 in cultured A549 cells resulted in a 5.5-fold decrease in the number of cells at 120 h, whereas decreasing NMHC II-A with siRNA does not affect cell proliferation. This decreased proliferation can be rescued by reintroducing NMHC II-C1 but not NMHC II-A or II-B into A549 cells, although noninserted NMHC II-C does rescue to a limited extent. Time lapse video microscopy revealed that loss of NMHC II-C1 leads to a delay in cytokinesis and prolongs it from 2 to 8-10 h. These findings are consistent with the localization of NMHC II-C1 to the intercellular bridge that attaches the two dividing cells during the late phases of cytokinesis. The results suggest a specific function for NMHC II-C1 in cytokinesis in the A549 tumor cell line.

Cell Line, Tumor↗
PubMed2005

Characterization of an RNA granule from developing brain.

In brain, mRNAs are transported from the cell body to the processes, allowing for local protein translation at sites distant from the nucleus. Using subcellular fractionation, we isolated a fraction from rat embryonic day 18 brains enriched for structures that resemble amorphous collections of ribosomes. This fraction was enriched for the mRNA encoding beta-actin, an mRNA that is transported in dendrites and axons of developing neurons. Abundant protein components of this fraction, determined by tandem mass spectrometry, include ribosomal proteins, RNA-binding proteins, microtubule-associated proteins (including the motor protein dynein), and several proteins described only as potential open reading frames. The conjunction of RNA-binding proteins, transported mRNA, ribosomal machinery, and transporting motor proteins defines these structures as RNA granules. Expression of a subset of the identified proteins in cultured hippocampal neurons confirmed that proteins identified in the proteomics were present in neurites associated with ribosomes and mRNAs. Moreover many of the expressed proteins co-localized together. Time lapse video microscopy indicated that complexes containing one of these proteins, the DEAD box 3 helicase, migrated in dendrites of hippocampal neurons at the same speed as that reported for RNA granules. Although the speed of the granules was unchanged by activity or the neurotrophin brain-derived neurotrophic factor, brain-derived neurotrophic factor, but not activity, increased the proportion of moving granules. These studies define the isolation and composition of RNA granules expressed in developing brain.

Actins↗
PubMed1999

Role of endothelium-derived vasodilators and K(+) channels in ischemic vasodilation of guinea-pig choroidal arterioles.

PURPOSE: To assess the roles of endothelium-derived vasodilators and K(+) channels on metabolic ischemia-induced vasodilation from diameter changes in choroidal arterioles of the guinea-pig. METHODS: The choroid was isolated from the guinea-pig eye-ball, pinned flat on a silicone rubber plate, and superfused with oxygenated warmed (35 degrees C) Krebs solution. Diameters of choroidal arterioles were measured using video microscopy and a computer program for analysis. Vasodilatory effects were examined after the choroid was exposed to glucose-free/NaCN solutions for 10 minutes. The effects of Nomega-nitro-L-arginine (nitroarginine), indomethacin, and K(+)<?Pub Caret> channel inhibitors (glibenclamide [Glib] and charybdotoxin [ChTX]) on ischemic vasodilation were assessed. RESULTS: Reversible vasodilation was observed when the choroid was exposed to glucose-free/NaCN (10(-3) M) solutions. Nitroarginine (10( -4) M), Glib (2x10(-5) M) and ChTX (10(-7) M) significantly inhibited glucose-free/NaCN (10(-3) M)-induced vasodilation by 47%, 62%, and 24%, respectively. No significant inhibitory effect was observed with indomethacin (10(-5) M). Simultaneous application of Glib and ChTX reduced vasodilation by 77%. When Glib and ChTX were added together to nitroarginine, dilation was reduced by 86%. With high K(+) ([K]o = 47.2 mM) Krebs solution, ischemia caused a slight vasodilation (11%), which was significantly inhibited by nitroarginine. CONCLUSIONS: In guinea-pig choroidal arterioles, glucose-free/ NaCN-induced ischemic vasodilation was mainly mediated by NO and K(ATP) channels. A part of NO-mediated vasodilation was induced independent of the opening of K(+) channels.

Animals↗
PubMed2001

Modeling the vertical motion of a soft contact lens.

PURPOSE: The motion of a soft contact lens in the up-down or vertical (inferior-superior) and in-out (anterior-posterior) directions drives mixing in the post lens tear film. Thus, it is important to obtain an accurate assessment of lens motion. The commonly used experimental technique to measure the vertical motion, video microscopy, only gauges motion during the interblink period. Since most of the eyelid force, which drives the motion, is exerted during the blink, it is reasonable to assume that the majority of the lens motion in the up-down direction takes place during the blink and is, therefore, hidden from view. Thus, experimentally measured values of vertical lens travel are currently underpredicted. METHODS: In this paper, we use a simple mechanical force balance on the lens to predict its motion in the vertical direction. The forces included in our model are due to the upper and the lower eyelids, gravity, elasticity, and viscous stresses. We input the lens physical parameters, the various tear film thicknesses, and the upper eyelid velocity. Then we integrate a macroscopic force balance to obtain the lens vertical position as a function of time. RESULTS: The proposed model predicts that the downward lens motion during a blink is about 2--3 times the downward motion during the interblink (centration). CONCLUSIONS: The up-down motion observable during the interblink period is only a small fraction of the total lens vertical travel.

Biomechanical Phenomena↗
PubMed2001

Chloride dependent intracellular pH effects of external ATP in cultured human non-pigmented ciliary body epithelium.

PURPOSE: To examine the effects of extracellular adenosine 5-triphosphate (ATP) on intracellular pH ([pH](i)) in cultured human non-pigmented ciliary body epithelium (HNPE). METHODS: Intracellular pH was measured using spectrofluorescence video microscopy in isolated HNPE cells loaded with the cell-permeable acetoxymethyl ester form of the fluorescent probe BCECF. RESULTS: In 5%CO(2)/HCO(3)(-) buffered Ringer's the resting [pH](i) was 7.25 +/- 0.006 (mean +/- SEM). Application of 10 microM ATP significantly decreased [pH](i) to 7.00 +/- 0.007 (P < 10(-5), n = 14). In the presence of 1 mM suramin, a P(2) receptor inhibitor, this process was significantly blocked. This [pH](i) effect required the presence of Cl(-) and was significantly inhibited by 0.1 mM diisothiocyanatostilbene-2-2'-disulfonic acid or acetazolamide (500 microM), indicating the involvement of a Cl(-)/HCO(3)( +) exchange mechanism. This response exhibited little dependence on external Na(+) and remained unaffected by the addition of the Na(+)/H( +) exchanger inhibitor amiloride (1 mM). Clamping intracellular calcium levels by incubation in the cell permeable calcium chelator, the acetoxymethyl ester form of BAPTA (100 microM) in low extracellular calcium solution (pCa9) did not affect the ATP-induced [pH](i) signal. In addition, the vacuolar H(+)-ATPase (V-ATPase) inhibitor, bafilomycin A(1) (1 microM), failed to alter the [pH](i) transient. CONCLUSION: We have demonstrated that extracellular ATP leads to a sustained increase in [H(+)](i) in HNPE cells via a purinergic receptor activated pathway which is independent of the intracellular calcium signaling system. This study demonstrates that the ATP induced [pH]( i) transient is mediated through an upregulation in Cl(-)/HCO( 3)(-) exchange across the plasmamembrane in HNPE cells.

Adenosine Triphosphate↗
PubMed2001

Extracellular ATP effects on calcium signaling in cultured human non-pigmented ciliary body epithelium.

PURPOSE: To determine the effects of extracellular ATP on calcium signaling in cultured human non-pigmented ciliary body epithelium (HNPE). METHODS: Intracellular calcium (Ca(2+)(i)) was measured using spectrofluorescence video microscopy in isolated HNPE cells loaded with the fluorescent dye Fura-2. RESULTS: Nucleotides caused a transient oscillatory increase in Ca(2+)(i) with a potency order of ATP = UTP > ADP > AMP> alpha,beta-methylene-ATP. Treatment with thapsigargin (100 nM), an inhibitor of endoplasmic Ca(2+)-ATPase pumps, produced a sustained increase in Ca(2+)(i). Subsequent exposure to ATP caused a rapid reduction in Ca(2+)(i) and this effect was reduced by pre-exposure to vanadate and to a lesser extent in sodium free solution. Prolonged exposure to ATP in the presence of thapsigargin caused a transient spike increase in Ca(2+)(i) which was prevented by exposure to low extracellular Ca(2+) (1 nmol/l), verapamil, nifedipine or the microfilament disrupting agent, cytochalasin B. CONCLUSIONS: These results provide evidence for ATP mobilisation of Ca(2+) from intracellular stores via P2Y2 receptor activation in HNPE cells. ATP also primarily activates a vanadate-sensitive Ca(2+ )-ATPase pump, in addition to having a smaller effect on the Na( +)/ Ca(2+) exchanger in terminating the calcium signal. Capacitative calcium entry, possibly via an L-type Ca(2+) channel, is implicated in generating a calcium signal following emptying of intracellular stores and is sensitive to cytoskeleton disruption. ATP can thus regulate a potent intracellular signal for secretion, suggest-ing that purinergic receptors may provide a therapeutic target in glaucoma.

Adenosine Triphosphate↗
PubMed2004

MCP-1 induces migration of adult neural stem cells.

As a model for brain inflammation we previously studied transcriptional profiles of tumor necrosis factor-alpha (TNF)treated U373 astroglioma cells. In previous work we were able to demonstrate that the chemokine monocyte chemoattractant protein-1 (MCP-1, SCYA2, CCL2, MCAF) expression in U373 cells was inducible by TNF-alpha treatment. Demonstrably MCP-1 mRNA and protein expression in U373 cells was sustainable over time and at the highest level of all genes analyzed (Schwamborn et al., BMC Genomics 4, 46, 2003). In the hematopoietic system MCP-1 is a CC chemokine that attracts monocytes, memory T lymphocytes, and natural killer cells. In search of further functions in brain inflammation we tested the hypothesis that MCP-1 acts as a chemokine on neural stem cells. Here we report that MCP-1 activates the migration capacity of rat-derived neural stem cells. The migration of stem cells in a Boyden chamber analysis was elevated after stimulation with MCP-1. Time-lapse video microscopy visualized the migration of single stem cells from neurospheres in MCP-1-treated cultures, whereas untreated cultures depicted no migration at all, but showed signs of sprouting. Expression of the MCP-1 receptor CCR2 in neurosphere cultures was verified by RT-PCR and immunofluorescence microscopy. Supernatants from TNF-treated U373 cells also induced migration of neural stem cells.

Animals↗
PubMed2002

Water fleas (Daphnia magna) provide a separate ventilatory mechanism for their brood.

The planktonic filter feeder Daphnia magna depends on a steady oxygen supply by convection. In the ventral carapace chamber, this convection is established by the feeding current which is generated by the movement of the thoracic limbs. The present study revealed that this movement can cause an additional flow of medium which passes through the brood chamber of the animal. To visualise this current, ink or fluorescent microspheres were released by a microcapillary near the posterior opening of the brood chamber. The tracks of these probes were monitored by video microscopy. Digital motion analysis was used for the determination of flow velocity and flow rate. Ambient medium entered the brood chamber at the abdomen of the animal and moved then to the anterior end of the brood chamber before entering the ventral carapace chamber. Two horizontal lamellae, which are attached at both sides of the trunk and project laterally to contact the carapace walls, almost completely separate the dorsal brood chamber from the ventral carapace chamber. Water can only pass these barriers through small depressions in these lamellae at the level of the 3rd and 4th appendages. Female daphnids with embryos at late developmental stages showed more rapid water currents in the brood chamber than those with younger embryos. Moreover, animals showed higher flow rates when exposed to hypoxic conditions. As the oxygen uptake rate of older embryos is approximately three times higher than that of younger embryos, the enhanced brood chamber current could improve the oxygen availability for both the mother and its brood under conditions of reduced oxygen availability.

Journal Article↗
PubMed1998

Gentacoll hampers epithelialisation and neovascularisation in excisional wounds in hairless mice.

Our aim was to analyse the effect of Gentacoll on the rate of epithelialisation and neovascularisation in wound healing. Standardised circular full thickness dermal wounds 2.25 mm in diameter were created on the dorsum of each ear on 24 hairless homozygous mice (n = 48). The cartilaginous layer was left intact. The wounds were treated in a randomised blinded fashion with bovine collagen implants with gentamicin (Gentacoll) (n = 17); bovine collagen implants without gentamicin (n = 15); and Silicone film (n = 16). Epithelialisation and neovascularisation were measured directly by intravital video-microscopy and computerised planimetry immediately after the wounds had been made and every third day until the wounds closed. Only five of the wounds treated with Gentacoll (n = 17) epithelialised completely; and their mean (SEM) epithelialisation time was 22.8 (1.6) days, significantly longer than controls without gentamicin (n = 15) for which the corresponding figures were 14.5 (0.6) days. In nine wounds treated with Gentacoll the ear cartilage in the wound bed perforated and two wounds developed severe inflammation, which was followed by self-mutilation. Neovascularisation was incomplete in all of the wounds in the Gentacoll group, whereas it was completed by 25.3 (0.7) days in the control group treated with implants without gentamicin. In the silicone treated group (n = 16), epithelialisation was completed by 12.7 (0.7) days and neovascularisation by 25.1 (0.5) days. None of wounds treated with collagen or silicone alone showed reactions similar to the Gentacoll-treated ears. Gentacoll hampers epithelialisation and neovascularisation, and might damage exposed cartilage.

Animals↗
PubMed2001

Two distinct mechanisms are involved in stent thrombosis under flow conditions.

The mechanism of stent thrombosis was evaluated by a flow chamber model. A Palmaz-Schatz stent was placed on glass surfaces coated with type I collagen in a parallel-plate flow chamber. Platelets were rendered fluorescent with mepacrine and whole blood was perfused through the chamber. Platelet thrombi formed around the implanted stents were continuously recorded by epi-fluorescent video-microscopy. Platelet thrombi appeared on the collagen surface within a few minutes in the absence of function blocking monoclonal antibodies. Antibody blocking vWF interaction with GP Ib alpha completely inhibited the platelet thrombi around the stent, but had no effect on platelet thrombus formation in the space between the stents and collagen, while both were inhibited by anti-GP IIb/IIIa. Our results suggest that two distinct mechanisms, one mediated by vWF interaction with GP Ib alpha occurring at high shear rates, and the other mediated exclusively by GP IIb/IIIa occurring at low shear rates, are involved in the process of platelet thrombus formation in the presence of stents.

Animals↗
PubMed2006

The free-radical scavenger, edaravone, augments NO release from vascular cells and platelets after laser-induced, acute endothelial injury in vivo.

In vitro and in vivo experimental models have demonstrated that vascular endothelial function is significantly impaired as a result of oxidative stress, mediated by the generation of oxygen-derived free radicals in response to chronic or acute inflammation. In particular, super-oxide () at specific concentrations leads to the impairment of nitric oxide (NO) bioactivity, and it is known that NO plays a fundamental role in the maintenance of vascular homeostasis. The relationship between reactive oxygen species (ROS) and NO release in thrombosis-related endothelial damage in the peripheral microvasculature remains unclear, however. The purpose of the present study was to investigate the effect of the free-radical scavenger, edaravone, on NO synthesis and thrombotic potential in arterioles after exposure to laser irradiation. Highly sensitive electrochemical NO microsensors were positioned in femoral arterioles of mice, and the kinetics of NO release were recorded in response to standardized laser irradiation in vivo. In addition, images of NO release from damaged vascular cells were investigated in a similar rat model using the NO-sensitive dye 4,5-diaminofluorescein diacetate (DAF-2DA). Thrombogenesis was assessed in carotid arterioles by continuous video microscopy using image analysis software. Laser irradiation led to NO release from perturbed endothelial cells and from platelet-rich thrombi. Edaravone had no significant effect on NO release in non-laser treated, intact endothelium compared with placebo. In contrast, edaravone demonstrated a dose-dependent effect on NO release and thrombogenicity. At a concentration of 10.5 mg/kg per h, edaravone promoted a 5-fold increase in NO and a reduction in platelet-rich thrombus volume to 58% of the placebo values. Our data provide direct evidence to confirm that acute endothelial damage in peripheral microvessels initially induces NO release and that the free-radical scavenger, edaravone, augments NO synthesis leading to suppression of platelet thrombus formation.

Animals↗
PubMedDate not supplied

Microvascular thrombosis models in venules and arterioles in vivo.

Platelets are intimately involved in hemostasis and thrombosis. Under physiological conditions, circulating platelets do not interact with microvascular walls. However, in response to microvascular injury, platelet adhesion and subsequent thrombus formation may be observed in venules and arterioles in vivo. Numerous intravital video microscopy techniques have been described to induce and monitor the formation of microvascular thrombi. The mechanisms of microvascular injury vary widely among different models. Some models induce platelet activation with minimal effects on endothelium, others induce endothelial inflammation or injury, while other models lead to thrombus formation associated with endothelial denudation. The molecular mechanisms mediating platelet-vessel wall adhesive interactions differ among various models. In some instances, differences in responses between venules and arterioles are described that cannot be explained solely by hemodynamic factors. Several models for induction of microvascular thrombosis in vivo are outlined in this review, with a focus on the mechanisms of injury and thrombus formation, as well as on differences in responses between venules and arterioles. Recognizing these characteristics should help investigators select an appropriate model for studying microvascular thrombosis in vivo.

Animals↗
PubMed2000

Plant cells on earth and in space.

Two quite different types of plant cells are analysed with regard to transduction of the gravity stimulus: (i) Unicellular rhizoids and protonemata of characean green algae; these are tube-like, tip-growing cells which respond to the direction of gravity. (ii) Columella cells located in the center of the root cap of higher plants; these cells (statocytes) perceive gravity. The two cell types contain heavy particles or organelles (statoliths) which sediment in the field of gravity, thereby inducing the graviresponse. Both cell types were studied under microgravity conditions (10(-4) g) in sounding rockets or spacelabs. From video microscopy of living Chara cells and different experiments with both cell types it was concluded that the position of statoliths depends on the balance of two forces, i.e. the gravitational force and the counteracting force mediated by actin microfilaments. The actomyosin system may be the missing link between the gravity-dependent movement of statoliths and the gravity receptor(s); it may also function as an amplifier.

Actins↗
PubMed2004

Endocytosis of connexin protein in adrenal cells.

The ability of adrenocorticotropin (ACTH) to affect gap junctions was examined in adrenal cells in vivo and in vitro. Treatment with ACTH increased the size and number of gap junction plaques on the cell membranes in hypophysectomized animals and in adrenal culture. Intracellular (cytoplasmic) annular gap junctions were observed in cells of the inner adrenal cortical zones and in adrenal cell cultures. To investigate the relationship of annular gap junctions to surface junctions, adrenal cells in culture were transfected with cDNA encoding a green fluorescent protein tagged connexin 43 construct (Cx43-GFP), and subsequently studied by time-lapse video microscopy, immunocytochemistry, and transmission electron microscopy (TEM). Internalization of part or all of a surface gap junction plaque resulted in annular gap junction formation. These studies support the hypothesis that cytoplasmic vesicles, initially described with TEM methods, can result from removal of gap junction plaques from the cell surface. It is suggested that hormones can play a regulatory role in cell-cell communication by influencing the availability of gap junction protein at the cell surface and that hormonally-sensitive processes might serve as a means of altering intercellular communication.

Adrenal Glands↗
PubMed1985

Cytoplasmic motions, rheology, and structure probed by a novel magnetic particle method.

The motions of magnetic particles contained within organelles of living cells were followed by measuring magnetic fields generated by the particles. The alignment of particles was sensed magnetometrically and was manipulated by external fields, allowing non-invasive detection of particle motion as well as examination of cytoplasmic viscoelasticity. Motility and rheology data are presented for pulmonary macrophages isolated from lungs of hamsters 1 d after the animals had breathed airborne gamma-Fe2O3 particles. The magnetic directions of particles within phagosomes and secondary lysosomes were aligned, and the weak magnetic field produced by the particles was recorded. For dead cells, this remanent field was constant, but for viable macrophages, the remanent field decreased rapidly so that only 42% of its initial magnitude remained 5 min after alignment. A twisting field was applied perpendicular to the direction of alignment and the rate at which particles reoriented to this new direction was followed. The same twisting was repeated for particles suspended in a series of viscosity standards. Based on this approach, the low-shear apparent intracellular viscosity was estimated to be 1.2-2.7 X 10(3) Pa.s (1.2-2.7 X 10(4) poise). Time-lapse video microscopy confirmed the alignment of ingested particles upon magnetization and showed persistent cellular motility during randomization of alignment. Cytochalasin D and low temperature both reduced cytoplasmic activity and remanent-field decay, but affected rheology differently. Magnetic particles were observed in association with the microtubule organizing center by immunofluorescence microscopy; magnetization did not affect microtubule distribution. However, both vimentin intermediate filaments and f-actin reorganized after magnetization. These data demonstrate that magnetometry of isolated phagocytic cells can probe organelle movements, rheology, and physical properties of the cytoskeleton in living cells.

Animals↗