Statistics of active transport in Xenopus melanophores cells

The transport of cell cargo, such as organelles and protein complexes in the cytoplasm, is determined by cooperative action of molecular motors stepping along polar cytoskeletal elements. Analysis of transport of individual organelles generated useful information about the properties of the motor proteins and underlying cytoskeletal elements. In this work, for the first time (to our knowledge), we study collective movement of multiple organelles using Xenopus melanophores, pigment cells that translocate several thousand of pigment granules (melanosomes), spherical organelles of a diameter of ∼1 μm. These cells disperse melanosomes in the cytoplasm in response to high cytoplasmic cAMP, while at low cAMP melanosomes cluster at the cell center. Obtained results suggest spatial and temporal organization, characterized by strong correlations between movement of neighboring organelles, with correlation length of ∼4 μm and pair lifetime ∼5 s. Furthermore, velocity statistics revealed strongly non-Gaussian velocity distribution with high velocity tails demonstrating exponential behavior suggestive of strong velocity correlations. Depolymerization of vimentin intermediate filaments using a dominant-negative vimentin mutant or actin with cytochalasin B reduced correlation of behavior of individual particles. Based on our analysis, we concluded that steric repulsion is dominant, but both intermediate filaments and actin microfilaments are involved in dynamic cross-linking organelles in the cytoplasm.     

Long-range nonanomalous diffusion of quantum dot-labeled aquaporin-1 water channels in the cell plasma membrane

Aquaporin-1 (AQP1) is an integral membrane protein that facilitates osmotic water transport across cell plasma membranes in epithelia and endothelia. AQP1 has no known specific interactions with cytoplasmic or membrane proteins, but its recovery in a detergent-insoluble membrane fraction has suggested possible raft association. We tracked the membrane diffusion of AQP1 molecules labeled with quantum dots at an engineered external epitope at frame rates up to 91 Hz and over times up to 6 min. In transfected COS-7 cells, >75% of AQP1 molecules diffused freely over ∼7 μm in 5 min, with diffusion coefficient, D_1-3 ∼ 9 × 10⁻¹⁰ cm²/s. In MDCK cells, ∼60% of AQP1 diffused freely, with D_1-3 ∼ 3 × 10⁻¹⁰ cm²/s. The determinants of AQP1 diffusion were investigated by measurements of AQP1 diffusion following skeletal disruption (latrunculin B), lipid/raft perturbations (cyclodextrin and sphingomyelinase), and bleb formation. We found that cytoskeletal disruption had no effect on AQP1 diffusion in the plasma membrane, but that diffusion was increased greater than fourfold in protein de-enriched blebs. Cholesterol depletion in MDCK cells greatly restricted AQP1 diffusion, consistent with the formation of a network of solid-like barriers in the membrane. These results establish the nature and determinants of AQP1 diffusion in cell plasma membranes and demonstrate long-range nonanomalous diffusion of AQP1, challenging the prevailing view of universally anomalous diffusion of integral membrane proteins, and providing evidence against the accumulation of AQP1 in lipid rafts.     

Oxygen Concentration Inside a Functioning Photosynthetic Cell

The excess oxygen concentration in the photosynthetic membranes of functioning oxygenic photosynthetic cells was estimated using classical diffusion theory combined with experimental data on oxygen production rates of cyanobacterial cells. The excess oxygen concentration within the plesiomorphic cyanobacterium Gloeobactor violaceus is only 0.025 μM, or four orders of magnitude lower than the oxygen concentration in air-saturated water. Such a low concentration suggests that the first oxygenic photosynthetic bacteria in solitary form could have evolved ∼2.8 billion years ago without special mechanisms to protect them against reactive oxygen species. These mechanisms instead could have been developed during the following ∼500 million years while the oxygen level in the Earth’s atmosphere was slowly rising. Excess oxygen concentrations within individual cells of the apomorphic cyanobacteria Synechocystis and Synechococcus are 0.064 and 0.25 μM, respectively. These numbers suggest that intramembrane and intracellular proteins in isolated oxygenic photosynthetic cells are not subjected to excessively high oxygen levels. The situation is different for closely packed colonies of photosynthetic cells. Calculations show that the excess concentration within colonies that are ∼40 μm or larger in diameter can be comparable to the oxygen concentration in air-saturated water, suggesting that species forming colonies require protection against reactive oxygen species even in the absence of oxygen in the surrounding atmosphere.     

Transport Theory for HIV Diffusion through In Vivo Distributions of Topical Microbicide Gels

Topical microbicide products are being developed for the prevention of sexually transmitted infections. These include vaginally-applied gels that deliver anti-HIV molecules. Gels may also provide partial barriers that slow virion diffusion from semen to vulnerable epithelium, increasing the time during which anti-HIV molecules can act. To explore the barrier function of microbicide gels, we developed a deterministic mathematical model for HIV diffusion through realistic gel distributions. We applied the model to experimental data for in vivo coating distributions of two vaginal gels in women. Time required for a threshold number of virions to reach the tissue surface was used as a metric for comparing different scenarios. Results delineated how time to threshold increased with increasing gel layer thickness and with decreasing diffusion coefficient. We note that for gel layers with average thickness >∼100 μm, the fractional area coated, rather than the gel layer thickness, was the primary determinant of time to threshold. For gel layers <∼100 μm, time to threshold was brief, regardless of fractional area coated. Application of the model to vaginal coating data showed little difference in time to threshold between the two gels tested. However, the protocol after gel application (i.e., with or without simulated coitus) had a much more significant effect. This study suggests that gel distribution in layers of thickness >100 μm and fractional area coated >0.8 is critical in determining the ability of the gel to serve as a barrier to HIV diffusion.     

Microimaging of Oxygen Concentration near Live Photosynthetic Cells by Electron Spin Resonance

We present what is, to our knowledge, a new methodology for high-resolution three-dimensional imaging of oxygen concentration near live cells. The cells are placed in the buffer solution of a stable paramagnetic probe, and electron spin-resonance microimaging is employed to map out the probe's spin-spin relaxation time (T₂). This information is directly linked to the concentration of the oxygen molecule. The method is demonstrated with a test sample and with a small amount of live photosynthetic cells (cyanobacteria), under conditions of darkness and light. Spatial resolution of ∼30 × 30 × 100 μm is demonstrated, with ∼μM oxygen concentration sensitivity and sub-fmol absolute oxygen sensitivity per voxel. The use of electron spin-resonance microimaging for oxygen mapping near cells complements the currently available techniques based on microelectrodes or fluorescence/phosphorescence. Furthermore, with the proper paramagnetic probe, it will also be readily applicable for intracellular oxygen microimaging, a capability which other methods find very difficult to achieve.     

Agnostic particle tracking for three-dimensional motion of cellular granules and membrane-tethered bead dynamics

The ability to detect biological events at the single-molecule level provides unique biophysical insights. Back-focal-plane laser interferometry is a promising technique for nanoscale three-dimensional position measurements at rates far beyond the capability of standard video. We report an in situ calibration technique for back-focal-plane, low-power (nontrapping) laser interferometry. The technique does not rely on any a priori model or calibration knowledge, hence the name “agnostic”. We apply the technique to track long-range (up to 100 μm) motion of a variety of particles, including magnetic beads, in three-dimensions with high spatiotemporal resolution (∼2 nm, 100 μs). Our tracking of individual unlabeled vesicles revealed a previously unreported grouping of mean-squared displacement curves at short timescales (<10 ms). Also, tracking functionalized magnetic beads attached to a live cell membrane revealed an anchorage-dependent nonlinear response of the membrane. The software-based technique involves injecting small perturbations into the probe position by driving a precalibrated specimen-mounting stage while recording the quadrant photodetector signals. The perturbations and corresponding quadrant photodetector signals are analyzed to extract the calibration parameters. The technique is sufficiently fast and noninvasive that the calibration can be performed on-the-fly without interrupting or compromising high-bandwidth, long-range tracking of a particle.     

Lateral Motion and Bending of Microtubules Studied with a New Single-Filament Tracking Routine in Living Cells

The cytoskeleton is involved in numerous cellular processes such as migration, division, and contraction and provides the tracks for transport driven by molecular motors. Therefore, it is very important to quantify the mechanical behavior of the cytoskeletal filaments to get a better insight into cell mechanics and organization. It has been demonstrated that relevant mechanical properties of microtubules can be extracted from the analysis of their motion and shape fluctuations. However, tracking individual filaments in living cells is extremely complex due, for example, to the high and heterogeneous background. We introduce a believed new tracking algorithm that allows recovering the coordinates of fluorescent microtubules with ∼9 nm precision in in vitro conditions. To illustrate potential applications of this algorithm, we studied the curvature distributions of fluorescent microtubules in living cells. By performing a Fourier analysis of the microtubule shapes, we found that the curvatures followed a thermal-like distribution as previously reported with an effective persistence length of ∼20 μm, a value significantly smaller than that measured in vitro. We also verified that the microtubule-associated protein XTP or the depolymerization of the actin network do not affect this value; however, the disruption of intermediate filaments decreased the persistence length. Also, we recovered trajectories of microtubule segments in actin or intermediate filament-depleted cells, and observed a significant increase of their motion with respect to untreated cells showing that these filaments contribute to the overall organization of the microtubule network. Moreover, the analysis of trajectories of microtubule segments in untreated cells showed that these filaments presented a slower but more directional motion in the cortex with respect to the perinuclear region, and suggests that the tracking routine would allow mapping the microtubule dynamical organization in cells.     

Effects of grinding processes on enzymatic degradation of wheat straw

The effectiveness of wheat straw fine to ultra-fine grindings at pilot scale was studied. The produced powders were characterised by their particle-size distribution (laser diffraction), crystallinity (WAXS) and enzymatic degradability (Trichoderma reesei enzymatic cocktail). A large range of wheat-straw powders was produced: from coarse (median particle size ∼800 μm) to fine particles (∼50 μm) using sieve-based grindings, then ultra-fine particles ∼20 μm by jet milling and ∼10 μm by ball milling. The wheat straw degradability was enhanced by the decrease of particle size until a limit: ∼100 μm, up to 36% total carbohydrate and 40% glucose hydrolysis yields. Ball milling samples overcame this limit up to 46% total carbohydrate and 72% glucose yields as a consequence of cellulose crystallinity reduction (from 22% to 13%). Ball milling appeared to be an effective pretreatment with similar glucose yield and superior carbohydrate yield compared to steam explosion pretreatment.     

The endothelial glycocalyx is hydrodynamically relevant in arterioles throughout the cardiac cycle

The existence of a hydrodynamically relevant endothelial glycocalyx of ∼0.5 μm in thickness is well established in capillaries and venules in vivo. Since the glycocalyx is likely to have implications for broad areas of vascular physiology and pathophysiology, including endothelial-cell mechanotransduction, vascular permeability, and atherosclerosis, it is necessary to determine the extent to which the glycocalyx is present on arteriolar endothelium. We applied microviscometric analysis to data obtained using microparticle image velocimetry in cremaster-muscle arterioles of wild-type mice. Due to the pulsatile nature of the flow regimes in arterioles, data acquisition was triggered with the electrocardiogram at specific time points in the cardiac cycle. Results show the existence of a hydrodynamically relevant glycocalyx having a mean thickness of 0.38 μm in arterioles ∼20-70 μm in diameter (n = 20), which is ∼0.13 μm thinner (p = 0.03) than that found previously in venules having a similar diameter range and under similar hemodynamic conditions. Results from data obtained at multiple time points in the cardiac cycle show that the glycocalyx remains hydrodynamically relevant in arterioles with statistically insignificant changes in mean thickness throughout the cardiac cycle, despite the inherent unsteadiness of the flow regimes in these microvessels. These results provide direct in vivo confirmation of the existence of a hydrodynamically relevant surface glycocalyx that essentially eliminates fluid shear stress on arteriolar endothelium throughout the entire cardiac cycle.     

Cell-Cell Adhesion and Cortical Actin Bending Govern Cell Elongation on Negatively Curved Substrates

Physiologically, cells experience and respond to a variety of mechanical stimuli such as rigidity and topography of the extracellular matrix. However, little is known about the effects of substrate curvature on cell behavior. We developed a novel, to our knowledge, method to fabricate cell culture substrates with semicylindrical grooves of negative curvatures (radius of curvature, R_c = 20–100 μm). We found that negative substrate curvatures induced elongation of mesenchymal and epithelial cells along the cylinder axis. As R_c decreases, mesenchymal National Institutes of Health 3T3 fibroblasts increasingly elongate along the long axis of the grooves, whereas elongation of epithelial Madin-Darby Canine Kidney (MDCK) cells is biphasic with maximal cell elongation when R_c = 40 μm. Addition of blebbistatin to MDCK cells to reduce cortical actin rigidity resulted in a decrease in cell elongation across all curvatures while preserving the biphasic trend. However, addition of calyculin A or ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid, to increase cortical rigidity or reduce intercellular adhesion, respectively, resulted in a monotonic increase in MDCK cell elongation with decreasing R_c. Using an energy minimization model, we showed that cell elongation in epithelial cell sheet is governed by the competition between two energies as R_c decreases: curvature-dependent intercellular adhesion that prevents elongation; and intracellular cortical actin bending that enhances elongation. Therefore, our results of cellular elongation induced by negatively curved substrates offer insights into how tubule elongation or growth of tubular structures such as kidney tubules can be controlled by the substrate curvature in vivo.     

Urea transport in MDCK cells that are stably transfected with UT-A1

Progress in understanding the cell biology of urea transporter proteins has been hampered by the lack of an appropriate cell culture system. The goal of this study was to create a polarized epithelial cell line that stably expresses the largest of the rat renal urea transporter UT-A isoforms, UT-A1. The gene for UT-A1 was cloned into pcDNA5/FRT and transfected into Madin-Darby canine kidney (MDCK) cells with an integrated Flp recombination target site. The cells from a single clone were grown to confluence on collagen-coated membranes until the resistance was >1,500 ·cm². Transepithelial [¹⁴C]urea fluxes were measured at 37°C in a HCO₃^–/CO₂ buffer, pH 7.4, with 5 mM urea. The baseline fluxes were not different between unstimulated UT-A1-transfected MDCK cells and nontransfected or sham-transfected MDCK cells. However, only in the UT-A1-transfected cells was UT-A1 protein expressed (as measured by Western blot analysis) and urea transport stimulated by forskolin or arginine vasopressin. Forskolin and arginine vasopressin also increased the phosphorylation of UT-A1. Thionicotinamide, dimethylurea, and phloretin inhibited the forskolin-stimulated [¹⁴C]urea fluxes in the UT-A1-transfected MDCK cells. These characteristics mimic those seen in rat terminal inner medullary collecting ducts. This new polarized epithelial cell line stably expresses UT-A1 and reproduces several of the physiological responses observed in rat terminal inner medullary collecting ducts. urea transporter-A1; arginine vasopressin; collecting duct; Madin-Darby canine kidney cells     

Temperature Dependences of Torque Generation and Membrane Voltage in the Bacterial Flagellar Motor

In their natural habitats bacteria are frequently exposed to sudden changes in temperature that have been shown to affect their swimming. With our believed to be new methods of rapid temperature control for single-molecule microscopy, we measured here the thermal response of the Na⁺-driven chimeric motor expressed in Escherichia coli cells. Motor torque at low load (0.35 μm bead) increased linearly with temperature, twofold between 15°C and 40°C, and torque at high load (1.0 μm bead) was independent of temperature, as reported for the H⁺-driven motor. Single cell membrane voltages were measured by fluorescence imaging and these were almost constant (∼120 mV) over the same temperature range. When the motor was heated above 40°C for 1–2 min the torque at high load dropped reversibly, recovering upon cooling below 40°C. This response was repeatable over as many as 10 heating cycles. Both increases and decreases in torque showed stepwise torque changes with unitary size ∼150 pN nm, close to the torque of a single stator at room temperature (∼180 pN nm), indicating that dynamic stator dissociation occurs at high temperature, with rebinding upon cooling. Our results suggest that the temperature-dependent assembly of stators is a general feature of flagellar motors.     

Translocation of MARCKS and reorganization of the cytoskeleton by PMA correlates with the ion selectivity,the confluence,and transformation state of kidney epithelial cell lines

The role of protein kinase C (PKC) in the regulation of the cytoskeleton of epithelial cells with tightly sealed contacts, poor contacts, and without contacts were investigated by incubating them with a protein kinase C activator phorbol myristoyl acetate (PMA). The morphology and organization of the membrane skeleton and stress fibers as well as the localization of an actin-bundling PKC substrate MARCKS in confluent MDCK cells originating from the distal tubulus of dog kidney, LLC-PK1 cells originating from the proximal tubulus of pig kidney, src-transformed MDCK cells, epidermoid carcinoma A431 cells, and MDCK cells grown in low calcium medium (LC medium) in low density were visualized with phase contrast and immunofluorescence microscopy. Four different responses to the PMA-treatment in actin-based structures of cultured epithelial cells were observed: 1) disintegration of the membrane skeleton in confluent MDCK cells; 2) depolymerization of the stress fibers in confluent MDCK and LLC-PK1 cells; 3) formation of the membrane skeleton in A431 cells, and 4) formation of the stress fibers and membrane skeleton in LC-MDCK cells. Thus, it seems that in fully confluent tightly sealed epithelium, activation of PKC has a deleterious effect on actin-based structures, whereas in cells without contacts or loose contacts, activation of PKC by PMA results in improvement of actin-based cytoskeletal structures. The main difference between the two kidney cell lines used is their selectivity to ion transport: the monolayer of LLC-PK1 cells is anion selective and MDCK cells cation selective. We propose a model where alterations in the ionic milieu within the MDCK cells by means of cation channels affect the disintegration of the membrane skeleton. The distribution of MARCKS followed the distribution of fodrin in both cell lines upon PMA-treatment, suggesting that phosphorylation of MARCKS by PKC may contribute in the regulation of the integrity of the membrane skeleton. J. Cell. Physiol. 181:83–95, 1999. © 1999 Wiley-Liss, Inc.     

Bovine colostrum supports the serum-free proliferation of epithelial cells but not of fibroblasts in long-term culture

Medium lacking serum but supplemented with milk will support the growth of sparse cells in culture. Milk obtained within 8 h after the birth of a calf (day 1 colostrum) is the most effective in supporting proliferation. In mixed cultures of early-passage bovine embryonic kidney (BEK) or early-passage calf kidney (CK) cells, both epithelial cells and fibroblasts grow in Dulbecco’s modified eagle’s medium (DMEM) supplemented with serum. However, only cells that appear to be epithelial-like grow in DMEM supplemented with colostrum. Sparse cultures of early-passage human and rat fibroblasts that grow readily in DMEM supplemented with serum do not grow in DMEM supplemented with colostrum. Canine kidney epithelial cells (MDCK), when plated sparsely, grow exponentially in DMEM supplemented with day 1 bovine colostrum. The generation time is 26 h, the same growth rate as in DMEM supplemented with calf serum. The MDCK cells can be subcultured and regrown to confluence repeatedly in colostrum-supplemented DMEM. Growth in DMEM supplemented with colostrum does not alter the morphological characteristics of the MDCK cells, which are polygonal, contain microvilli at the apical surface, and are connected by tight junctions and desmosomes. MDCK cells do not proliferate in DMEM supplemented with milk obtained 1 wk after the birth of a calf.     

Quercetin is a substrate for the transmembrane oxidoreductase Dcytb

Duodenal cytochrome b (Dcytb) is a transmembrane oxidoreductase protein found in apical membranes of duodenal enterocytes, as well as human erythrocytes, with the capacity to transport electrons donated by cytosolic ascorbate to extracellular electron receptors such as Fe(III), dehydroascorbate, or molecular O₂. We have investigated the capacity of the flavonoid quercetin to act as an electron donor for Dcytb in a manner similar to that of ascorbate by observing the reduction of extracellular Fe(III) to Fe(II) in either Madin–Darby canine kidney (MDCK) cells overexpressing Dcytb (Dcytb⁺) or Dcytb-null MDCK cells. In Dcytb⁺ cells there is a saturable increase in extracellular Fe(III) reduction in response to increasing intracellular quercetin concentrations (K_m = 6.53 ± 1.57 μM), in addition to a small linear response, whereas in Dcytb-null cells there is only a small linear increase in extracellular Fe(III) reduction. No extracellular Fe(III) reduction occurs in Dcytb-null cells when the cells are preloaded with ascorbate. Flavonoids such as quercetin at their physiological concentrations can therefore function as modulators of ferric reductases, enhancing the import of Fe(II) and also providing extracellular reducing potential.     

Effect of corn preparation methods on dry-grind ethanol production by granular starch hydrolysis and partitioning of spent beer solids

Two corn preparation methods, rollermill flaking and hammermill grinding, were compared for efficient processing of corn into ethanol by granular starch hydrolysis and simultaneous fermentation by yeast Saccharomyces cerevisiae. Corn was either ground in a hammermill with different size screens or crushed in a smooth-surfaced rollermill at different roller gap settings. The partitioning of beer solids and size distribution of solids in the thin stillage were compared. The mean particle diameter d₅₀ for preparations varied with set-ups and ranged between 210 and 340 μm for ground corn, and 1180-1267 μm for flaked corn. The ethanol concentrations in beer were similar (18-19% v/v) for ground and flaked preparations, however, ethanol productivity increased with reduced particle size. Roller versus hammermilling of corn reduced solids in thin stillage by 28%, and doubled the volume percent of fines (d₅₀ ∼ 7 μm)in thin stillage and decreased coarse (d₅₀ ∼ 122 μm) by half compared to hammermilling.     

Phenotypic transformation of normal rat kidney cells by transforming growth factor beta is not paralleled by enhanced production of a platelet-derived growth factor.

Phenotypic transformation of normal rat kidney (NRK) cells requires the concerted action of multiple polypeptide growth factors. Serum-deprived NRK cells cultured in the presence of epidermal growth factor (EGF) become density-inhibited at confluence, but they can be restimulated by a number of defined polypeptide growth factors, resulting in phenotypic cellular transformation. Kinetic data show that restimulation by transforming growth factor beta (TGF-beta) and retinoic acid is delayed when compared to induction by platelet-derived growth factor (PDGF), indicating that both TGF beta and retinoic acid may exert their growth-stimulating action by an indirect mechanism. Northern blot analysis shows that NRK cells express the genes for various polypeptide growth factors, including TGF beta 1, PDGF A-chain and basic fibroblast growth factor, but that the levels of expression are not affected by TGF beta or retinoic acid treatment. NRK cells also secrete low amounts of a PDGF-like growth factor into their extracellular medium, but the levels of secretion are insufficient to induce mitogenic stimulation and are unaffected by agents inducing phenotypic transformation. In combination with studies on the effects of anti-PDGF antibodies, it is concluded that phenotypic transformation of NRK cells by TGF beta and retinoic acid is not the result of enhanced production of a PDGF-like growth factor.     

Characterization of azaspiracids in plankton size-fractions and isolation of an azaspiracid-producing dinoflagellate from the North Sea

Azaspiracids (AZAs) are a group of lipophilic polyether toxins implicated in incidents of shellfish poisoning in humans, particularly in northern Europe. In an attempt to establish the biogeographical distribution of AZA toxins, their association with plankton size-fractions, and to confirm the identity of the causative species responsible for human poisoning, a month-long oceanographic study was undertaken in coastal North Sea waters. The occurrence and abundance of AZA analogues was measured by on board triple quadrupole mass spectrometry coupled to liquid chromatography (LC-MS/MS). In size-fractionated plankton samples collected by net tows (20 μm mesh-size), by pumping from discrete depths and from Niskin entrapment bottle casts to fixed depths, AZA-1 was consistently the major azaspiracid component. In eastern Scottish coastal waters, the highest amounts of AZA-1 in net tow samples were in the 50–200 μm fractions, with lesser amounts detected in the >200 μm and 50–20 μm fractions. At these stations, the 50–200 μm fractions were rich in the ciliate Favella ehrenbergii. Cells of F. ehrenbergii isolated by microcapillary indeed contained AZA-1, but isolated cells grown and fed the non-toxic dinoflagellate Scrippsiella trochoidea for one week failed to contain any detectable AZA-1—evidence that F. ehrenbergii is merely a vector for AZA. Detailed analysis of plankton from Niskin bottle samples from around the North Sea typically showed highest amounts of AZA in the 3–20 μm fraction. From this fraction, a large number of crude cultures were established and subsequently screened for the presence of AZAs. A small photosynthetic thecate dinoflagellate, provisionally designated as strain 3D9, was isolated by microcapillary and brought into pure culture. This dinoflagellate strain was found to produce AZA-1, AZA-2 and an isomer of AZA-2. Sequence comparisons by molecular genetic techniques also indicated that this genotype was present in field samples rich in AZA. This discovery of a novel causative dinoflagellate for AZA toxicity essentially explains the lack of correlation of AZA with the abundance and distribution of the previously postulated culprit species Protoperidinium crassipes. We instead propose that such large phagotrophic dinoflagellates can act as an AZA vector following grazing upon a proximal source, such as the dinoflagellate 3D9 strain.     

Rapid turnover rate of phosphoinositides at the front of migrating MDCK cells

Phosphoinositides (PtdInss) play key roles in cell polarization and motility. With a series of biosensors based on Förster resonance energy transfer, we examined the distribution and metabolism of PtdInss and diacylglycerol (DAG) in stochastically migrating Madin-Darby canine kidney (MDCK) cells. The concentrations of phosphatidylinositol (4,5)-bisphosphate, phosphatidylinositol (3,4,5)-trisphosphate (PIP₃), phosphatidylinositol (3,4)-bisphosphate, and DAG were higher at the plasma membrane in the front of the cell than at the plasma membrane of the rear of the cell. The difference in the concentrations of PtdInss was estimated to be less than twofold between the front and rear of the migrating MDCK cells. To decode the spatial activities of PtdIns metabolic enzymes from the obtained concentration maps of PtdInss, we developed a one-dimensional reaction diffusion model of PtdIns metabolism. In this model, the activities of phosphatidylinositol monophosphate 5-kinase, phosphatidylinositol 3-kinase, phospholipase C, and PIP₃ 5-phosphatases were higher at the plasma membrane of the front than at the plasma membrane of the rear of the cell. This result suggests that, although the difference in the steady-state level of PtdInss is less than twofold, PtdInss were more rapidly turned over at the front than the rear of the migrating MDCK cells.     

Cytochalasin D-mediated hyperinduction of the substrate-associated 52-kilodalton protein p52 in rat kidney fibroblasts

Regulation of certain differentiated and housekeeping functions in cultured mammalian cells is significantly influenced by cell shape. The shape-modulating agent cytochalasin D (CD) was used, therefore, to elucidate potential cytoarchitectural influences affecting synthesis of a major 52 kDa secreted/substrate-associated protein (p52) of normal rat kidney (NRK) fibroblasts. Biosynthetic labeling experiments indicated that treatment of NRK cells with CD increased, by 10-18-fold, the medium content of an Mr 52,000 protein. Two-dimensional gel electrophoresis and peptide fragment mapping confirmed that the 52 kDa protein produced in abundance as a consequence of CD treatment was identical to p52 constitutively expressed by NRK cells. A lower mw protein (p50; Mr 50,000) was also resolved which, based on pl microheterogeneity, protease fragmentation profile, and sensitivity to tunicamycin, could be identified as a less-glycosylated form of p52. p50 and p52 were both detected in the matrix and medium compartments of NRK and NRK/CD cells. The matrix p52 content of CD-induced and uninduced cells, however, was significantly greater (by 200-500-fold) than the corresponding medium levels. This differential compartmentalization, the time course of p52 accumulation in the matrix of NRK/CD cells compared to its appearance in the medium, and the kinetics of p52 pulse-chase from the matrix collectively indicated that the matrix is the initial site of p52 deposition. Low levels of CD (1 microM) produced extensive disruptions of cellular microfilaments but did not result in an overall cell shape change nor a hyperinduction of p52. Morphologic rounding (seen in 10-100 microM CD) coincided with augmented p52 production. Transition from a flat to a round phenotype in NRK cells, or at least the generation of sufficient microfilament fragmentation to compromise cell-substrate adhesivity, appears to be an essential aspect of CD-mediated p52 hyperinduction.