Receptor-mediated endocytosis of alpha 2-macroglobulin in living intact 3T3 cells

Internalization process of the fluoresceinisothiocyanate-labeled alpha 2-macroglobulin (MG-F) in Swiss 3T3 cells has been studied. The presence of internalized MG-F, formation of endocytotic vesicles and endosomal pH value were estimated visually on living monolayer cells with TV microscope, on the base of supervidicon LI-702. The use of the TV method allowed to obtain a series of photographs of cell images without any significant dye fading. A brief characteristics of microprocess control system is presented. The influence of various chemical agents on MG-F internalization and endosomal pH value was investigated. It was shown that dansylcadaverine (150-200 microM) and monensin (10-20 microM) inhibited MG-F internalization. Methylamine, chloroquine and monensin quickly enhanced the endosomal pH value, while other amines did not, for example dansylcadaverine.     

High-affinity cytochalasin B binding to normal and transformed BALB/3T3 cells.

To study the molecular basis of changes in sugar uptake rate in cultured mouse fibroblasts with different physiological states, we have measured the high affinity binding of [3H] cytochalsin B, a potent sugar transport inhibitor, to actively growing and contact inhibited Balb/3T3 cells as well as to 3T12 and SV3T3 cells. Binding was the same whether the cells were detached from dishes with EDTA or trypsin. The amount of drug bound to intact cells measured with a centrifugation assay was essentially the same as that bound to cell sonicates measured with equilibrium dialysis. Cytochalasin B binding to intact cells was extremely rapid and reversible over a wide range of drug concentrations, and was not affected by 0.1 M D--glucose in the assay medium. Actively growing and contact inhibited 3T3 cells had a similar number of high affinity cytochalasin B binding sites per cell, while 3T12 and SV3T3 cells had one third to one fourth the number of sites per cell. However, the number of sites per mug cellular protein appeared to be similar for cells in all of the physiological states examined.     

Dissociation by cytochalasin B of movement, DNA synthesis and transport in 3T3 cells.

Cytochalasin B was used as a tool to study the inter-relationships between cell movement, the reinitiated DNA synthesis and the enhanced transport of specific small molecules stimulated by serum in quiescent 3T3 cells. Cytochalasin at concentrations of less than 1 mug/ml inhibits serum-stimulated movement within the monolayer and migration into a wound. Even at ten times this concentration there is little effect on the increase in DNA in the culture, indicating that movement away from neighboring cells is not required for the initiation of DNA synthesis. While DNA synthesis is not inhibited by concentrations of cytochalasin up to 10 mug/ml, the increased thymidine transport which is associated with the onset of the S phase of the cell cycle is inhibited and DNA synthesis cannot be measured by the labelling of nuclei with radioactive thymidine. Cytochalasin has a differential effect on the early transport changes produced by serum addition. Glucose transport is inhibited by low concentrations of the drug (less than 1 mug/ml) while the enhanced uptake of phosphate and uridine is unaffected by a 10-fold increase in concentration. Although the doses of cytochalasin required for 50% inhibition of hexose uptake and of cell movement are the same, no causal relationship between sugar transport and locomotion can be demonstrated. Cytochalasin affects membrane functions in at least two different ways. The drug inhibits the uptake of glucose directly but affects only the S-phase associated increase in thymidine transport.     

Early effect of growth factors (EGF + insulin) upon ATP turnover in 3T3 cells. Inhibition by cytochalasin B and D

We have demonstrated previously a rapid increase in ATP turnover soon after adding epidermal growth factor (EGF) and insulin to quiescent cultures of Swiss 3T3 cells. In the present work, we tried to determine whether this increase could be correlated with the early stimulation by growth factors of cell movements. We showed that cytochalasin B (CB), in complete or glucose-free medium, inhibited this early increase caused by growth factors, in phosphate incorporation in small organic acid-soluble compounds (Po). Cytochalasin D (CD) specifically inhibited the stimulation caused by growth factors of Po labelling and ATP turnover, but lacked all inhibitory effect on unstimulated cells. The inhibitory effect of CD was transient. We hypothesize that addition of EGF and insulin to quiescent 3T3 cell cultures induces a rapid and transient change in cell movements, which could be responsible for about half of the early increase in ATP degradation and turnover.     

Visualization of gap junction mobility in living cells

In order to study the dynamics of gap junctions in living cells, a cDNA was expressed in hepatocellular carcinoma-derived PLC cells coding for chimerical polypeptide Cx.EGFP-1, which consists of rat connexin32 and enhanced green fluorescent protein (EGFP). Cx.EGFP-1 was integrated into gap junctions, and the emitted epifluorescence reliably reported the distribution of the chimera. Therefore, stably transfected PLC clone PCx-9 was used to examine the dynamic behavior of gap junctions by time-lapse fluorescence microscopy. The pleomorphic fluorescent junctional plaques were highly motile within the plasma membrane. They often fused with each other or segregated into smaller patches, and fluctuation of fluorescence was detected within individual gap junctions. Furthermore, the uptake of junctional fragments into the cytoplasm of live cells was documented as originating from dynamic invaginations that form long tubulovesicular structures that pinch off. Endocytosis and subsequent lysosomal degradation, however, appeared to contribute only a little to the rapid gap junction turnover (determined half-life of 3.3 h for Cx.EGFP-1), since most cytoplasmic Cx.EGFP-1 fluorescence did not colocalize with the endocytosed fluid phase marker horseradish peroxidase or the receptor-specific endocytotic ligand transferrin and since it was distinct from lysosomes. Disassembly of gap junctions was monitored in the presence of the translation-inhibitor cycloheximide and showed increased endocytosis and continuous reduction of junctional plaques. Highly motile cytoplasmic microvesicles, which were detectable as multiple, weakly fluorescent puncta in all movies, are proposed to contribute significantly to gap junction morphogenesis by the transport of small subunits between biosynthetic, degradative, and recycling compartments.     

Effect of cytochalasin D on DNA synthesis in cultured cells

The effect of cytochalasin D (CD), an agent specifically destroying actin cytoskeleton, on DNA replication of cultured mouse embryonic fibroblasts (MEF) and BALB/3T3 strain cells was studied. Incubation of normal cells with CD resulted in progressive inhibition of DNA synthesis: in the first 16-20 h the percentage of cells pulse-labelled with 3H-thymidine was similar to that in control cultures, on day 8 the percentage of labelled cells was 7-8 times lower than in the control. The transfer of cells into fresh medium upon 8-day incubation in the presence of CD resulted in the recovery of DNA synthesis. Similar curves of DNA synthesis inhibition in the presence of CD and of DNA synthesis recovery in fresh medium were observed both in mononuclear and binuclear cells. Thus, CD-induced reorganization of actin cytoskeleton can have an abrupt but reversible disturbing effect on normal cell cycle.     

Behavior of a fluorescent analogue of calmodulin in living 3T3 cells

We have prepared and partially characterized a lissamine-rhodamine B fluorescent analogue of calmodulin, LRB-CM. The analogue had a dye/protein ratio of approximately 1.0 and contained no free dye or contaminating labeled proteins. LRB-CM was indistinguishable from native calmodulin upon SDS PAGE and in assays of phosphodiesterase and myosin light chain kinase. The emission spectrum of LRB-CM was insensitive to changes in pH, ionic strength, and temperature over the physiological range, but the apparent quantum yield was influenced somewhat by divalent cation concentration. LRB-CM injected into living Swiss 3T3 fibroblasts became associated with nitrobenzoxadiazole-phallacidin staining stress fibers in some interphase cells. LRB-CM and acetamidofluorescein-labeled actin co-injected into the same cell both became associated with fibers in some cells, but in most cases association of the two analogues with fibers was mutually exclusive. This suggests that calmodulin may differ from actin in the timing of incorporation into stress fibers or that we have distinguished distinct populations of stress fibers. We were able to detect no direct interaction of LRB-CM with actin by fluorescence photobleaching recovery (FRAP) of aqueous solutions. Interaction of LRB-CM with myosin light chain kinase also was not detected by FRAP. This suggests that the mean lifetime of the calmodulin-myosin light chain kinase complex is too short to affect the diffusion coefficient of calmodulin. We examined various fluorescent derivatives of proteins and dextrans as suitable control molecules for quantitative fluorescent analogue cytochemistry in living cells. Fluorescein isothiocyanate-dextrans were found to be preferable to all the proteins tested, since their mobilities in cytoplasm were inversely dependent on molecular size and there was no evidence of binding to intracellular components. In contrast, FRAP of LRB-CM in the cytoplasm of living 3T3 cells suggested that the analogue interacts with intracellular components with a range of affinities. The mobility of LRB-CM in the cytoplasm was sensitive to treatment of the cells with trifluoperazine, which suggests that at least some of the intracellular binding sites are specific for calmodulin in the calcium-bound form. FRAP of LRB-CM in the nuclei of living 3T3 cells indicated that the analogue was highly mobile within the nucleus but entered the nucleus from the cytoplasm much more slowly than fluorescein isothiocyanate-dextran of comparable molecular size and much more slowly than predicted from its mobility in cytoplasm.     

Analysis of hexose transport in untransformed and sarcoma virus-transformed mouse 3T3 cells by photoaffinity binding of cytochalasin B

The effect of simian virus 40 transformation on the hexose transport system in mouse embryo fibroblast Swiss 3T3 cells was examined. The concentration of hexose transporters was estimated by measuring D-glucose-inhibitable cytochalasin B binding. The binding of cytochalasin B to the plasma membranes of simian virus 40-transformed mouse 3T3 cells (SV3T3 cells) was significantly greater than that of 3T3 cells. On the other hand, cytochalasin B binding to the microsomal membranes of SV3T3 cells was decreased, and the total amount of binding to plasma and microsomal membranes was not significantly changed in both cell lines. The electrophoretic analysis demonstrated that both hexose-transporter components of Mr 46 000 and Mr 58 000 affinity labeled were responsible for an increase in the hexose transport by viral transformation. These results suggested that the higher hexose-transport activity of transformed cells is caused by a redistribution of transporter from intracellular membranes to plasma membranes.     

Vitamin D3 metabolites modulate dihydropyridine-sensitive calcium currents in clonal rat osteosarcoma cells

A slowly inactivating inward calcium current was identified in the rat osteosarcoma cell line ROS 17/2.8 using a combination of ion flux and electrophysiological techniques. Voltage dependence, dihydropyridine sensitivity, divalent cation selectivity, and single channel properties identified this current as a high threshold, "L-type" calcium current. Ion flux experiments using 45Ca2+ confirmed that calcium uptake through these channel represents a major pathway for calcium entry into osteosarcoma cells. In resting cells, i.e. at negative membrane potentials, stimulation of both calcium current and rapid 45Ca2+ influx could be elicited by concentrations of 1,25-(OH)2-vitamin D3 between 0.1 and 3 nM. At these concentrations, 1,25-(OH)2-vitamin D3 shifted the threshold for activation of inward calcium current to more negative potentials. At higher concentrations (5-10 nM), inhibitory effects became predominant. These opposing effects are functionally similar to those of the dihydropyridine BAY K 8644. Other vitamin D3 metabolites (25-(OH)-D3 and 24,25-(OH)2-D3) exhibited less potent stimulatory effects and greater inhibition of calcium current than 1,25-(OH)2-D3. These results suggest that (i) vitamin D3 acts as a potent modulator of calcium channel function in osteosarcoma cells, and (ii) intracellular Ca2+-dependent signaling processes may be affected acutely by physiological concentrations of vitamin D3 metabolites.     

Oligomerization of adenosine A2A and dopamine D2 receptors in living cells

We investigated whether oligomerization of adenosine A(2A) receptor (A(2A)R) and dopamine D(2) receptor (D(2)R) exists in living cells using modified bioluminescence resonance energy transfer (BRET(2)) technology. Fusion of these receptors to a donor, Renilla luciferase (Rluc), and to an acceptor, modified green fluorescent protein (GFP(2)), did not affect the ligand binding affinity, subcellular distribution, and coimmunoprecipitation of the receptors. BRET was detected not only between Myc-D(2)R-Rluc and A(2A)R-GFP(2) but also between HA-tagged A(2A)R-Rluc and A(2A)R-GFP(2). These results indicate A(2A)R, either homomeric or heteromeric with D(2)R, exists as an oligomer in living cells.     

Microdetermination of 2-Deoxyglucose and 2-Deoxyglucose 6-Phosphate to Determine Glucose Utilization Rates in Single Neurons and Small CNS Regions After Injecting Nontracer Amounts of 2-Deoxyglucose

A nontracer amount (0.25 mmol/kg of body weight) of 2-deoxyglucose (DG) was intravenously injected into rats, which were frozen 2 and 4 min later in liquid nitrogen. Freeze-dried samples of CNS regions and cell bodies of spinal motor neurons were prepared, and the concentrations of glucose, glucose 6-phosphate, DG, and DG 6-phosphate (DG6P) in them were microassayed after 3,000-1,500,000-fold amplification using an enzymatic amplification reaction, NADP cycling. Based on the time course of glucose, DG, and DG6P concentrations in arterial plasma and the anterior horn of the spinal cord, the Sokoloff-type rate equations for DG and DG6P concentrations were mathematically solved, and the resultant DG and DG6P concentration functions were fitted to the data points using the nonlinear least-squares fitting SALS package program. This fitting provided four rate constants for the functions and supported the theoretical basis for our calculations of glucose utilization rate (GUR) when DG was administered in nontracer amounts. The GUR was highest in the spinal motor neurons and lowest in the white matter of the cerebellum. Neuron-rich structures, such as the cerebellar molecular and granular layers and the anterior horn of the spinal cord, had higher GUR values than the white matter of the cerebellum and spinal cord.     

Determination of protein mobility in mitochondrial membranes of living cells

Molecular mobility in membranes of intracellular organelles is poorly understood, due to the lack of experimental tools applicable for a great diversity of shapes and sizes such organelles can acquire. Determinations of diffusion within the plasma membrane or cytosol are based mostly on the assumption of an infinite flat space, not valid for curved membranes of smaller organelles. Here we extend the application of FRAP to mitochondria of living cells by application of numerical analysis to data collected from a small region inside a single organelle. The spatiotemporal pattern of light pulses generated by the laser scanning microscope during the measurement is reconstructed in silico and consequently the values of diffusion parameters best suited to the particular organelle are found. The mobility of the outer membrane proteins hFis and Tom7, as well as oxidative phosphorylation complexes COX and F₁F₀ ATPase located in the inner membrane is analyzed in detail. Several alternative models of diffusivity applied to these proteins provide insight into the mechanisms determining the rate of motion in each of the membranes. Tom7 and hFis move along the mitochondrial axis in the outer membrane with similar diffusion coefficients (D = 0.7 μm²/s and 0.6 μm²/s respectively) and equal immobile fraction (7%). The notably slower motion of the inner membrane proteins is best represented by a dual-component model with approximately equal partitioning of the fractions (F₁F₀ ATPase: 0.4 μm²/s and 0.0005 μm²/s; COX: 0.3 μm²/s and 0.007 μm²/s). The mobility patterns specific for the membranes of this organelle are unambiguously distinguishable from those of the plasma membrane or artificial lipid environments: The parameters of mitochondrial proteins indicate a distinct set of factors responsible for their diffusion characteristics.     

Five-parameter fluorescence imaging: wound healing of living Swiss 3T3 cells

Cellular functions involve the temporal and spatial interplay of ions, metabolites, macromolecules, and organelles. To define the mechanisms responsible for completing cellular functions, we used methods that can yield both temporal and spatial information on multiple physiological parameters and chemical components in the same cell. We demonstrated that the combined use of selected fluorescent probes, fluorescence microscopy, and imaging methods can yield information on at least five separate cellular parameters and components in the same living cell. Furthermore, the temporal and spatial dynamics of each of the parameters and/or components can be correlated with one or more of the others. Five parameters were investigated by spectrally isolating defined regions of the ultraviolet, visible, and near-infrared spectrum based on five distinct fluorescent probes. The parameters included nuclei (Hoechst 33342), mitochondria (diIC1-[5] ), endosomes (lissamine rhodamine B-dextran), actin (fluorescein), and the cell volume Cy7-dextran). Nonmotile, confluent Swiss 3T3 cells did not show any detectable polarity of cell shape, or distribution of nuclei, endosomes, or mitochondria. These cells also organized a large percentage of the actin into stress fibers. In contrast, cells migrating into an in vitro wound exhibited at least two stages of reorganization of organelles and cytoplasm. During the first 3 h after wounding, the cells along the edge of the wound assumed a polarized shape, carried the nuclei in the rear of the cells, excluded endosomes and mitochondria from the lamellipodia, and lost most of the highly organized stress fibers. The cell showed a dramatic change between 3 and 7 h after producing the wound. The cells became highly elongated and motile; both the endosomes and the mitochondria penetrated into the lamellipodia, while the nuclei remained in the rear and the actin remained in less organized structures. Defining the temporal and spatial dynamics and interplay of ions, contractile proteins, lipids, regulatory proteins, metabolites, and organelles should lead to an understanding of the molecular basis of cell migration, as well as other cellular functions.     

2-Deoxyglucose transport by intestinal epithelial cells isolated from the chick

Summary Characteristics of 2-deoxyglucose uptake (2DG) by intestinal epithelial cells isolated from chickens were evaluated as a means of discriminating between the concentrative transport system for monosaccharides, associated with the mucosal brush border, and other possible routes of monosaccharide entry. 2DG was chosen as it is not a substrate for the mucosal transport system. The deoxysugar enters via a saturable pathway which is not Na⁺-dependent, is not inhibited by K⁺, does not accumulate solute against a concentration gradient; exhibits a high sensitivity to inhibition by phloretin; is relatively insensitive to phlorizin inhibition; and has low affinity [but high capacity relative to Na⁺-dependent mucosal transport of 3-O-methylglucose (3-OMG) and other monosaccharides]. These characteristics confirm those established in an earlier report for Na⁺-independent uptake of 3-OMG. Complications encountered in the use of 2DG as a test sugar include significant rates of metabolic conversion to an anionic form which presumably is a phosphorylated species. Methods for distinguishing between transport and subsequent metabolism are described. Inhibition of 2DG entry by several other sugars is described and inhibitory constants (K's) given for each.     

Blockage of prereplicative progression by cytochalasin D in serum-stimulated GC-7 cells

When growth-arrested GC-7 cells, a cell line from African green monkey kidney, are stimulated with 10% calf serum, they enter S phase 14-15 h later. Cytochalasin D at 0.6 micrograms/ml blocks the entrance into S phase, and inhibits, though only partially, the increase in protein synthesis after serum stimulation. Since partial inhibition of protein synthesis by cycloheximide interferes with accumulation of labile proteins and thus blocks the entrance of serum-stimulated cells into S phase, the effects of these two inhibitors are compared. Cytochalasin D at lower concentrations reduced the rate of entry into S phase without affecting the length of the prereplicative phase, whereas cycloheximide extended the prereplicative phase dose dependently without affecting the rate of entry into S phase. Cytochalasin D affected neither individual [35S]methionine-labeled spots on two-dimensional polyacrylamide-gel nor degradation of cellular proteins. These results indicate that cytochalasin D, though it interferes with protein synthesis, blocks prereplicative progression of serum-stimulated GC-7 cells in a different manner than cycloheximide.