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PubMed2006

HPMA based macromolecular therapeutics: internalization, intracellular pathway and cell death depend on the character of covalent bond between the drug and the peptidic spacer and also on spacer composition.

Polymeric conjugates based on N-(2-hydroxypropyl)methacrylamide (HPMA) have been tested as potential carrier for anticancer drug - doxorubicin (Dox). Two types of conjugates were synthesized: (a) conjugates containing Dox bound through an amidic bond to an oligopeptidic side-chain (usually GFLG) and (b) hydrolytically cleavable conjugates wherein Dox is bound to the polymeric carrier through a pH sensitive bond. The mechanism of action of both conjugates is different and reflects the diverse way and intensity of their intracellular accumulation. All conjugates containing doxorubicin bound via an amidic bond directly penetrate the plasma membrane and are detectable in all associated cellular membranes, i.e. membranes of the endocytic compartment, a nuclear membrane as well as membranes of Golgi and endoplasmic reticulum. We have never been able to detect released doxorubicin inside the nuclei of the treated cells. The cytotoxicity of these conjugates seems to be primarily caused by the damage of cellular membranes. Necrosis is the main mechanism of the cell death. Conjugates containing hydrolytically bound doxorubicin are internalized by endocytosis and fluid phase pinocytosis and doxorubicin is cleaved from the polymeric carrier at low pH in late endosomes and lysosomes. An apoptosis is the main mechanism of the cell death. The spacer influences the rate of the intracellular release of the drug rather than the rate of internalization.

3T3 Cells↗
PubMed2006

Effect of static electric fields in root cells of Vicia faba (Fabaceae).

Serial electron microscopic sections were prepared from half-ripened meristematic root cells of Vicia faba (Fabaceae) which had been exposed gradually to 700, 1000, 2500, 3500, and 5000 V/m static electric fields during seven days with and without Zn and Cd electrodes. At the end of five weeks, wall loosenings and very small nuclei were observed in those root cells which were exposed to static electric currents from the lower side of the medium without electrodes, while abnormalities in cell formation, e.g., two cells with one nucleus, and GER occurrence were present in an electrolytic (Cd upward and Zn downward) medium. The cells exposed to a static current from the upper side of the medium had small nuclei and abnormal cell divisions in the electrolyte, but in a non-electrolyte very large nuclei and thicker cell walls were observed, the cytoplasm was dense with GER, pinocytosis was seen filled with mitochondria, and protoplast formation with big nuclei was seen in exocytosis.

Cadmium↗
PubMed1985

Membrane and cytoskeletal changes associated with IgE-mediated serotonin release from rat basophilic leukemia cells.

Binding of antigen to IgE-receptor complexes on the surface of RBL-2H3 rat basophilic leukemia cells is the first event leading to the release of cellular serotonin, histamine, and other mediators of allergic, asthmatic, and inflammatory responses. We have used dinitrophenol-conjugated bovine serum albumin (DNP-BSA) as well as the fluorescent antigen, DNP-B-phycoerythrin, and the electron-dense antigen, DNP-BSA-gold, to investigate dynamic membrane and cytoskeletal events associated with the release of [3H]serotonin from anti-DNP-IgE-primed RBL-2H3 cells. These multivalent antigens bind rapidly to cell surface IgE-receptor complexes. Their distribution is initially uniform, but within 2 min DNP-BSA-gold is found in coated pits and is subsequently internalized. Antigen internalization occurs in the presence and absence of extracellular Ca2+. The F-actin content of the detergent-extracted cell matrices analyzed by SDS PAGE decreases during the first 10-30 s of antigen binding and then increases by 1 min to almost double the control levels. A rapid and sustained increase is also observed when total F-actin is quantified by flow cytometry after binding of rhodamine-phalloidin. The antigen-stimulated increase in F-actin coincides with (and may cause) the transformation of the cell surface from a finely microvillous to a highly folded or plicated topography. Other early membrane responses include increased cell spreading and a 2-3-fold increase in the uptake of fluorescein-dextran by fluid pinocytosis. The surface and F-actin changes show the same dependence on DNP-protein concentration as stimulated [3H]serotonin release; and both the membrane responses and the release of mediators are terminated by the addition of the non-cross-linking monovalent ligand, DNP-lysine. These data indicate that the same antigen-stimulated transduction pathway controls both the membrane/cytoskeletal and secretory events. However, the membrane and actin responses to IgE-receptor cross-linking are independent of extracellular Ca2+ and are mimicked by phorbol myristate acetate, whereas ligand-dependent mediator release depends on extracellular Ca2+ and is mimicked by the Ca2+ ionophore A23187.

Actin Cytoskeleton↗
PubMed1986

Limited and selective transfer of plasma membrane glycoproteins to membrane of secondary lysosomes.

Radioactive galactose, covalently bound to cell surface glycoconjugates on mouse macrophage cells, P388D1, was used as a membrane marker to study the composition, and the kinetics of exchange, of plasma membrane-derived constituents in the membrane of secondary lysosomes. Secondary lysosomes were separated from endosomes and plasma membrane on self-forming Percoll density gradients. Horseradish peroxidase, taken up by fluid-phase pinocytosis, served as a vesicle contents marker to monitor transfer of endosomal contents into secondary lysosomes. Concurrently, the fraction of plasma membrane-derived label in secondary lysosomes increased by first order kinetics (k = [56 min]-1) from less than 0.1% (background level) to a steady-state level of approximately 2.5% of the total label. As analyzed by NaDodSO4 PAGE, labeled molecules of Mr 160-190 kD were depleted and of Mr 100-120 kD were enriched in lysosome membrane compared with the relative composition of label on the cell surface. No corresponding selectivity was observed for the degradation of label, with all Mr classes being affected to the same relative extent. The results indicate that endocytosis-derived transfer of plasma membrane constituents to secondary lysosomes is a limited and selective process, and that only approximately 1% of internalized membrane is recycled via a membrane pool of secondary lysosomes.

Animals↗
PubMed1989

Prelysosomal acidic vacuoles in Dictyostelium discoideum.

We have examined the ameba Dictyostelium discoideum for evidence of a discrete, prelysosomal, acidic receiving compartment in endocytosis. We observed in the cytoplasm abundant round vacuoles with diameters up to 2 microns that concentrated acridine orange by a process inhibited by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl). They were therefore taken to be acidic. The vacuoles were observed to fuse nearly quantitatively with primary phagosomes over 30 min and thereby to confer upon them the ability to accumulate acridine orange. The entry into lysosomes of phagocytic cargo occurred later. In the absence of phagocytosis, almost all of the acidic vacuoles rapidly accumulated fluorescent markers that had either been covalently coupled to the cell surface or fed as the soluble dextran conjugate. Therefore, these vacuoles also lie on the pathway of pinocytosis. A prominent subcellular ATPase activity inhibited by 25 microM NBD-Cl co-distributed on sucrose equilibrium density gradients with vacuoles capable of concentrating acridine orange in vitro. The peak was broad and more buoyant than that bearing lysosomal acid hydrolases, which contained only a minor amount of this ATPase. Also migrating in the buoyant peak were internalized plasma membrane markers; e.g., 3H-galactose had been covalently coupled to the surface of intact cells and allowed to enter pinosomes. We conclude that in D. discoideum an extensive prelysosomal vacuolar compartment provides the proton pumps that acidify both phagosomes and pinosomes.

Acid Phosphatase↗
PubMed1993

Macropinosome maturation and fusion with tubular lysosomes in macrophages.

Macropinosomes formed by addition of recombinant macrophage colony-stimulating factor (rM-CSF) to mouse macrophages migrate centripetally and shrink, remaining detectable by phase microscopy for up to 15 min. This longevity allowed us to study how macropinosomes age. Macropinosomes were pulse labeled for 1 min with fixable fluorescein dextran (FDx10f), a probe for fluid phase pinocytosis, and chased for various times. To quantify changes in their antigenic profile, pulse-labeled macropinosomes of different ages were fixed and stained for immunofluorescence with a panel of antibodies specific for the transferrin receptor (TfR), the late endosome-specific, GTP-binding protein rab 7 or lysosomal glycoprotein A (lgp-A), and the percentage of antibody positive, FDx10f-labeled macropinosomes was scored. Some newly formed macropinosomes were positive for TfR, but few were rab 7 or lgp-A-positive. With intermediate chase times (2-4 min), staining for rab 7 and lgp-A increased to > 60%, while TfR staining declined. After a long chase (9-12 min), rab 7 staining returned to low levels while lgp-A staining remained at a high level. Thus, macropinosomes matured by progressive acquisition and loss of characteristic endocytic vesicle markers. However, unlike a maturation process, their merger with the tubular lysosomal compartment more nearly resembled the incorporation of a transient vesicle into a pre-existing, stable compartment. Shortly after their formation, FDx10f-labeled macropinosomes contacted and merged with Texas red dextran (TRDx10)-labeled tubular lysosomes. This occurred in two steps: macropinosomes acquired lgp-A first, and then several minutes later the cation-independent mannose-6-phosphate receptor (CI-MPR) and markers of lysosomal content (cathepsin L or pre-loaded TRDx10), all apparently derived from tubular lysosomes. Thus, macropinosome progress through macrophages showed features of both the maturation and vesicle shuttle models of endocytosis, beginning with a maturation process and ending by merger into a stable, resident lysosomal compartment.

Animals↗
PubMed1994

Clathrin heavy chain functions in sorting and secretion of lysosomal enzymes in Dictyostelium discoideum.

The clathrin heavy chain is a major component of clathrin-coated vesicles that function in selective membrane traffic in eukaryotic cells. We disrupted the clathrin heavy chain gene (chcA) in Dictyostelium discoideum to generate a stable clathrin heavy chain-deficient cell line. Measurement of pinocytosis in the clathrin-minus mutant revealed a four-to five-fold deficiency in the internalization of fluid-phase markers. Once internalized, these markers recycled to the cell surface of mutant cells at wild-type rates. We also explored the involvement of clathrin heavy chain in the trafficking of lysosomal enzymes. Pulse chase analysis revealed that clathrin-minus cells processed most alpha-mannosidase to mature forms, however, approximately 20-25% of the precursor molecules remained uncleaved, were missorted, and were rapidly secreted by the constitutive secretory pathway. The remaining intracellular alpha-mannosidase was successfully targeted to mature lysosomes. Standard secretion assays showed that the rate of secretion of alpha-mannosidase was significantly less in clathrin-minus cells compared to control cells in growth medium. Interestingly, the secretion rates of another lysosomal enzyme, acid phosphatase, were similar in clathrin-minus and wild-type cells. Like wild-type cells, clathrin-minus mutants responded to starvation conditions with increased lysosomal enzyme secretion. Our study of the mutant cells provide in vivo evidence for roles for the clathrin heavy chain in (a) the internalization of fluid from the plasma membrane; (b) sorting of hydrolase precursors from the constitutive secretory pathway to the lysosomal pathway; and (c) secretion of mature hydrolases from lysosomes to the extracellular space.

Acid Phosphatase↗
PubMed1996

A role for phosphoinositide 3-kinase in the completion of macropinocytosis and phagocytosis by macrophages.

Phosphoinositide 3-kinase (PI 3-kinase) has been implicated in growth factor signal transduction and vesicular membrane traffic. It is thought to mediate the earliest steps leading from ligation of cell surface receptors to increased cell surface ruffling. We show here that inhibitors of PI 3-kinase inhibit endocytosis in macrophages, not by interfering with the initiation of the process but rather by preventing its completion. Consistent with earlier studies, the inhibitors wortmannin and LY294002 inhibited fluid-phase pinocytosis and Fc receptor-mediated phagocytosis, but they had little effect on the receptor-mediated endocytosis of diI-labeled, acetylated, low density lipoprotein. Large solute probes of endocytosis reported greater inhibition by wortmannin than smaller probes did, indicating that macropinocytosis was affected more than micropinocytosis. Since macropinocytosis and phagocytosis are actin-mediated processes, we expected that their inhibition by wortmannin resulted from deficient signaling from macrophage colony-stimulating factor (M-CSF) receptors or Fc receptors to the actin cytoskeleton. However, video microscopy showed cell surface ruffling in wortmannin-treated cells, and increased ruffling after addition of M-CSF or phorbol myristate acetate. Quantitative measurements of video data reported slightly diminished ruffling in wortmannin-treated cells. Remarkably, the ruffles that formed in wortmannin-treated macrophages all receded into the cytoplasm without closing into macropinosomes. Similarly, wortmannin and LY294002 did not inhibit the extension of actin-rich pseudopodia along IgG-opsonized sheep erythrocytes, but instead prevented them from closing into phagosomes. These findings indicate that PI 3-kinase is not necessary for receptor-mediated stimulation of pseudopod extension, but rather functions in the closure of macropinosomes and phagosomes into intracellular organelles.

Androstadienes↗
PubMed1997

Inactivation of two Dictyostelium discoideum genes, DdPIK1 and DdPIK2, encoding proteins related to mammalian phosphatidylinositide 3-kinases, results in defects in endocytosis, lysosome to postlysosome transport, and actin cytoskeleton organization.

Phosphatidylinositide 3-kinases (PI3-kinases) have been implicated in controlling cell proliferation, actin cytoskeleton organization, and the regulation of vesicle trafficking between intracellular organelles. There are at least three genes in Dictyostelium discoideum. DdPIK1, DdPIK2, and DdPIK3, encoding proteins most closely related to the mammalian 110-kD PI-3 kinase in amino acid sequence within the kinase domain. A mutant disrupted in DdPIK1 and DdPIK2 (delta ddpik1/ddpik2) grows slowly in liquid medium. Using FITC-dextran (FD) as a fluid phase marker, we determined that the mutant strain was impaired in pinocytosis but normal in phagocytosis of beads or bacteria. Microscopic and biochemical approaches indicated that the transport rate of fluid-phase from acidic lysosomes to non-acidic postlysosomal vacuoles was reduced in mutant cells resulting in a reduction in efflux of fluid phase. Mutant cells were also almost completely devoid of large postlysosomal vacuoles as determined by transmission EM. However, delta ddpik1/ddpik2 cells functioned normally in the regulation of other membrane traffic. For instance, radiolabel pulse-chase experiments indicated that the transport rates along the secretory pathway and the sorting efficiency of the lysosomal enzyme alpha-mannosidase were normal in the mutant strain. Furthermore, the contractile vacuole network of membranes (probably connected to the endosomal pathway by membrane traffic) was functionally and morphologically normal in mutant cells. Light microscopy revealed that delta ddpik1/ddpik2 cells appeared smaller and more irregularly shaped than wild-type cells; 1-3% of the mutant cells were also connected by a thin cytoplasmic bridge. Scanning EM indicated that the mutant cells contained numerous filopodia projecting laterally and vertically from the cell surface, and fluorescent microscopy indicated that these filopodia were enriched in F-actin which accumulated in a cortical pattern in control cells. Finally, delta ddpik1/ddpik2 cells responded and moved more rapidly towards cAMP. Together, these results suggest that Dictyostelium DdPIK1 and DdPIK2 gene products regulate multiple steps in the endosomal pathway, and function in the regulation of cell shape and movement perhaps through changes in actin organization.

Actins↗
PubMed1962

Studies on protein uptake by isolated tumor cells. II. Quantitative data on the adsorption and uptake of I-131-serum albumin by Ehrlich ascites tumor cells.

Surface adsorption is studied in some detail because it is believed to be a major artifact in measurements of protein uptake by mammalian cells. Adsorption increases linearly with the I(131)-albumin concentration between 0.001 and 300 mg/ml. After short exposure to 300 mg/ml and two cell washings, the adsorption amounts to 38 mg albumin per gm cell proteins. Further washings remove 80 per cent of this value, leaving a small irreversibly bound residue. At equilibrium, adsorbed albumin can be labeled by a simple albumin exchange. This labeling reaches a steady state within seconds and stays at constant level over 30 minutes. Significant increases above this initial level are measured over periods of 2 hours. In our experimental conditions these increases can be considered due to albumin uptake. This uptake rises linearly with the albumin concentration between 0.5 and 50.0 mg/ml, and reaches 0.2 mg/gm cell protein or 4 x 10(5) molecules per cell. Compared to the incorporation of free amino acids in similar conditions, this value does not appear to contribute significantly to the N-metabolism of the tumor cells. Adsorption was generally greater than uptake. Both processes are linear functions of the same variable over the whole range of concentration tested. It is suggested that albumin is taken up by pinocytosis.

Adsorption↗
PubMed1963

THE DIFFERENTIATION OF WHITE ADIPOSE CELLS. AN ELECTRON MICROSCOPE STUDY.

Differentiating white adipose tissue from presumptive and developing fat pads of newborn and young rats was fixed in buffered osmium tetroxide, embedded in Vestopal W, and examined in an electron microscope. Pre-adipose cells were found to be fibroblasts characterized by their spindle shape, long tenuous cytoplasmic extensions, and profuse endoplasmic reticulum. The developmental stages traced from fibroblast to mature adipose cell show a gradual change in cell shape, an accumulation of cytoplasm and non-membrane-bounded lipid, a decrease in the endoplasmic reticulum, and a change in shape of mitochondria. Transitory glycogen appears at mid-differentiation. Numerous smooth-membraned vesicles occur in the cytoplasm throughout differentiation. Pinocytosis is constantly evident. Cells of the multilocular stage are shown to differ from brown fat cells, particularly with respect to cytoplasmic membrane systems and mitochondria. No transport of particulate lipid from the lumen of the capillary to, or within, the adipose cell was detected, nor could any cell organelle be demonstrated to be visibly related to lipid synthesis and/or deposition.

Adipose Tissue↗
PubMed1963

UPTAKE OF MAMMALIAN CHROMOSOMES BY MAMMALIAN CELLS.

Chromosomes isolated from mouse leukemia L1210 cells were taken up by mouse macrophages, HeLa cells, and rat embryo fibroblasts following simple exposure in vitro. The process, which resembles pinocytosis or phagocytosis, was traced by autoradiography of chromosomes prelabeled with thymidine-H(3), and by staining techniques and phase contrast microscopy. During the first six hours, the uptake of chromosomes was restricted to the cytoplasm, but there was some evidence of penetration into the nucleus after 16 and 26 hours of exposure. Treatment of rat fibroblasts with glucose and insulin markedly enhanced the uptake of chromosomes, whereas iodoacetate inhibited their penetration.

Animals↗
PubMed1965

The distribution within the brain of ferritin injected into cerebrospinal fluid compartments. I. Ependymal distribution.

From 10 minutes to 3(1/2) hours after the intraventricular injection into rats of 15 to 100 mg of ferritin, an appreciable fraction of the protein, visualized electron microscopically, traverses the ependymal epithelium by diffusing along the dense intercellular substance of the luminal open junction and thence, by circumventing discrete intercellular fusions which partition rather than seal the interspace. These partitions shunt additional protein into the cell, where ferritin is transported within pinocytotic vesicles to the lateral and basal plasma-lemma and, presumably, back into the interspace again. The basal interspace is irregularly distended by pools of moderately dense "filler" within which ferritin accumulates. The larger fraction of protein enters the ependyma by pinocytosis and is eventually segregated within membrane-enclosed organelles such as vacuoles, multivesicular bodies, and dense bodies, where the molecules may assume a crystalline packing. As a result of the accumulation of ferritin within these inclusions and within filler substance, only a small amount of protein remains to enter the underlying parenchyma. Presentation of ferritin to prefixed cells leads to a random dispersion of free cytoplasmic ferritin. This artifactual distribution in both prefixed and postfixed cells is concurrent with disruption of cell membranes.

Animals↗
PubMed1957

Iron particles in normal erythroblasts and normal and pathological erythrocytes.

Iron-containing granules (40 to 100 A in diameter), in groups or isolated, are present in normal erythroblasts, normoblasts, reticulocytes, and certain pathological erythrocytes. Similar granules are present in macrophages and have been noted in the stroma of erythrocytes following phagocytosis and hemolysis by macrophages. Incorporation of the granules from the macrophage into erythroblasts is presented as a process similar to pinocytosis. The presence of the iron-containing granules within the erythroid cells parallels hemoglobin synthesis.

Electrons↗
PubMed1957

Electron microscopy of HeLa cells after the ingestion of colloidal gold.

Tissue cultures of HeLa cells were grown in media containing colloidal gold, and after various intervals, the cells were fixed, embedded, and sectioned for electron microscopy. Uncoated grids with small holes were used in many of the experiments. Intracellular particles of gold were identified in areas surrounded by single membranes, in moderately dense granules, in globoid bodies, and in the cytoplasmic matrix. Gold particles were not found in typical mitochondria, Golgi complex, ergastoplasm (granular forms of endoplasmic reticulum), or nuclei. The phenomenon of pinocytosis was considered to be the most likely means by which the gold particles were ingested, and the locations of gold particles appeared to have significance concerning theories that membranous organelles of the cytoplasm may be derived from the cell membrane.

Animals↗
PubMed1967

Metabolic and morphological observations on the effect of surface-active agents of leukocytes.

Morphological and metabolic observations have been made on the effects of endotoxin, deoxycholate, and digitonin (at less than 50 microg/ml) on polymorphonuclear leukocytes and mononuclear cells. The agents stimulate the respiration and glucose oxidation of these cells in a manner similar to that seen during phagocytosis. Electron microscopy revealed no morphological changes with the first two agents, but dramatic membrane changes were seen in the case of digitonin. Here tubular projections of characteristic size and shape formed on and split off the membrane. All the agents stimulated uptake of inulin, but efforts to demonstrate increased pinocytosis by electron microscopy have not so far succeeded, probably due to limitations in present experimental techniques.

Animals↗
PubMed1967

Intestinal triglyceride absorption in the rat. An electron microscopical study.

This report provides information on the morphology of fat absorption in rat intestinal epithelial cells. Three types of experiments were performed: (a) intubation of corn oil into fasted rats, (b) injection of physiological fatty-chyme prepared from fat-fed donor rats into ligated segments of jejunum of fasted animals, and (c) administration of electron-opaque particles in corn oil and markers given concurrently with the fat. These results support the hypothesis that fat is absorbed by selective diffusion of monoglycerides and fatty acids from micelles rather than by pinocytosis of unhydrolized triglycerides. Evidence is presented that the pits between the microvilli, previously believed to function in the transport of fat, are not involved in this process. Instead they appear to contribute their contents to lysosomes in the apical cytoplasm. Arguments are offered that the monoglycerides and fatty acids diffuse from the micelle while the latter is associated with the microvillous membrane of the absorptive cell. These micellar components penetrate the plasma membrane and diffuse into the cytoplasmic matrix where they encounter the SER. Triglyceride synthesis occurs in the SER and results in the deposition of fat droplets within its lumina. The synthesis of triglycerides and their sequestration into the SER establishes an inward diffusion gradient of monoglycerides and fatty acids.

Animals↗
PubMed1968

Pinocytotic response of circulating erythrocytes to specific blood grouping antibodies.

Human blood samples from adults and newborns of blood groups O, A, and B were treated with either anti-A blood grouping serum, ferritin-conjugated anti-A serum, free ferritin, or saline and then prepared for electron microscopy. Morphological differences were observed between the untreated erythrocytes of infants and adults. Circulating red cells of newborns were frequently vesiculated (25.5%), whereas those of adults only occasionally showed vesicles (5.5%). On the basis of morphology and incidence, the majority of these vesiculated cells seemed to be mature erythrocytes. The introduction of anti-A serum to group A erythrocytes of infants appeared to stimulate vesicle formation, but anti-A serum did not have a similar effect on group O or B cells of infants or on group A cells of adults. Vesicles which formed in response to antiserum treatment appeared to be the result of pinocytosis. In contrast to the well dispersed ferritin along the membrane of agglutinated adult cells, the ferritin particles on the infants' cells were frequently clustered at irregular intervals. These accumulations seemed to lead to invaginations of the cell membrane, resulting in ferritin-lined intracytoplasmic vesicles. The addition of free ferritin or ferritin-conjugated antibodies of the wrong specificity to red cells did not increase vesicle formation.

Adult↗