Pinosome Formation Research Articles

Dynein Light Chain 1 Phosphorylation Controls Macropinocytosis*[boxs

Recent studies have identified dynein light chain-1 (DLC1), a component of the dynein motor, as a p21-activated kinase 1 (Pak1)-interacting substrate with binding sites mapped to amino acids 61-89 of DLC1 and phosphorylation site at serine 88. Here we investigated the role of DLC1 phosphorylation by Pak1 upon the process of macropinocytosis. We found that Pak1 associates with dynein motor and that Pak1-DLC1 interaction starts at the initiation of pinosome formation and persists in early and late endosomes. Pak1 phosphorylation of DLC1 on Ser-88 controls vesicle formation and trafficking functions, as Ser-88 substitution for alanine prevents macropinocytosis. A peptide spanning the C-terminal 19-amino acid region of DLC1 efficiently blocked Ser-88 phosphorylation and macropinocytosis. These results suggest that the regulation of DLC1 by Pak1 is a novel mechanism by which a signaling kinase might influence macropinocytosis.

Membrane retrieval in neutrophils during phagocytosis: inhibition by M protein-expressing S. pyogenes bacteria.

During phagocytosis and phagosome maturation, complex membrane traffic events must be coordinated. We have observed, using fluorescent fluid-phase and membrane markers, that in the human neutrophil, internalization of nonopsonized, Gram-positive bacteria, but not of latex beads, is accompanied by a rapid and localized formation of pinosomal structures. This pinocytic response is calcium-dependent but insensitive to actin cytoskeleton disruption and wortmannin treatment. Contrary to what we observe, endosomal structures usually are considered to participate in phagosome formation by providing necessary membrane to forming phagosomes. Instead, our results show a coupling between neutrophil secretory and membrane-retrieval processes during phagosome maturation, and we suggest that the observed, localized pinocytic response is linked to the secretion of azurophilic granules toward nascent phagosomes. Accordingly, M and M-like protein-expressing Streptococcus pyogenes bacteria, which are able to survive inside neutrophil phagosomes, inhibit both the secretion of azurophilic granules to phagosomes and pinosome formation.

Annexin 2 Binding to Phosphatidylinositol 4,5-Bisphosphate on Endocytic Vesicles Is Regulated by the Stress Response Pathway

Annexin 2 is a Ca(2+)-binding protein that has an essential role in actin-dependent macropinosome motility. We show here that macropinosome rocketing can be induced by hyperosmotic shock, either alone or synergistically when combined with phorbol ester or pervanadate. Rocketing was blocked by inhibitors of phosphatidylinositol-3-kinase(s), p38 mitogen-activated protein (MAP) kinase, and calcium, suggesting the involvement of phosphoinositide signaling. Since various phosphoinositides are enriched on inwardly mobile vesicles, we examined whether or not annexin 2 binds to any of this class of phospholipid. In liposome sedimentation assays, we show that recombinant annexin 2 binds to phosphatidylinositol 4,5-bisphosphate (PtdIns-4,5P(2)) but not to other poly- and mono-phosphoinositides. The affinity of annexin 2 for PtdIns-4,5P(2) (K(D) approximately 5 microm) is comparable with those reported for a variety of PtdIns-4,5P(2)-binding proteins and is enhanced in the presence of Ca(2+). Although annexin 1 also bound to PtdIns-4,5P(2), annexin 5 did not, indicating that this is not a generic annexin property. To test whether annexin 2 binds to PtdIns-4,5P(2) in vivo, we microinjected rat basophilic leukemia cells stably expressing annexin 2-green fluorescent protein (GFP) with fluorescently tagged antibodies to PtdIns-4,5P(2). Annexin 2-GFP and anti-PtdIns-4,5P(2) IgG co-localize at sites of pinosome formation, and annexin 2-GFP relocalizes to intracellular membranes in Ptk cells microinjected with Arf6Q67L, which has been shown to stimulate PtdIns-4,5P(2) synthesis on pinosomes through activation of phosphatidylinositol 5 kinase. These results establish a novel phospholipid-binding specificity for annexin 2 consistent with a role in mediating the interaction between the macropinosome surface and the polymerized actin tail.

Dynamics of rab5 activation in endocytosis and phagocytosis

Fluid-phase endocytosis is stimulated by H-ras-linked growth factor receptors and this stimulation requires activation of rab5. We utilized a GFP-rab5a:wt fusion protein to monitor GFP-rab5a:wt activation in living fibroblasts and in J774 macrophages. Control CHO cells that expressed GFP-rab5a:wt were cultured in serum-free conditions and showed GFP-rab5a:wt localized to endosomal vesicles with a mean diameter of 0.3 +/- 0.1 microm. Endosome fusion, membrane ruffling, and pinosome formation were rarely detected in these cells. Coexpression of H-ras:G12V, a constitutively active H-ras mutant that activates rab5a, in cells resulted in marked enlargement of labeled endosomes (mean diameter 0.7 +/- 0.2 microm) and large numbers of giant GFP-rab5a:wt-positive endosomes were present. Time-lapse recordings showed abundant fusion among giant labeled endosomes, and membrane ruffling and pinosome formation were commonly observed. Alterations in GFP-rab5a:wt endosome structure and activity in cells expressing H-ras:G12V were linked to rab5a activation because these changes were identical to those found in cells expressing GFP-rab5a:Q79L, a constitutively activated rab5a mutant. Furthermore, cells co-expressing H-ras:G12V and GFP-rab5a:S34N, an inactive rab5a mutant, exhibited no evidence of H-ras:G12V-induced endosome enlargement. To observe changes in endosome structure and activity that directly followed activation of GFP-rab5a:wt, we performed time-lapse recordings of cells cultured overnight in serum-free media after addition of EGF. EGF caused a rapid increase in endosome fusion and in membrane ruffling activity. Membrane ruffling was often associated with GFP-rab5a:wt-positive vesicle (pinosome) formation at the base of membrane ruffles. Endosome and pinosome fusion were common in EGF-stimulated cells. Phagocytosis is also regulated by GFP-rab5a:wt. J774 macrophages that expressed GFP-rab5a:wt showed transiently activation and recruitment of GFP-rab5a:wt to newly formed phagosomes that contained rhodamine-labeled Escherichia coli. These studies show that GFP-rab5a:wt activation results in dynamic alterations in the structure and activity of the early endosomal and early phagosomal elements.

CISPLATIN INHIBITS PINOCYTOSIS INDICTYOSTELIUM DISCOIDEUM

The effects ofcis-diamminedichloroplatinum(II) [cisplatin], a potential anticancer drug, were studied on pinocytotic functions in the cellular slime mouldDictyostelium discoideumby administering FITC-dextran as a fluid phase marker. Cisplatin treatment at a concentration of 100 and 200 μg/ml for 1 h causes inhibition in pinocytotic uptake in growingDictyosteliumcells in a dose-dependent manner. Cisplatin treatment induced the association of more actin with the cell cortex, thereby presumably restricting the flexibility of the cell membrane and inhibiting the formation of pinosomes. Ultrastructural analysis of cisplatin-treated cells showed a lower number of pinosomes. These results have been discussed in the light of cisplatin's known actions that affect various cellular functions.

Effect of the amino acid analog hadacidin on intracellular membrane flow and the cell surface in Dictyostelium discoideum: A transmission- and scanning-electron-microscope study

We examined the effect of the amino acid analog hadacidin ( N-formyl- N-hydroxy glycine) on the process of endocytosis in the slime mold Dictyostelium discoideum. Endocytosis was followed using iron-dextran and transmission electron microscopy. In cells taken from the mid-log growth stage, iron-dextran was found to be distributed in small, medium, and large vesicles at a density lower than that present in the incubation medium, thus suggesting the fusion of small, iron-dextran-containing pinosomal vesicles with intracellular vesicles not containing iron-dextran. In cells treated with hadacidin, more small vesicles were present than in untreated cells, there being a reduction in the number of larger-sized vesicles; in these vesicles, iron-dextron was present at a density similar to that of the medium. This result is consistent with the conclusion that, while pinocytosis had continued, the fusion of vesicles and dilution of the vesicle contents had been inhibited. Also, the large number of small pinosomal vesicles in the drug-treated cells suggested that the recycling of vesicles to the surface had been inhibited. The observation that pinocytosis but not recycling continued after drug treatment raised the question of the origin of the membrane needed for the formation of pinosomes. Measurements of the cell surface revealed no difference between drug-treated and untreated cells, indicating that, when the membrane was internalized for pinosomes, the cell size remained constant. Scanning electron microscopy of the surface of hadacidin-treated cells revealed that the filopodia normally seen on their surface had disappeared, suggesting that the filopodial membrane may serve as a reservoir of the cellular membrane destined to replace the membrane utilized in pinosome formation.

Pinocytosis in Dictyostelium discoideum cells : A possible implication of cytoskeletal actin for pinocytotic activity

Pinocytosis in Dictyostelium discoideum axenic strain (Ax-2) cells in the growth phase is progressively inhibited at higher Ca 2+ concentrations, the activity being maximal at submicromolar Ca 2+ concentrations. The cytoskeletal actin content is also markedly reduced in the presence of 10 mM EGTA. This was confirmed by electronmicroscopy using intact cells and Triton X-100-insoluble cell cortices. Interestingly, the pinocytotic activity seemed to be somewhat increased in response to cytochalasin B (CB). Aggregation-competent Ax-2 cells which are usually devoid of pinocytotic activity can resume their activity considerably following treatment with 10 mM EGTA. Under these conditions, cytoskeletal actin declines markedly, as also was the case for growing Ax-2 cells. Our findings indicate a correlation between the pinocytotic activity and presence of cytoskeletal actin: reduced amounts of actin in the cell cortex seem to favour pinocytosis. Conceivably, membrane-associated actin filaments may function as a powerful anchor, restricting the flexibility of the cell membrane and thereby inhibiting the pinosome formation. Other properties of pinocytosis like a developmental change as well as the effects of pH and temperature are also described and were compared with the properties of wild-type strain, NC-4.

Reduced pinosome formation in a cell-fusion-impaired mutant of mouse lymphocytic cell line L5178Y.

Accumulation and release of a fluid-phase marker ([14C]-sucrose) were studied in a subline of the mouse lymphocytic cell line L5178Y and in a polyethylene glycol-resistant, intercellular-fusion-impaired mutant of this line. The mutant was found to accumulate [14C]-sucrose at a significantly slower rate than the parent. Analysis of release of preloaded label shows that the reduced rate of accumulation is due to a correspondingly low level of internalization, i.e., pinosome formation, rather than to a reduction in delivery of label to lysosomes. Cell-to-cell fusion and pinosome formation both involve a fusion event initiated at the extracellular surface of the plasma membrane, and we propose that the coordinate reduction in both processes suggests that they are mechanistically related.

Taenia crassiceps: Temperature, polycations, polyanions, and cysticercal endocytosis

The effect of various temperatures, poly- l-lysine, and poly- l-glutamic acid on endocytosis of smooth micropinocytotic vesicles (pinosomes) in the tegument of the cysticercus of Taenia crassiceps has been investigated stereologically. The temperature regimes used were 0, 5, 10, 20, 30, and 40 C. Maximum volume, surface density, and number per unit volume were found at 40 C, and minimum surface-to-volume ratio and numbers at 10 C. At 10 C, mean pinosome volume and mean surface area per pinosome were maximal, but volume and surface density did not differ significantly from 40 C. It is proposed that this anomalous finding for 10 C incubations was due to this being a critical temperature at which a slower rate of pinosome formation was compensated for by the formation of larger individual pinosomes. Poly- l-lysine was shown to be a stimulant of pinosome formation, leading to a significant increase in numbers per unit volume. However, volume and surface density, surface-to-volume ratio, mean volume, and mean surface area per pinosome were not significantly different in poly- l-lysine-incubated samples, when compared to controls (fresh from the mouse) or incubations in medium only or samples returned to medium after poly- l-lysine incubation, the only exception being surface to volume ratio and mean volume of pinosomes in the 75-min incubation. These anomalous results were explained by a marked reduction in the form ellipse values, which indicated the production of more elliptical-shaped pinosomes under poly- l-lysine stimulation. Incubation in poly- l-glutamine acid did not have any significant effect at any incubation time.

37] Ligand-induced patching and capping of surface immunoglobulins

This chapter discusses the patching and capping phenomena that has been described for a variety of membrane components in a number of different cell types. The basic model remains, however, the patching and capping of membrane immunoglobulin (mIg) la of B lymphocytes. With other membrane components, the conditions for obtaining such redistribution phenomena may be significantly different. The chapter states that a detailed model for the mechanisms of patching and capping, and related membrane phenomena such as pinosome formation, microvilli formation, endocytosis, and shedding, has also been developed. The chapter reviews the general characteristics of the capping phenomenon, particularly the energy requirements, temperature dependence, inhibition, reversion by microfilament-directed drugs, and the accumulation of microfilaments under the cap that have led to the suggestion that capping is probably a cellular contractile phenomenon. The role of microtubules in the capping phenomenon is more complex and they do not appear to enhance microfilament mobility but rather to inhibit it. Capping of any membrane component probably occurs as a consequence of microprecipitation resulting in entrapment within the lattice formed in the plasma membrane, of a microfilament-associated membrane component: the “membrane capping component” or “integral protein X.” The hypothesis has been formulated, however, that not only clustered components but also whole domains of membrane might be brought into the cap.

Phorbol myristate acetate stimulates pinocytosis and membrane spreading in mouse peritoneal macrophages.

Phorbol myristate acetate (PMA) at a concentration of 0.01 microgram/ml causes an approximately threefold increase in surface area of resident, proteose-peptone-elicited, and thioglycolate-broth-elicited mouse peritoneal macrophages. Resident and proteose-peptone-elicited macrophages, cultured for 24 h in the presence of PMA, increase their pinocytic rate twofold in response to addition of PMA (0.01 microgram/ml) to the medium. Thioglycolate-broth-elicited macrophages, cultured for 24 h in the absence of PMA, immediately increase their pinocytic rate 2- to 3.5-fold in response to a single challenge with PMA (0.01 microgram/ml). Cytochalasin B, colchicine, and podophyllotoxin have only modest inhibitory effects on the basal rate of pinocytosis and on PMA-induced cellular spreading, but completely block the stimulatory effects of PMA on pinocytosis in thioglycolate-broth-elicited macrophages. Cytochalasin D markedly inhibits both basal and PMA-stimulated pinocytosis in these cells. Thus, PMA is a useful tool for studying mechanisms of macrophage spreading and for enhancing the overall rate of pinosome formation.

Hydrolysis of an exogenous 125I-labelled protein by rat yolk sacs. Evidence for intracellular degradation within lysosomes.

When added to the serum-free medium in which 17.5-day rat yolk sacs were incubated, formaldehyde-denatured 125I-labelled bovine serum albumin was rapidly degraded. More than 80% of the radiolabelled digestion products appearing in the incubation medium consisted of [125I]iodo-L-tyrosine; larger digestion products were found only in association with the yolk-sac tissue. In the early stages of an incubation, low-molecular-weight digestion products began to appear in the incubation medium only after they could be detected within the tissue, and progressive association of trichloroacetic acid-insoluble radioactivity with the tissue preceded both these events. None of the observed proteolysis could be attributed to proteinases released into the incubation medium. Tissue-associated acid-insoluble radioactivity showed a lysosomal distribution on sub-cellular fractionation, and cell-free homogenates of yolk sacs degraded albumin only at acid pH values. Progressively decreasing the rat of pinosome formation (either by progressively lowering the incubation temperature or by the use of increasing concentrations of the metabolic inhibitor rotenone) caused a corresponding decrease in the rate of degradation of albumin. These findings indicate that, in vitro, formaldehyde-denatured 125I-labelled bovine serum albumin is digested by rat yolk sacs exclusively intracellularly, within lysosomes.

Rate of pinocytic capture of macromolecular substrates by rat yolk sac incubated in serum-free culture medium

Pinocytic activity was quantified for rat yolk sacs incubated in a medium that was either serum-free or contained 10% (v/v) of calf serum. Absence of serum from the medium caused a small increase in the rate of pinosome formation, as determined by the rates of capture of both 125I-labelled poly(vinylpyrrolidone) and [14C]sucrose. In contrast, the rates of uptake of substrates ingested by adsorptive pinocytosis were greatly enhanced when serum proteins, which compete for the same binding sites on the plasma membrane as used by adsorbing substrates, were absent. Elimination of such competition greatly simplifies the quantitative analysis of the binding process, and permitted a detailed study of the binding to the plasma membrane of formaldehyde-denatured bovine serum albumin, a protein that is rapidly digested within the lysosomal system after its pinocytic capture. Binding obeyed Michaelis-Menten kinetics and showed a dissociation constant of approx. 1 micron, indicating the high affinity of this protein for binding sites on the surface of actively pinocytosing yolk-sac cells.

Pinocytosis in the rat visceral yolk sac effects of temperature, metabolic inhibitors and some other modifiers

Low temperature, 2,4-dinitrophenol and moniodoacetate could each completely abolish the pinocytic uptake of 125I-labelled polyvinylpyrrolidone, 125I-labelled bovine serum albumin or colloidal 198Au by 17.5-day rat visceral yolk sac cultured in vitro. Cytochalasin B and colchicine caused a partial and dose-dependent inhibition. It is concluded that the mechanism of pinocytic uptake of these substrates is not micropinocytosis as conventionally defined. Removal of extracellular calcium or the oresence of theophylline inhibited liquid-phase pinocytosis by the rat yolk sac, whereas addition of ouabain caused a biphasic response: a slight stimulation of pinosome formation at a low concentration, and an inhibitory effect at a higher concentration.

The effect of chemical treatments of albumin and orosomucoid on rate of clearance from the rat bloodstream and rate of pinocytic capture of rat yolk sac cultured in vitro.

Portions of a 125I-iodinated bovine serum albumin preparation were exposed to freezing, acetic acid (pH 3.5, 3.0 or 2.5), urea or formaldehyde, and the effect of these treatments on the rates of pinocytic uptake by yolk sacs from 17.5-day-pregnant rats cultured in vitro and of clearance from the rat bloodstream were studied. Uptake of albumin by the yolk sac was followed by rapid release of [125I]iodo-L-tyrosine into the culture medium. Similarly clearance of albumin in vivo was accompanied by the appearance of trichloroacetic acid-soluble radioactivity in the bloodstream. In both systems the rates of uptake of modified albumin preparations formed a series: formaldehyde or urea greater than acetic acid greater than freezing. The increased rates of uptake of modified albumin preparations could not be ascribed to the formation of aggregates nor, in the yolk-sac system, to an increase in the rate of pinosome formation. It is concluded that the various treatments to which the albumin was subjected increase to varying degrees the affinity of the albumin molecule for binding sites on that region of the plasma membrane from which pinocytic vesicles are formed. Some comparable experiments with native and desialylated human orosomucoid indicate that the rat yolk-sac epithelial cells do not possess the recognition system for uptake of asialoglycoproteins that exists on the surface of hepatic parenchymal cells.

The regulation of pinocytosis in mouse macrophages. II. Factors inducing vesicle formation.

The pinocytosis-inducing effect of a number of molecular species was studied in cultures of mouse macrophages. Agents were added to a basal medium containing 1% NBCS-No. 199 and allowed to interact with cells for 150 min. Vesicle counts were then performed and compared to control cells in the basal medium. Certain proteins, i.e. albumin and fetuin, with isoelectric points of five and below were found to be potent stimulators of vesicle formation. Basic proteins including lysozyme, histone, and protamine had little influence at sublethal concentrations. The pinocytosis-stimulating activity of bovine plasma albumin could be markedly depressed by removal of bound fatty acids. The addition of either oleic or linoleic acid to de-fatted albumin restored its inducing properties to initial levels. The activity of fetuin could be abolished by either mild acid hydrolysis or neuraminidase digestion. Both procedures removed the majority of the sialic acid content of fetuin. The D and L isomers of polyglutamic acid were found to produce a marked increase in pinosome production. In contrast, poly-DL-lysine was not effective. Neutral and basic amino acids were without significant effect on pinocytosis, whereas aspartic and glutamic acids were stimulatory. The amides of glutamic and aspartic acid did not induce pinocytosis. The unnatural D isomers of glutamic, aspartic, leucine, and phenylalanine inhibited pinocytosis. The inhibition by D-glutamic acid could be reversed with the L isomer. A number of acid mucopolysaccharides, including heparin, hyaluronic acid, and chondroitin sulfate, were excellent inducers. High molecular weight dextran was without significant stimulatory effect whereas dextran sulfate was very active. Both desoxyribonucleic acid and ribonucleic acid enhanced pinosome formation. A number of low molecular weight anions including N-acetylneuraminic acid were found to enhance vesicle formation. In general, anionic molecules were better inducers than either neutral or cationic species. The minimum effective dose of macroanions was a function of molecular weight and their activity appeared unrelated to specific chemical groupings.

The regulation of pinocytosis in mouse macrophages. I. Metabolic requirements as defined by the use of inhibitors.

A method is described to study the formation of pinocytic vesicles in cultivated mouse macrophages. Vesicles which arise in the peripheral cytoplasm and are in transit to the centrosphere region are enumerated by the phase-contrast microscopy of glutaraldehyde-fixed cells. Under these conditions there is a prompt reversible response of vesicle formation to calf serum factors in the external environment. The reduction of pinosome formation by a variety of metabolic inhibitors was then studied in a medium containing 50% newborn calf serum. Inhibitors of both glycolysis and respiration reduced vesicle formation to low levels. This influence was most striking with cyanide, antimycin A, and anaerobiosis. This indicates an important contribution of an intact respiratory pathway in pinocytosis. Both 2,4-dinitrophenol and oligomycin inhibited vesicle formation at low concentrations. These data suggest a central role of ATP as an energy source for vesicle formation. Inhibitors of protein synthesis, such as puromycin and p-fluorophenylalanine, produced a prompt reduction in vesicle formation. The action of p-fluorophenylalanine was effectively reversed with L-phenylalanine. Actinomycin D depressed pinocytosis to low levels at concentrations of 0.1 to 0.003 microg/ml. This effect was observed only after a 120 min lag phase. A 10 degrees C reduction in ambient temperature lowered vesicle counts to 30% of control preparations at 37 degrees C.