Membrane cholesterol removal changes mechanical properties of cells and induces secretion of a specific pool of lysosomes.

In a previous study we had shown that membrane cholesterol removal induced unregulated lysosomal exocytosis events leading to the depletion of lysosomes located at cell periphery. However, the mechanism by which cholesterol triggered these exocytic events had not been uncovered. In this study we investigated the importance of cholesterol in controlling mechanical properties of cells and its connection with lysosomal exocytosis. Tether extraction with optical tweezers and defocusing microscopy were used to assess cell dynamics in mouse fibroblasts. These assays showed that bending modulus and surface tension increased when cholesterol was extracted from fibroblasts plasma membrane upon incubation with MßCD, and that the membrane-cytoskeleton relaxation time increased at the beginning of MßCD treatment and decreased at the end. We also showed for the first time that the amplitude of membrane-cytoskeleton fluctuation decreased during cholesterol sequestration, showing that these cells become stiffer. These changes in membrane dynamics involved not only rearrangement of the actin cytoskeleton, but also de novo actin polymerization and stress fiber formation through Rho activation. We found that these mechanical changes observed after cholesterol sequestration were involved in triggering lysosomal exocytosis. Exocytosis occurred even in the absence of the lysosomal calcium sensor synaptotagmin VII, and was associated with actin polymerization induced by MßCD. Notably, exocytosis triggered by cholesterol removal led to the secretion of a unique population of lysosomes, different from the pool mobilized by actin depolymerizing drugs such as Latrunculin-A. These data support the existence of at least two different pools of lysosomes with different exocytosis dynamics, one of which is directly mobilized for plasma membrane fusion after cholesterol removal.

Unifying nomenclature for the isoforms of the lysosomal membrane protein LAMP-2.

The present nomenclature of the splice variants of the lysosome-associated membrane protein type 2 (LAMP-2) is confusing. The LAMP-2a isoform is uniformly named in human, chicken, and mouse, but the LAMP-2b and LAMP-2c isoforms are switched in human as compared with mouse and chicken. We propose to change the nomenclature of the chicken and mouse b and c isoforms to agree with that currently used for the human isoforms. To avoid confusion in the literature, we further propose to adopt the use of capital letters for the updated nomenclature of all the isoforms in all three species: LAMP-2A, LAMP-2B, and LAMP-2C.

Plant lysozymes.

Structural and functional features of plant lysozymes are reviewed. All lysozymes also have chitinase activity, but not all plant chitinases are also lysozymes. However, for many chitinases it is not yet known if they also possess lysozyme activity. Enzymes with lysozyme activity occur in different, structurally unrelated, families of chitinases. Plant chitinases with lysozyme activity are basic enzymes with high isoionic points. Their lysozyme activities have a shart pH optimum around pH 4.5-5.0, while they show chitinase activities in a much broader pH range. High lysozyme activities are observed at low ionic strength values (0.05). The X-ray structure of a lysozyme/chitinase from latex of the rubber tree, Hevea brasiliensis, is presented. This enzyme is also known under the name hevamine. It belongs to the family 18 or h-type chitinases (also called class III chitinases). The structure consists of an alpha/beta barrel fold, which has not been found in other chitinase or lysozyme structures. A glutamic acid residue may be catalytically active in the substrate-binding cleft of the enzyme. Other plant lysozymes are homologous with the family 19 or b-type chitinases (class I, II and IV). The X-ray structure of barley chitinase, a representative of this family with negligible lysozyme activity, has a similar folding as found in animal and phage lysozymes.

Heterogeneity of lysosomes in human fibroblasts.

Lysosomes are defined traditionally with the marker enzyme acid phosphatase. We showed recently that lysosomes from human fibroblasts can be separated into a light and dense fraction as well as prelysosomal population. We now provide evidence that although acid phosphatase is enriched in all three fractions, the marker enzyme in the prelysosomal compartment is qualitatively distinct from that of the lysosomes. Ultrastructural analysis showed that the acid phosphatase in the prelysosomal vesicles deposited an extremely electron-dense reaction product, entirely obliterating the lumen of the vesicle, in contrast to that of the light and dense lysosomes which deposited a fine and diffuse product scattered throughout the luminal space. Biochemical analysis showed that only 51% of the acid phosphatase in the prelysosomes was inhibited by tartrate, while 80% of that in the lysosomes was tartrate-inhibitable. Immunoprecipitation with antibodies specific for various isozymes of acid phosphatase showed that 39% of the acid phosphatase in the prelysosomes was of the 'lysosomal' type whereas over 50% of the acid phosphatase in the lysosomes was of this type. These results showed that acid phosphatase in the prelysosomes of human cultured fibroblasts can be distinguished from that of the lysosomes cytochemically, biochemically, and immunologically and that lysosomes, as marked by acid phosphatase, are a heterogeneous organelle.

Isolation and characterization of crinosomes--a subclass of secondary lysosomes.

A technique is presented for isolating a subclass of lysosomes, designated crinosomes, by one-step floating-up centrifugation in a cesium-containing sucrose gradient. Rats were treated with vinblastine in order to induce the formation of crinosomes. Vinblastine blocked exocytosis of secretory granules at the cell border. Later on, lipoprotein-containing granules were seen throughout the cytoplasm. Immunolabeling with polyclonal antibodies against albumin demonstrated a severalfold greater presence of gold particles over crinosomes and Golgi cisternae than over the surrounding organelles. The induction of crinosomes by vinblastine made it possible to isolate thenm on a sucrose gradient. These organelles contained marker enzymes for the Golgi complex (galactosyl transferase) and lysosomes (cathepsins). The purity of the fraction was high. The crinosomes were proteolytically and lipolytically very active. The crinosomes were positive to cytochemical acid phosphatase staining. It is concluded that crinosomes develop from fusion between lysosomes and secretory granules and that they are active in degrading retained secretory material.

Characterization of argentosomes, a type of secondary lysosomes, and the sub-organellar distribution of a lysosomotropic basic compound.

Secondary lysosomes (argentosomes) were isolated by centrifugation in a discontinuous sucrose density gradient from livers of rats administered colloidal silver. Compared to the crude homogenate, the purities of the argentosome preparations were 17.4- and 18.5-fold in terms of acid phosphatase and N-acetyl-beta-glucosaminidase activities, respectively. By lipid analysis, the argentosomes were shown to have intermediate properties between normal lysosomes and tritosomes with regard to the contents of triglyceride and cholesterol. The phospholipid content in the argentosomes was also different from that in these two organelles. The cross-point of argentosomes shifted more to the acidic side than that of normal lysosomes. The data on the binding of tritiated p- biphenylmethyl-(dl-tropyl-alpha-tropiniun)bromide [( 3H]BTTB) to argentosomes indicated that the degree of binding and/or incorporation of this basic compound to the organelles was much higher than that to normal lysosomes. These results suggested that the distribution of BTTB on or within argentosomes might be under the control of the surface charge of the argentosomal membranes.

Identification of two subpopulations of thyroid lysosomes: relation to the thyroglobulin proteolytic pathway.

Using a combination of differential centrifugation and isopycnic centrifugation in Percoll gradients, we obtained a highly purified preparation of thyroid lysosomes [Alquier, Guenin, Munari-Silem, Audebet & Rousset (1985) Biochem. J. 232, 529-537] in which we identified thyroglobulin. From this observation, we postulated that the isolated lysosome population could be composed of primary lysosomes and of secondary lysosomes resulting from the fusion of lysosomes with thyroglobulin-containing vesicles. In the present study, we have tried to characterize these lysosome populations by (a) subfractionation of purified lysosomes using iterative centrifugation on Percoll gradients and (b) by functional studies on cultured thyroid cells. Thyroglobulin analysed by soluble phase radioimmunoassay, Western blotting or immunoprecipitation was used as a marker of secondary lysosomes. The total lysosome population separated from other cell organelles on a first gradient was centrifuged on a second Percoll gradient. Resedimented lysosomes were recovered as a slightly asymmetrical peak under which the distribution patterns of acid hydrolase activities and immunoreactive thyroglobulin did not superimpose. This lysosomal material (L) was separated into two fractions: a light (thyroglobulin-enriched) fraction (L2) and a dense fraction (L1). L1 and L2 subfractions centrifuged on a third series of Percoll gradients were recovered as symmetrical peaks at buoyant densities of 1.12-1.13 and 1.08 g/ml, respectively. In each case, protein and acid hydrolase activities were superimposable. The specific activity of acid phosphatase was slightly lower in L2 than in L1. In contrast, the immunoassayable thyroglobulin content of L2 was about 4-fold higher than that of L1. The overall polypeptide composition of L, L1 and L2 analysed by polyacrylamide-gel electrophoresis was very similar, except for thyroglobulin which was more abundant in L2 than in either L or L1. The functional relationship between L1 and L2 lysosome subpopulations has been studied in cultured thyroid cells reassociated into follicles. Thyroid cells, prelabelled with 125I-iodide to generate 125I-thyroglobulin, were incubated in the absence of in the presence of inhibitors of intralysosomal proteolysis. The fate of 125I-thyroglobulin, and especially its appearance in the lysosomal compartment, was studied by Percoll gradient fractionation and immunoprecipitation. Treatment of prelabelled thyroid cells with chloroquine and leupeptin induced the accumulation of immunoprecipitable 125I-thyroglobulin into a lysosome fraction corresponding to the L2 subpopulation. In control cells, in which intralysosomal proteolysis was n

Effect of ammonium chloride on subcellular distribution of lysosomal enzymes in human fibroblasts.

Three subcellular fractions enriched in lysosomal enzyme activities have been isolated recently from human cultured fibroblasts with Percoll gradients: the dense lysosomes (DL), light lysosomes (LL), and light membranous vesicles (LM). They were shown to have different morphological, cytochemical, biochemical, and immunological properties. We now report on the dramatic but different effects of a primary amine, NH4Cl, on these subfractions. The lysosomes, as detected with a specific ultrastructural cytochemical stain for the lysosomal enzyme, arylsulfatase A, were swollen significantly in all these fractions, increasing their volumes by 64% (DL), 53% (LL), and 95% (LM), respectively. When arylsulfatase A enzyme activity was monitored, about half of the DL content was diverted to the LL. However, when newly synthesized arylsulfatase A enzyme protein was monitored with metabolic labeling and immunoprecipitation, about 80% of the enzyme protein was depleted from both the DL and LL. In contrast, neither the enzyme activity nor the newly synthesized enzyme protein of arylsulfatase A was greatly altered in the LM fraction by the treatment. Since primary amines caused newly synthesized lysosomal enzymes to diverge from the lysosomal route to a secretory pathway, it was deduced that (i) the LM fraction corresponded to a prelysosomal compartment whose lysosomal enzyme content was not affected by the amine and was thus proximal to the point of diversion between the secretory and lysosomal pathways; (ii) the LL and DL fractions were distal to the point of diversion since both fractions were depleted of their newly synthesized lysosomal enzyme; and (iii) the sorting of newly synthesized lysosomal enzyme may be different from that of the preexisting pool of the same enzyme since the LL fraction was depleted of its newly synthesized enzyme protein while accumulating excessive enzyme activity.

Intracellular transport of asialoglycoproteins in rat hepatocytes. Evidence for two subpopulations of lysosomes.

The intracellular transport and degradation of asialoorosomucoid (AOM) in isolated rat hepatocytes was studied by means of subcellular fractionation in Nycodenz gradients. The asialoglycoprotein was labelled by covalent attachment of a radioiodinated tyramine-cellobiose adduct ( [125I]TC) which leads to labelled degradation products being trapped intracellularly and thus serving as markers for the degradative organelles. The ligand was initially (1 min) in a slowly sedimenting (small) vesicle and subsequently in larger endosomes. Acid-soluble, radioactive degradation products were first found in a relatively light lysosome whose distribution coincided in the gradient with that of the larger endosome. Later (30 min) degradation products were found in denser lysosomes which banded in the same region of the gradient as the lysosomal enzyme, beta-acetylglucosaminidase. Colchicine, monensin and leupeptin all inhibited degradation of [125I]tyramine-cellobiose asialoorosomucoid ( [125I]TC-AOM) and reduced the formation of degradation products in both the light and the dense lysosomes. In presence of monensin and colchicine no undegraded ligand was seen in the dense lysosome, suggesting that uptake in these vesicles was inhibited. Leupeptin allowed accumulation of undegraded ligand in the dense lysosome. Therefore, transfer from light to dense lysosomes is not dependent on degradation as such. In the presence of monensin two peaks of undegraded ligand were found in the gradients. It seems possible that in the monensin-sensitive endosomes, dissociation of the ligand-receptor complex is inhibited, allowing ligand to recycle with the receptors in small vesicles.

Presence of monocytes in systemic lupus erythematosus-associated glomerulonephritis: marker study and significance.

The presence and significance of monocytes in the glomerular lesions of 13 renal specimens obtained from ten patients with systemic lupus erythematosus-associated nephritis were studied. The monocytes were identified by the presence of intracytoplasmic lysozyme, as demonstrated by immunoperoxidase staining on paraffin-embedded tissue. Eight specimens obtained from six patients displayed monocytes. These were found most often associated with mesangial hypercellularity, and their appearance fluctuated with it. There was a positive correlation between the number of lysozyme-positive cells and the amount of proteinuria. We conclude that monocytes contribute to the endocapillary hypercellularity and affect protein filtration in lupus nephritis.

The resolution of two populations of lysosomal organelles containing endocytosed Wistaria floribunda agglutinin from murine fibroblasts.

Subcellular fractionation of Balb/c 3T3 fibroblasts exposed to Wistaria floribunda agglutinin was performed to localize fractions containing internalized lectin. Employing two sequential self-generating silica sol density gradients, the postnuclear supernatant of cell homogenates was resolved into five distinct cellular components. Lysosome enzyme activities were displayed by two populations of vesicles, each separated from plasma membrane, golgi, and mitochondria markers. The more dense of these fractions exhibited morphological and biochemical properties ascribed to secondary lysosomes. The more buoyant population was similar to that reported by Rome et al [11] who noted that it may be a product of vesiculated golgi endoplasmic reticulum lysosome (GERL). Treatment with W floribunda agglutinin of cells, surface radioiodinated demonstrated that plasma membrane proteins were localized within both the buoyant and dense lysosome populations in as little as 10 min after exposure to lectin. Prolonged incubation of the cells with W floribunda agglutinin resulted in maintenance of this distribution. However, when nonradiactive cells were exposed to 125I-labeled W floribunda agglutinin for 10 min, radioactivity was detected only in the buoyant population of lysosomes as well as the plasma membrane/golgi fraction. Treatment of cells with W floribunda agglutinin for 30 min resulted in appearance of lectin associated radioactivity in the dense lysosome fraction in addition to those populations containing radioactivity seen after a 10-min incubation. These data indicate that the endocytosis of W floribunda agglutinin differed substantially from the internalization of a portion of the plasma membrane proteins. Furthermore, we found radioactivity associated with both plasma membrane proteins W floribunda agglutinin in regions of the density gradient fractionation that virtually lacked golgi and lysosome markers. These fractions may have represented populations of nonlysosome vesicles formed during the process of endocytosis.