Lysosomal enzyme secretory mutants of Dictyostelium discoideum

1990 ◽  
Vol 96 (3) ◽  
pp. 491-500
Author(s):  
D.L. Ebert ◽  
K.B. Jordan ◽  
R.L. Dimond
Keyword(s):  
Dictyostelium Discoideum ◽  
Lysosomal Enzyme ◽  
High Frequency ◽  
Lysosomal Enzymes ◽  
Enzyme Modification ◽  
Regulation Of Secretion ◽  
Low Ionic Strength ◽  
Enzyme Targeting ◽  
Axenic Growth

Dictyostelium discoideum secretes a number of lysosomal enzymes during axenic growth and upon suspension in a low ionic strength, non-nutrient buffer (standard secretion conditions). These secretory characteristics have allowed us to identify 74 lysosomal enzyme secretory mutants generated by N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis. The majority of these mutants fell into one of four classes, on the basis of their secretory characteristics in non-nutrient buffer. The four mutant classes indicate that a minimum of three distinct sets of genes are necessary for proper secretion of lysosomal enzymes from D. discoideum cells under standard secretion conditions: one set of genes that is involved in general lysosomal enzyme secretion, one that is involved in glycosidase type secretion, and a third that is involved in acid phosphatase type secretion. These three classes likely reflect heterogeneity in the intracellular destination of lysosomal enzymes, the secretory mechanism, or both. A fourth set of genes may be necessary for proper secretion during growth, but plays no role under standard secretion conditions. These are likely altered in the regulation of secretion or in lysosomal enzyme targeting. Of the 74 secretory mutants, 36 were also modification mutants resulting in decreased pI, thermolability, or in vivo instability of lysosomal enzyme activities. The high frequency of modification mutants indicates an integral relationship between lysosomal enzyme modification, and lysosomal enzyme targeting and secretion in D. discoideum.

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

1994 ◽  
Vol 126 (2) ◽  
pp. 343-352 ◽  
Author(s):  
T Ruscetti ◽  
J A Cardelli ◽  
M L Niswonger ◽  
T J O'Halloran
Keyword(s):  
Dictyostelium Discoideum ◽  
Heavy Chain ◽  
Lysosomal Enzyme ◽  
Lysosomal Enzymes ◽  
Secretory Pathway ◽  
Chain Gene ◽  
Wild Type ◽  
Clathrin Heavy Chain ◽  
Mutant Cells

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.

Microtubule modulation of biliary excretion of endogenous and exogenous hepatic lysosomal constituents

1984 ◽  
Vol 246 (1) ◽  
pp. G8-G15 ◽  
Author(s):  
R. B. Sewell ◽  
S. S. Barham ◽  
A. R. Zinsmeister ◽  
N. F. LaRusso
Keyword(s):  
Lysosomal Enzyme ◽  
Biliary Excretion ◽  
Lysosomal Enzymes ◽  
Male Rats ◽  
Enzyme Secretion ◽  
Initial Increase ◽  
Acute Administration ◽  
Subsequent Decrease ◽  
Before And After

We tested the hypothesis that hepatocyte microtubules modulate the biliary excretion of endogenous and exogenous constituents of hepatocyte lysosomes. We collected bile via bile fistulas from male rats before and after acute administration of colchicine and vinblastine, agents known to bind to hepatocyte microtubules; rats were then killed and livers were homogenized for biochemical analyses or processed for electron microscopy. Colchicine caused biphasic, parallel alterations in the biliary excretion of three lysosomal enzymes compared with control rats given saline or lumicolchicine; a peak rise in enzyme outputs of approximately 175% at 45-60 min after colchicine administration was followed by a sustained fall to approximately 25% of control values, which persisted for 2-4 h. When hepatocyte lysosomes were prelabeled in vivo by administration of [3H]Triton WR-1339, a nonionic detergent that is sequestered in hepatic lysosomes, the biliary excretion of radiolabel in response to colchicine paralleled the biliary excretion of the three lysosomal enzymes. Vinblastine also induced a biphasic response in biliary lysosomal enzyme output that was similar to that produced by colchicine administration. Morphometric analysis of electron micrographs of rat livers demonstrated changes in the number of lysosomelike vesicles in the vicinity of bile canaliculi after colchicine and vinblastine administration; the initial increase in lysosomal enzyme secretion was associated with a significant decrease in the number of pericanalicular lysosomes after both agents, while the subsequent decrease in enzyme secretion coincided with an increase in the number of pericanalicular lysosomes after vinblastine.(ABSTRACT TRUNCATED AT 250 WORDS)

Dictyostelium Discoideum Mutants Resistant to the Toxic Action of Methylene Diphosphonate are Defective in Endocytosis

1992 ◽  
Vol 101 (1) ◽  
pp. 139-144 ◽  
Author(s):  
MIREILLE BOF ◽  
FRANÇOISE BRÉNOT ◽  
CARLOS GONZALEZ ◽  
GÉRARD KLEIN ◽  
JEAN-BAPTISTE MARTIN ◽  
...  
Keyword(s):  
Dictyostelium Discoideum ◽  
Lysosomal Enzymes ◽  
Fluid Phase ◽  
Screening Strategy ◽  
31P Nmr Spectroscopy ◽  
Lysosomal Hydrolases ◽  
Methylene Diphosphonate ◽  
Endosomal Pathway ◽  
Enzyme Markers

Methylene diphosphonate is taken up in Dictyostelium discoideum amoebae by fluid-phase pinocytosis, and it inhibits growth through the production of methylene analogs of adenosine triphosphate and diadenosine tetraphosphate. Methylene diphosphonate resistance was thus used as the basis of a screening strategy for the isolation of endocytosis mutants. Fifteen Dictyostelium mutants, whose growth was resistant to 7.5 mM methylene diphosphonate, were obtained and three of them were characterized in more detail. They were partially defective in fluid-phase pinocytosis (both the rate and extent of FITC-dextran entry were reduced to 40–50% of the parent type activity) and they had smaller amounts of several lysosomal enzymes, such as acid phosphatase, N-acetylhexosaminidase, α-mannosidase (20–60% of the parent type activities). In contrast to the lysosomal hydrolases, the mutants had unchanged activities for enzyme markers selective for other compartments. They appeared phenotypically similar to the Dictyostelium mutant HMW570, which is defective in fluid-phase pinocytosis and oversecretes lysosomal enzymes. The methylene diphosphonate-resistant mutants were found to be unable to acidify fully their endosomal compartments and they have an increased endosomal pH, as shown by the use of the pH-sensitive fluorescence of FITC-dextran. Furthermore, the hypothesis proposing a defective acidification of the endosomal pathway was supported by the measurement of ATP-dependent vesicular acidification with acridine orange, and by in vivo 31P NMR spectroscopy with aminomethylphosphonate as a pH probe.

scholarly journals A Dictyostelium discoideum mutant that missorts and oversecretes lysosomal enzyme precursors is defective in endocytosis.

1989 ◽  
Vol 109 (4) ◽  
pp. 1445-1456 ◽  
Author(s):  
D L Ebert ◽  
H H Freeze ◽  
J Richardson ◽  
R L Dimond ◽  
J A Cardelli
Keyword(s):  
Dictyostelium Discoideum ◽  
Mutant Strain ◽  
Lysosomal Enzyme ◽  
Fluid Phase ◽  
Wild Type ◽  
Lysosomal Hydrolases ◽  
Chase Period ◽  
Secondary Lysosomes ◽  
Beta Glucosidase ◽  
Enzyme Targeting

A mutant strain of Dictyostelium discoideum, HMW570, oversecretes several lysosomal enzyme activities during growth. Using a radiolabel pulse-chase protocol, we followed the synthesis and secretion of two of these enzymes, alpha-mannosidase and beta-glucosidase. A few hours into the chase period, HMW570 had secreted 95% of its radiolabeled alpha-mannosidase and 86% of its radiolabeled beta-glucosidase as precursor polypeptides compared to the secretion of less than 10% of these forms from wild-type cells. Neither alpha-mannosidase nor beta-glucosidase in HMW570 were ever found in the lysosomal fractions of sucrose gradients consistent with HMW570 being defective in lysosomal enzyme targeting. Also, both alpha-mannosidase and beta-glucosidase precursors in the mutant strain were membrane associated as previously observed for wild-type precursors, indicating membrane association is not sufficient for lysosomal enzyme targeting. Hypersecretion of the alpha-mannosidase precursor by HMW570 was not accompanied by major alterations in N-linked oligosaccharides such as size, charge, and ratio of sulfate and phosphate esters. However, HMW570 was defective in endocytosis. A fluid phase marker, [3H]dextran, accumulated in the mutant at one-half of the rate of wild-type cells and to only one-half the normal concentration. Fractionation of cellular organelles on self-forming Percoll gradients revealed that the majority of the fluid-phase marker resided in compartments in mutant cells with a density characteristic of endosomes. In contrast, in wild-type cells [3H]dextran was predominantly located in vesicles with a density identical to secondary lysosomes. Furthermore, the residual lysosomal enzyme activity in the mutant accumulated in endosomal-like vesicles. Thus, the mutation in HMW570 may be in a gene required for both the generation of dense secondary lysosomes and the sorting of lysosomal hydrolases.

Vanadate, an inhibitor of growth, development and endocytosis in Dictyostelium discoideum amoebae

1989 ◽  
Vol 94 (1) ◽  
pp. 127-134
Author(s):  
G.É. KLEIN ◽  
DAVID A. COTTER ◽  
JEAN-BAPTISTE MARTIN ◽  
MICHEL SATRE
Keyword(s):  
Dictyostelium Discoideum ◽  
Lysosomal Enzymes ◽  
Fluid Phase ◽  
Nucleoside Triphosphate ◽  
Cytosolic Ph ◽  
Developmental Program ◽  
Slime Mold Dictyostelium Discoideum ◽  
Vanadate Inhibition ◽  
Massive Cell ◽  
Axenic Growth

Axenic growth of amoebae of the slime mold Dictyostelium discoideum was found to be reversibly inhibited by vanadate. Pinocytosis, when measured with fluorescein-labeled dextran as a fluorescent fluid-phase marker was strongly inhibited by vanadate. Inhibition was observable at vanadate concentrations as low as 0*2 mM. Sucrose entry through pinocytosis induced massive cell vacuolation and this effect was blocked by vanadate. Secretion of soluble lysosomal enzymes is another aspect of membrane traffic in Dictyostelium. Secretion of two typical lysosomal enzymes, acid phosphatase and hexosaminidase, was inhibited by concentrations of vanadate in the same range as for pinocytosis inhibition. Vanadate also prevented the morphogenetic developmental program that follows nutrient starvation. In contrast, vanadate did not prevent heat-induced spore germination. Vanadate had no significant action on the intracellular nucleoside triphosphate level or on the cytosolic pH. It is suggested that the particular effect of vanadate in Dictyostelium is to inhibit the fusion of endosomes with lysosomes. Our results provide a probe that could be useful to clarify the mechanisms of endocytosis.

scholarly journals The isolation and characterization of lysosomal particles from myxamoebae of the cellular slime mould Dictyostelium discoideum

1970 ◽  
Vol 118 (3) ◽  
pp. 505-512 ◽  
Author(s):  
Edith Wiener ◽  
J. M. Ashworth
Keyword(s):  
Cell Differentiation ◽  
Dictyostelium Discoideum ◽  
Lysosomal Enzyme ◽  
Lysosomal Enzymes ◽  
Cellular Slime Mould ◽  
Slime Mould ◽  
Isolation And Characterization ◽  
Cellular Slime ◽  
Specific Activities

1. The myxamoebae of the cellular slime mould Dictyostelium discoideum possess several typically lysosomal enzyme activities. 2. These enzymes are present in the cell in association and in a lysosome-like particle. 3. The lysosomes of myxamoebae grown axenically have a different enzymic composition and a different density from those grown on bacteria. 4. During cell differentiation the specific activities of the lysosomal enzymes change. 5. It is suggested that both during growth and differentiation the amounts of lysosomal enzymes present in the cell are regulated.

Release of β-glucuronidase from peritoneal macrophages of normal and endotoxin-tolerant mice

1979 ◽  
Vol 25 (11) ◽  
pp. 1245-1251 ◽  
Author(s):  
Stephen L. Snyder ◽  
Sharyn K. Eklund ◽  
Richard I. Walker
Keyword(s):  
Lysosomal Enzyme ◽  
Lysosomal Enzymes ◽  
Lethal Dose ◽  
In Vitro Release ◽  
Peritoneal Macrophages ◽  
Endotoxin Shock ◽  
Enzyme Release ◽  
Endotoxin Challenge

Lysosomal enzyme release from cells involved in inflammatory response could play a central role in the pathogenesis of endotoxin shock. Therefore we have studied the release of the lysosomal enzyme, β-glucuronidase, from peritoneal macrophages obtained from normal and endotoxin-tolerant B6CBF1 mice both before and after challenge with lethal doses of endotoxin. Unstimulated cells from tolerant mice spontaneously released a smaller percentage of their total β-glucuronidase content in culture than cells from normal mice during a 5-h incubation period. In support of the lysosomal enzyme release hypothesis, it was found that the in vitro release of β-glucuronidase was accelerated when cells were collected from the mouse peritoneum 3 h after i.v. challenge with a lethal dose (1.0 mg) of endotoxin. The increased in vitro "leakiness" of peritoneal macrophages following endotoxin challenge was less marked when tolerance was induced in mice by prior repeated injections of endotoxin. Furthermore, measurements of the total enzyme activities of peritoneal cells revealed a significant reduction in the β-glucuronidase content of cells from normal mice 3 h after endotoxin challenge but no such decrease for cells from tolerant mice. These results suggest that macrophages in endotoxin-sensitive mice release their lysosomal enzymes in vivo during endotoxemia, whereas cells found in tolerant mice do not.In related experiments, the phagocytosis of latex particles and inhibition of bacterial growth by macrophages from normal and tolerant mice were compared. These studies suggest that cells from tolerant mice may also release a smaller percentage of their lysosomal enzymes during phagocytosis.

scholarly journals Mouse mutants lacking the cation-independent mannose 6-phosphate/insulin-like growth factor II receptor are impaired in lysosomal enzyme transport: comparison of cation-independent and cation-dependent mannose 6-phosphate receptor-deficient mice

1998 ◽  
Vol 330 (2) ◽  
pp. 903-908 ◽  
Author(s):  
Istvan SOHAR ◽  
David SLEAT ◽  
Chang-Gong LIU ◽  
Thomas LUDWIG ◽  
Peter LOBEL
Keyword(s):  
Growth Factor ◽  
Lysosomal Enzyme ◽  
Enzyme Activities ◽  
Lysosomal Enzymes ◽  
Insulin Like Growth Factor ◽  
Factor Ii ◽  
Solid Tissues ◽  
Mannose 6 Phosphate ◽  
Deficient Mice

Two proteins have been implicated in the mannose 6-phosphate-dependent transport of lysosomal enzymes to lysosomes: the 300 kDa cation-independent and the 46 kDa cation-dependent mannose 6-phosphate receptors (CI- and CD-MPRs). The mammalian CI-MPR also mediates endocytosis and clearance of insulin-like growth factor II (IGF-II). Mutant mice that lack the CD-MPR are viable, mice that lack the CI-MPR accumulate high levels of IGF-II and usually die perinatally, whereas mice that lack both IGF-II and CI-MPR are viable. To investigate the relative roles of the MPRs in the targeting of lysosomal enzymes in vivo, we analysed the effect of a deficiency of either MPR on lysosomal enzyme activities in animals lacking IGF-II. In CD-MPR-deficient mice, most activities were relatively normal in solid tissues and some were marginally elevated in serum. In CI-MPR-deficient mice, some enzyme activities were moderately decreased in solid tissues and multiple enzymes were markedly elevated in serum. Finally, total levels of serum mannose 6-phosphorylated glycoproteins were ~ 45-fold and ~ 15-fold higher than wild type in CI- and CD-MPR-deficient mice respectively, and there were specific differences in the pattern of these proteins when comparing CI- and CD-MPR deficient animals. These results indicate that while lack of the CI-MPR appears to perturb lysosome function to a greater degree than lack of the CD-MPR, each MPR has distinct functions for the targeting of lysosomal enzymes in vivo.

Аpplying of high-frequency monopolar diathermocoagulation in the treatment of chronic pulpitis

2020 ◽  
Vol 1 (12) ◽  
pp. 40-42
Author(s):  
F. Yu. Daurova ◽  
D. I. Tomaeva ◽  
S. V. Podkopaeva ◽  
Yu. A. Taptun
Keyword(s):  
Root Canal ◽  
High Frequency ◽  
Antibacterial Effect ◽  
Comparison Group ◽  
Poor Quality ◽  
Endodontic Treatment ◽  
Root Canals ◽  
The Third ◽  
Before And After

Relevance: the reason for the development of complications in endodontic treatment is poor-quality instrumental treatment root canals.Aims: a study of the animicrobial action and clinical efficacy of high-frequency monopolar diathermocoagulation in the treatment of chronic forms of pulpitis.Materials and methods: 102 patients with various chronic forms of pulpitis were divided into three groups of 34 patients each. In the first two groups, high-frequency monopolar diathermocoagulation was used in endodontic treatment in different modes. In the third group, endodontic treatment was carried out without the use of diathermocoagulation (comparison group). The root canal microflora in chronic pulpitis in vivo was studied twice-before and after diathermocoagulation.Results: it was established that high-frequency monopolar diathermocoagulation in the effect mode is 3, power is 4 (4.1 W) and effect is 4, power is 4 (5.4 W) with an exposure time of 3 seconds, it has a pronounced antibacterial effect on all presented pathogenic microflora obtained from the root canals of the teeth.

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