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.

Kinetics of endosomal pH evolution in Dictyostelium discoideum amoebae. Study by fluorescence spectroscopy

1993 ◽  
Vol 105 (3) ◽  
pp. 861-866 ◽  
Author(s):  
L. Aubry ◽  
G. Klein ◽  
J.L. Martiel ◽  
M. Satre
Keyword(s):  
Dictyostelium Discoideum ◽  
Fluid Phase ◽  
Lag Phase ◽  
Nutritive Medium ◽  
Chase Period ◽  
Endosomal Acidification ◽  
Fluid Phase Endocytosis ◽  
Endosomal Ph ◽  
Efflux Kinetics ◽  
Secondary Lysosomes

The evolution of endo-lysosomal pH in Dictyostelium discoideum amoebae was examined during fluid-phase endocytosis. Pulse-chase experiments were conducted in nutritive medium or in non-nutritive medium using fluorescein labelled dextran (FITC-dextran) as fluid-phase marker and pH probe. In both conditions, efflux kinetics were characterized by an extended lag phase lasting for 45–60 min and corresponding to intracellular transit of FITC-dextran cohort. During the chase period, endosomal pH decreased during approximately 20 min from extracellular pH down to pH 4.6-5.0, then, it increased within the next 20–40 min to reach pH 6.0-6.2. It was only at this stage that FITC-dextran was released back into the medium with pseudo first-order kinetics. A vacuolar H(+)-ATPase is involved in endosomal acidification as the acidification process was markedly reduced in mutant strain HGR8, partially defective in vacuolar H(+)-ATPase and in parent type strain AX2 by bafilomycin A1, a selective inhibitor of this enzyme. Our data suggest that endocytic cargo is channeled from endosomes to secondary lysosomes that are actively linked to the plasma membrane via recycling vesicles.

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.

scholarly journals Evidence for a recycling role for Rab7 in regulating a late step in endocytosis and in retention of lysosomal enzymes in Dictyostelium discoideum.

1997 ◽  
Vol 8 (7) ◽  
pp. 1343-1360 ◽  
Author(s):  
G Buczynski ◽  
J Bush ◽  
L Zhang ◽  
J Rodriguez-Paris ◽  
J Cardelli
Keyword(s):  
Dictyostelium Discoideum ◽  
Cell Lines ◽  
Lysosomal Enzymes ◽  
Amino Acid Level ◽  
Active Form ◽  
Fluid Phase ◽  
Retrograde Transport ◽  
Dominant Negative ◽  
Lysosomal Hydrolases ◽  
Small Molecular Weight

The mammalian small molecular weight GTPase Rab7 (Ypt7 in yeast) has been implicated in regulating membrane traffic at postinternalization steps along the endosomal pathway. A cDNA encoding a protein 85% identical at the amino acid level to mammalian Rab7 has been cloned from Dictyostelium discoideum. Subcellular fractionation and immunofluorescence microscopy indicated that Rab7 was enriched in lysosomes, postlysosomes, and maturing phagosomes. Cell lines were generated that overexposed Rab7 wild-type (WT), Rab7 Q67L (constitutively active form), and Rab7 T22N (dominant negative form) proteins. The Rab7 T22N cell line internalized fluid phase markers and latex beads (phagocytosis) at one-third the rate of control cells, whereas Rab7 WT and Rab7 Q67L cell lines were normal in uptake rates but exocytosed fluid phase faster than control cells. In contrast, fluid phase markers resided in acidic compartments for longer periods of time and were more slowly exocytosed from Rab7 T22N cells as compared with control cells. Light microscopy indicated that Rab7-expressing cell lines contained morphologically altered endosomal compartments. Compared with control cells, Rab7 WT- and Rab7 Q67L-expressing cells contained a reduced number of vesicles, the size of postlysosomes (> 2.5 microns) and an increased number of smaller vesicles, many of which were nonacidic; in control cells, > 90% of the smaller vesicles were acidic. In contrast, Rab7 T22N cells contained an increased proportion of large acidic vesicles relative to nonacidic vesicles. Radiolabel pulse-chase experiments indicated that all of the cell lines processed and targeted lysosomal alpha-mannosidase normally, indicating the lack of a significant role for Rab7 in the targeting pathway; however, retention of mature lysosomal hydrolases was affected in Rab7 WT and Rab7 T22N cell lines. Contrary to the results observed for the fluid phase efflux experiments, Rab7 T22N cells oversecreted alpha-mannosidase, whereas Rab7 WT cells retained this hydrolase as compared with control cells. These data support a model that Rab7 may regulate retrograde transport of lysosomal enzymes and the V-type H(+)-ATPase from postlysosomes to lysosomes coupled with the efficient release of fluid phase from cells.

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 Lysosomal enzymes in Dictyostelium discoideum are transported to lysosomes at distinctly different rates.

1986 ◽  
Vol 102 (4) ◽  
pp. 1264-1270 ◽  
Author(s):  
J A Cardelli ◽  
G S Golumbeski ◽  
R L Dimond
Keyword(s):  
Dictyostelium Discoideum ◽  
Golgi Complex ◽  
Lysosomal Enzymes ◽  
Time Course ◽  
Molecular Mechanisms ◽  
Subcellular Fractionation ◽  
Enzyme Digestion ◽  
Transport Pathway ◽  
Chase Period ◽  
Beta Glucosidase

We are investigating the molecular mechanisms involved in the localization of lysosomal enzymes in Dictyostelium discoideum, an organism that lacks any detectable mannose-6-phosphate receptors. The lysosomal enzymes alpha-mannosidase and beta-glucosidase are both initially synthesized as precursor polypeptides that are proteolytically processed to mature forms and deposited in lysosomes. Time course experiments revealed that 20 min into the chase period, the pulse-labeled alpha-mannosidase precursor (140 kD) begins to be processed, and 35 min into the chase 50% of the polypeptides are cleaved to mature 60 and 58-kD forms. In contrast, the pulse-labeled beta-glucosidase precursor (105 kD) begins to be processed 10 min into the chase period, and by 30 min of the chase all of the precursor has been converted into mature 100-kD subunits. Between 5 and 10% of both precursors escape processing and are rapidly secreted from cells. Endoglycosidase H treatment of immunopurified radioactively labeled alpha-mannosidase and beta-glucosidase precursor polypeptides demonstrated that the beta-glucosidase precursor becomes resistant to enzyme digestion 10 min sooner than the alpha-mannosidase precursor. Moreover, subcellular fractionation studies have revealed that 70-75% of the pulse-labeled beta-glucosidase molecules move from the rough endoplasmic reticulum (RER) to the Golgi complex less than 10 min into the chase. In contrast, 20 min of chase are required before 50% of the pulse-labeled alpha-mannosidase precursor exits the RER. The beta-glucosidase and alpha-mannosidase precursor polypeptides are both membrane associated along the entire transport pathway. After proteolytic cleavage, the mature forms of both enzymes are released into the lumen of lysosomes. These results suggest that beta-glucosidase is transported from the RER to the Golgi complex and ultimately lysosomes at a distinctly faster rate than the alpha-mannosidase precursor. Thus, our results are consistent with the presence of a receptor that recognizes the beta-glucosidase precursor more readily than the alpha-mannosidase precursor and therefore more quickly directs these polypeptides to the Golgi complex.

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 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

1997 ◽  
Vol 136 (6) ◽  
pp. 1271-1286 ◽  
Author(s):  
Greg Buczynski ◽  
Bryon Grove ◽  
Anson Nomura ◽  
Maurice Kleve ◽  
John Bush ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Dictyostelium Discoideum ◽  
Mutant Strain ◽  
Fluorescent Microscopy ◽  
Membrane Traffic ◽  
Fluid Phase ◽  
Cytoskeleton Organization ◽  
Actin Cytoskeleton Organization ◽  
Endosomal Pathway ◽  
Mutant Cells

Phosphatidylinositide 3-kinases (PI 3-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 (Δddpik1/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, Δddpik1/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 α-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 Δddpik1/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, Δddpik1/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.

scholarly journals The Monomeric Clathrin Assembly Protein, AP180, Regulates Contractile Vacuole Size in Dictyostelium discoideum

2006 ◽  
Vol 17 (12) ◽  
pp. 5381-5389 ◽  
Author(s):  
Irene Stavrou ◽  
Theresa J. O'Halloran
Keyword(s):  
Plasma Membrane ◽  
Dictyostelium Discoideum ◽  
Mutant Strain ◽  
Adaptor Protein ◽  
Contractile Vacuole ◽  
Unique Contribution ◽  
Wild Type ◽  
Unique Role ◽  
Contractile Vacuoles ◽  
Assembly Proteins

AP180, one of many assembly proteins and adaptors for clathrin, stimulates the assembly of clathrin lattices on membranes, but its unique contribution to clathrin function remains elusive. In this study we identified the Dictyostelium discoideum ortholog of the adaptor protein AP180 and characterized a mutant strain carrying a deletion in this gene. Imaging GFP-labeled AP180 showed that it localized to punctae at the plasma membrane, the contractile vacuole, and the cytoplasm and associated with clathrin. AP180 null cells did not display defects characteristic of clathrin mutants and continued to localize clathrin punctae on their plasma membrane and within the cytoplasm. However, like clathrin mutants, AP180 mutants, were osmosensitive. When immersed in water, AP180 null cells formed abnormally large contractile vacuoles. Furthermore, the cycle of expansion and contraction for contractile vacuoles in AP80 null cells was twice as long as that of wild-type cells. Taken together, our results suggest that AP180 plays a unique role as a regulator of contractile vacuole morphology and activity in Dictyostelium.

scholarly journals The Chymotrypsin-Like Protease Complex of Treponema denticola ATCC 35405 Mediates Fibrinogen Adherence and Degradation

2007 ◽  
Vol 75 (9) ◽  
pp. 4364-4372 ◽  
Author(s):  
Caroline V. Bamford ◽  
J. Christopher Fenno ◽  
Howard F. Jenkinson ◽  
David Dymock
Keyword(s):  
Cell Surface ◽  
Mutant Strain ◽  
Surface Protein ◽  
Fluid Phase ◽  
Treponema Denticola ◽  
Wild Type ◽  
Chromatography Analysis ◽  
Fibrinogen Binding ◽  
In The Wild ◽  
Major Surface

ABSTRACT Treponema denticola is an anaerobic spirochete strongly associated with human periodontal disease. T. denticola bacteria interact with a range of host tissue proteins, including fibronectin, laminin, and fibrinogen. The latter localizes in the extracellular matrix where tissue damage has occurred, and interactions with fibrinogen may play a key role in T. denticola colonization of the damaged sites. T. denticola ATCC 35405 showed saturable binding of fluid-phase fibrinogen to the cell surface and saturable adherence to immobilized fibrinogen. Levels of fibrinogen binding were enhanced in the presence of the serine protease inhibitor phenylmethylsulfonyl fluoride. The Aα and Bβ chains of fibrinogen, but not the γ chains, were specifically recognized by T. denticola. Following fibrinogen affinity chromatography analysis of cell surface extracts, a major fibrinogen-binding component (polypeptide molecular mass, ∼100 kDa), which also degraded fibrinogen, was purified. Upon heating at 100°C, the polypeptide was dissociated into three components (apparent molecular masses, 80, 48, and 45 kDa) that did not individually bind or degrade fibrinogen. The native 100-kDa polypeptide complex was identified as chymotrypsin-like protease (CTLP), or dentilisin. In an isogenic CTLP− mutant strain, CKE, chymotrypsin-like activity was reduced >90% compared to that in the wild type and fibrinogen binding and hydrolysis were ablated. Isogenic mutant strain MHE, deficient in the production of Msp (major surface protein), showed levels of CTLP reduced 40% relative to those in the wild type and exhibited correspondingly reduced levels of fibrinogen binding and proteolysis. Thrombin clotting times in the presence of wild-type T. denticola cells, but not strain CKE (CTLP−) cells, were extended. These results suggest that interactions of T. denticola with fibrinogen, which may promote colonization and modulate hemostasis, are mediated principally by CTLP.

Sign in / Sign up

Export Citation Format

Share Document