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.

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 Rab11-like GTPase associates with and regulates the structure and function of the contractile vacuole system in Dictyostelium

2001 ◽  
Vol 114 (16) ◽  
pp. 3035-3045 ◽  
Author(s):  
Edward Harris ◽  
Kunito Yoshida ◽  
James Cardelli ◽  
John Bush
Keyword(s):  
Proton Pump ◽  
Membrane Trafficking ◽  
Amino Acid Level ◽  
Fluid Phase ◽  
Membrane System ◽  
Structure And Function ◽  
Dominant Negative ◽  
Contractile Vacuole ◽  
And Function ◽  
Mutant Cells

Screening of a cDNA library revealed the existence of a Dictyostelium cDNA encoding a protein 80% identical at the amino acid level to mammalian Rab11. Subcellular fractionation and immunofluorescence studies revealed that DdRab11 was exclusively associated with the ATPase proton pump-rich contractile vacuole membrane system, consisting of a reticular network and bladder-like vacuoles. Video microscopy of cells expressing GFP-DdRab11 revealed that this Rab was associated with contractile vacuolar bladders undergoing formation, fusion and expulsion of water. The association of DdRab11 with contractile vacuole membranes was disrupted when cells were exposed to either hypo-osmotic conditions or an inhibitor of the ATPase proton pump. Cells that overexpressed a dominant negative form of DdRab11 were analyzed biochemically and microscopically to measure changes in the structure and function of the contractile vacuole system. Compared with wild-type cells, the dominant negative DdRab11-expressing cells contained a more extensive contractile vacuole network and abnormally enlarged contractile vacuole bladders, most likely the result of defects in membrane trafficking. In addition, the mutant cells enlarged, detached from surfaces and contained large vacuoles when exposed to water, suggesting a functional defect in osmotic regulation. No changes were observed in mutant cells in the rate of fluid phase internalization or release, suggesting the DdRab11-mediated membrane trafficking defects were not general in nature. Surprisingly, the rate of phagocytosis was increased in the dominant negative DdRab11-expressing cells when compared with control cells. Our results are consistent with a role for DdRab11 in regulating membrane traffic to maintain the normal morphology and function of the contractile vacuole.

scholarly journals Examination of the endosomal and lysosomal pathways in Dictyostelium discoideum myosin I mutants

1996 ◽  
Vol 109 (3) ◽  
pp. 663-673 ◽  
Author(s):  
L.A. Temesvari ◽  
J.M. Bush ◽  
M.D. Peterson ◽  
K.D. Novak ◽  
M.A. Titus ◽  
...  
Keyword(s):  
Acid Phosphatase ◽  
Dictyostelium Discoideum ◽  
Cell Lines ◽  
Double Mutant ◽  
Fluid Phase ◽  
Endocytic Pathway ◽  
Contractile Vacuole ◽  
C Cell ◽  
Myosin I ◽  
Lysosomal System

The role of myosin Is in endosomal trafficking and the lysosomal system was investigated in a Dictyostelium discoideum myosin I double mutant myoB-/C-, that has been previously shown to exhibit defects in fluid-phase endocytosis during growth in suspension culture (Novak et al., 1995). Various properties of the endosomal pathway in the myoB-/C- double mutant as well as in the myoB- and myoC- single mutants, including intravesicular pH, and intracellular retention time and exocytosis of a fluid phase marker, were found to be indistinguishable from wild-type parental cells. The intimate connection between the contractile vacuole complex and the endocytic pathway in Dictyostelium, and the localization of a myosin I to the contractile vacuole in Acanthamoeba, led us to also examine the structure and function of this organelle in the three myosin I mutants. No alteration in contractile vacuole structure or function was observed in the myoB-, myoC- or myoB-/C- cell lines. The transport, processing, and localization of a lysosomal enzyme, alpha-mannosidase, were also unaltered in all three mutants. However, the myoB- and myoB-/C- cell lines, but not the myoC- cell line, were found to oversecrete the lysosomal enzymes alpha-mannosidase and acid phosphatase, during growth and starvation. None of the mutants oversecreted proteins following the constitutive secretory pathway. Two additional myosin I mutants, myoA- and myoA-/B-, were also found to oversecrete the lysosomally localized enzymes alpha-mannosidase and acid phosphatase. Taken together, these results suggest that these myosins do not play a role in the intracellular movement of vesicles, but that they may participate in controlling events that occur at the actin-rich cortical region of the cell. While no direct evidence has been found for the association of myosin Is with lysosomes, we predict that the integrity of the lysosomal system is tied to the fidelity of the actin cortex, and changes in cortical organization could influence lysosomal-related membrane events such as internalization or transit of vesicles to the cell surface.

scholarly journals A role for a Rab4-like GTPase in endocytosis and in regulation of contractile vacuole structure and function in Dictyostelium discoideum.

1996 ◽  
Vol 7 (10) ◽  
pp. 1623-1638 ◽  
Author(s):  
J Bush ◽  
L Temesvari ◽  
J Rodriguez-Paris ◽  
G Buczynski ◽  
J Cardelli
Keyword(s):  
Dictyostelium Discoideum ◽  
Cell Lines ◽  
Structure And Function ◽  
Dominant Negative ◽  
Contractile Vacuole ◽  
Vacuole Membrane ◽  
Endosomal Pathway ◽  
Transformed Cell Lines ◽  
And Function ◽  
Mutant Cells

The small Mr Rab4-like GTPase, RabD, localizes to the endosomal pathway and the contractile vacuole membrane system in Dictyostelium discoideum. Stably transformed cell lines overexpressing a dominant negative functioning RabD internalized fluid phase marker at 50% of the rate of wild-type cells. Mutant cells were also slower at recycling internalized fluid. Microscopic and biochemical approaches indicated that the transport of fluid to large postlysosome vacuoles was delayed in mutant cells, resulting in an accumulation in acidic smaller vesicles, probably lysosomes. Also, RabD N121I-expressing cell lines missorted a small but significant percentage of newly synthesized lysosomal alpha-mannosidase precursor polypeptides. However, the majority of the newly synthesized alpha-mannosidase was transported with normal kinetics and correctly delivered to lysosomes. Subcellular fractionation and immunofluorescent microscopy indicated that in mutant cells contractile vacuole membrane proteins were associated with compartments morphologically distinct from the normal reticular network. Osmotic tests revealed that the contractile vacuole functioned inefficiently in mutant cells. Our results suggest that RabD regulates membrane traffic along the endosomal pathway, and that this GTPase may play a role in regulating the structure and function of the contractile vacuole system by facilitating communication with the endosomal pathway.

Rab7 regulates phagosome maturation in Dictyostelium

2001 ◽  
Vol 114 (13) ◽  
pp. 2449-2460
Author(s):  
Adam Rupper ◽  
Bryon Grove ◽  
James Cardelli
Keyword(s):  
Fluid Phase ◽  
Integral Membrane Protein ◽  
Dominant Negative ◽  
Cysteine Proteinases ◽  
Lysosomal Hydrolases ◽  
Phagosomal Maturation ◽  
Lysosomal System ◽  
Phagocytic Cups ◽  
In Cells

A Dictyostelium Rab7 homolog has been demonstrated to regulate fluid-phase influx, efflux, retention of lysosomal hydrolases and phagocytosis. Since Rab7 function appeared to be required for efficient phagocytosis, we sought to further characterize the role of Rab7 in phagosomal maturation. Expression of GFP-Rab7 resulted in labeling of both early and late phagosomes containing yeast, but not forming phagocytic cups. In order to determine if Rab7 played a role in regulating membrane traffic between the endo/lysosomal system and maturing phagosomes, latex bead containing (LBC) phagosomes were purified from wild-type cells at various times after internalization. Glycosidases, cysteine proteinases, Rab7 and lysosomally associated membrane proteins were delivered rapidly to nascent phagosomes in control cells. LBC phagosomes isolated from cells overexpressing dominant negative (DN) Rab7 contained very low levels of LmpA (lysosomal integral membrane protein) and α-mannosidase was not detectable. Interestingly, cysteine proteinases were delivered to phagosomes as apparent pro-forms in cells overexpressing DN Rab7. Despite these defects, phagosomes in cells overexpressing DN Rab7 matured to form multi-particle spacious phagosomes, except that these phagosomes remained significantly more acidic than control phagosomes. These results suggested that Rab7 regulates both an early and late steps of phagosomal maturation, similar to its role in the endo/lysosomal system.

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.

scholarly journals Lysosome biogenesis requires Rab9 function and receptor recycling from endosomes to the trans-Golgi network.

1994 ◽  
Vol 125 (3) ◽  
pp. 573-582 ◽  
Author(s):  
M A Riederer ◽  
T Soldati ◽  
A D Shapiro ◽  
J Lin ◽  
S R Pfeffer
Keyword(s):  
Lysosomal Enzyme ◽  
Lysosomal Enzymes ◽  
Secretory Pathway ◽  
Protein Transport ◽  
Fluid Phase ◽  
Dominant Negative ◽  
Lysosome Biogenesis ◽  
Dominant Negative Form ◽  
Golgi Network

Newly synthesized lysosomal enzymes bind to mannose 6-phosphate receptors (MPRs) in the TGN, and are carried to prelysosomes, where they are released. MPRs then return to the TGN for another round of transport. Rab9 is a ras-like GTPase which facilitates MPR recycling to the TGN in vitro. We show here that a dominant negative form of rab9, rab9 S21N, strongly inhibited MPR recycling in living cells. The block was specific in that the rates of biosynthetic protein transport, fluid phase endocytosis and receptor-mediated endocytosis were unchanged. Expression of rab9 S21N was accompanied by a decrease in the efficiency of lysosomal enzyme sorting. Cells compensated for the presence of the mutant protein by inducing the synthesis of both soluble and membrane-associated lysosomal enzymes, and by internalizing lysosomal enzymes that were secreted by default. These data show that MPRs are limiting in the secretory pathway of cells expressing rab9 S21N and document the importance of MPR recycling and the rab9 GTPase for efficient lysosomal enzyme delivery.

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.

THE ROLE OF LYSOSOMAL ENZYMES IN THE PATHOGENESIS OF SHOCK. 2, THE ORIGIN OF PLASMA LYSOSOMAL HYDROLASES IN SHOCK

1979 ◽  
Vol 29 (1) ◽  
pp. 33-38
Author(s):  
RYO OGAWA ◽  
TAKASUKE IMAI ◽  
TATSUSHI FUJITA
Keyword(s):  
Lysosomal Enzymes ◽  
Lysosomal Hydrolases
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