Characterization of endosome-endosome fusion in a cell-free system using Dictyostelium discoideum.

Lenhard, James M.; Mayorga, Luis S.; Stahl, Philip D.

doi:10.1016/s0021-9258(18)46031-0

Cited by 30 publications

(2 citation statements)

References 46 publications

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“…A significantly higher level of fusion between endosomes from Leishmania was observed at its optimal growth temperature of 23°C than at 37°C (data not shown). This is not surprising since Dictyostelium , which grows optimally at 21−28°C, exhibits significant fusion at a similar temperature (Lenhard et al ., 1992). It has been shown in several systems that Rab‐GDI in the presence of GDP specifically depletes Rab proteins from the membrane (Funato et al ., 1997) and exhibits broad substrate specificity across species (Attal and Langsley, 1996; Chaturvedi et al ., 1999).…”

Section: Discussionmentioning

confidence: 93%

Rab5-mediated endosome-endosome fusion regulates hemoglobin endocytosis in Leishmania donovani

Singh

2003

The EMBO Journal

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contributed equally to this workTo understand the traf®cking of endocytosed hemoglobin (Hb) in Leishmania, we investigated the characteristics of in vitro fusion between endosomes containing biotinylated Hb (BHb) and avidin±horse-radish peroxidase (AHRP). We showed that early endosome fusion in Leishmania is temperature and cytosol dependent and is inhibited by ATP depletion, ATPgS, GTPgS and N-ethylmaleimide treatment. The Rab5 homolog from Leishmania donovani, LdRab5, was cloned and expressed. Our results showed that homotypic fusion between the early endosomes in Leishmania is Rab5 dependent. Early endosomes containing BHb fused ef®ciently with late endosomes in a process regulated by Rab7, whereas no fusion between early and late endosomes was detected usinḡ uid phase markers. Pre-treatment of early endosomes containing BHb with monoclonal antibody speci®c for the C-terminus of the Hb receptor (HbR) or the addition of the C-terminal cytoplasmic fragment of the HbR speci®cally inhibited the fusion with late endosomes, suggesting that signal(s) mediated through the HbR cytoplasmic tail promotes the fusion of early endosomes containing Hb with late endosomes.

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Section: Discussionmentioning

confidence: 93%

Rab5-mediated endosome-endosome fusion regulates hemoglobin endocytosis in Leishmania donovani

Singh

2003

The EMBO Journal

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“…It was also possible that the defect observed in indyl reflected a primary block in the delivery of internalized material to lysosomes, a situation that might lead to a reduced capacity to accumulate endocytic tracers. This was tested by loading cells with FITC-BSA at 21°C for 5 min and then chasing them in the absence of the marker at 26.5°C for <2 h. It has been shown previously that at 5 min, the internalized material has not yet reached a hydrolase-containing compartment (Lenhard et al, 1992). After 0, 1, and 2 h, cells and media were collected, and the percentage of extracellular fluorescence that was not TCA precipitable was taken as the degree of degradation of the protein.…”

Section: Transport To the Lysosomes Is Not Impaired In Indyl Cellsmentioning

confidence: 99%

Dictyostelium discoideum mutants with temperature-sensitive defects in endocytosis.

Bacon

Cohen

Lewin

et al. 1994

The Journal of Cell Biology

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Abstract. We have isolated and characterized temperature-sensitive endocytosis mutants in Dictyostelium discoideum. Dictyostelium is an attractive model for genetic studies of endocytosis because of its high rates of endocytosis, its reliance on endocytosis for nutrient uptake, and tractable molecular genetics. Endocytosis-defective mutants were isolated by a fluorescence-activated cell sorting (FACS TM) as cells unable to take up a fluorescent marker. One temperature-sensitive mutant (indyl) was characterized in detail and found to exhibit a complete block in fluid phase endocytosis at the restrictive temperature, but normal rates of endocytosis at the permissive temperature. Likewise, a potential cell surface receptor that was rapidly internalized in wild-type cells and indyl cells at the permissive temperature was poorly internalized in indyl under restrictive conditions. Growth was also completely arrested at the restrictive temperature. The endocytosis block was rapidly induced upon shift to the restrictive temperature and reversed upon return to normal conditions. Inhibition of endocytosis was also specific, as other membranetrafficking events such as phagocytosis, secretion of lysosomal enzymes, and contractile vacuole function were unaffected at the restrictive temperature. Because recycling and transport to late endocytic compartments were not affected, the site of the defect's action is probably at an early step in the endocytic pathway. Additionally, indyl cells were unable to proceed through the normal development program at the restrictive temperature. Given the tight functional and growth phenotypes, the indyl mutant provides an opportunity to isolate genes responsible for endocytosis in Dictyostelium by complementation cloning.NDOCYTOSIS plays an important role in a wide variety of functions in eukaryotic cells, including nutrient uptake, internalization of receptors, antigen processing, and retrieval of membrane inserted into the cell surface during secretion. In animal cells, endocytosis typically involves the budding of clathrin-coated pits from the plasma membrane to yield coated vesicles that rapidly uncoat and fuse with early endosomes, a heterogeneous population of tubules and vesicles scattered throughout the cytoplasm. In early endosomes, internalized plasma membrane components and extracellular macromolecules are sorted such that proteins to be recycled back to the cell surface are separated from those destined for transport to late endosomes and lysosomes. A great deal is known about the kinetics and morphology of the intraceUular compartments involved in mammalian cells, but the molecular mechanisms for these various steps remain poorly understood.A number of proteins that play important roles during endocytosis have been identified. These include low molecular weight GTPases of the rab and ARF families, members of which have been implicated in regulating the formation or

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In vitro microtubule-based organelle transport in wild-typeDictyostelium and cells overexpressing a truncated dynein heavy chain

Pollock

Koonce

Hostos

et al. 1998

Cell Motil. Cytoskeleton

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Characterization of endosome-endosome fusion in a cell-free system using Dictyostelium discoideum.

Cited by 30 publications

References 46 publications

Rab5-mediated endosome-endosome fusion regulates hemoglobin endocytosis in Leishmania donovani

Rab5-mediated endosome-endosome fusion regulates hemoglobin endocytosis in Leishmania donovani

Dictyostelium discoideum mutants with temperature-sensitive defects in endocytosis.

In vitro microtubule-based organelle transport in wild-typeDictyostelium and cells overexpressing a truncated dynein heavy chain

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