Inhibition Of Endosome Fusion Research Articles

Haploinsufficiency Of SAMD9L, An Endosome Fusion Facilitator, In Mice Induces The Development Of Myeloid Malignancies Mimicking Human Diseases With Monosomy 7

Monosomy 7 is a common chromosomal abnormality found frequently in MDS and AML. We previously identified a common microdeletion cluster in 7q21.3 in juvenile myelomonocytic leukemia patients. This cluster contains three poorly characterized genes: sterile alpha motif (SAM) domain-9 (SAMD9) which is absent in mice, samd9-like (SAMD9L) and Miki (LOC253012). Although mutations have rarely been seen in these genes, the genes reside proximal to the 7q22 band that is deleted in single copy in nearly 25% of AML and MDS patients. We recently reported that Miki, a centrosomal protein that promotes alignment of chromosomes at metaphase, is a candidate gene responsible for mitotic/nuclear abnormalities observed in MDS patients (Mol Cell 2012).In this study, we established and characterized SAMD9L-deficient mice, along with the analysis of molecular function of SAMD9L protein. Among SAMD9L-/- (n=15) and SAMD9L+/- (n=15) littermates, 13 mice developed myeloid dysplasia, 2 mice developed myeloid leukemia and one mouse developed myeloproliferative disease after the age of 18 months, while all but one SAMD9L+/+ mice (n=23) maintained normal hematopoiesis throughout the 24-month observation period. Infection of MOL4070A retrovirus into newborn mice developed myeloid leukemia within 15 months preferentially in SAMD9L-deficient genetic background with Evi1 and Fbxl10 (encoding a H3K36 demethylase) genes as common virus integration sites.While bone marrow (BM) cells from SAMD9L+/+ mice (12 weeks old) formed fewer colonies by the third replating, cells from SAMD9L-deficient mice continued to form similar numbers and sizes of well-differentiated colonies beyond the 7th plating. The excess number of colonies formed was reduced by retrovirus-mediated forced expression of Samd9L. These data suggested enhanced self-renewal and/or delays in differentiation of SAMD9L-deficient stem cells. In addition, enhanced reconstitution ability of SAMD9L-deficient stem cells was demonstrated by competitive repopulation assay using the Ly5 congenic mouse system, where irradiated Ly5.1 mice were transplanted with long term-LSK cells from SAMD9L+/+ or SAMD9L-deficient Ly5.2 mice (10 weeks old) together with BM cells from SAMD9L+/+Ly5.1 mice. This was confirmed by limiting-dilution transplants, results of which showed a higher frequency of multi-lineage repopulating cells at 8 weeks in SAMD9L-/- donor BM.Moreover, growth advantage in the presence of cytokines was evident in liquid cultures of SAMD9L-deficient BM progenitor cells. Hypersensitivity of SAMD9L-deficient BM progenitors to cytokines was also shown in in vivo experiments, in which SAMD9L-deficient mice injected with cyclophosphamide (day 0) and G-CSF (days 1-4) showed significantly higher WBC counts than SAMD9L+/+ mice at the nadir (day 3). These findings suggested that SAMD9L-deficiency sensitizes hematopoietic progenitors to cytokines. Immunostaining using SAMD9L antibody showed a vesicular pattern of SAMD9L localization in approximately 15% of BM progenitor cells that overlapped with the localization of EEA1, an early endosomal protein. In SAMD9L-/- fibroblasts, while rapid endocytosis of PDGF-receptor (PDGFR) by PDGF stimulation occurred in a time-course similar to that in SAMD9L+/+ cells, homotypic fusion of endosomes containing PDGFR delayed. Inhibition of endosome fusion in SAMD9L-/- cells lead to the accumulation of PDGFR that were remained to be phosphorylated in early endosome, resulting in the prolonged activation of cytokine signals. Accumulation of cytokine receptors in early endosome and persistent cytokine signals were also found in BM progenitors obtained from SAMD9L-deficient mice. These observations suggest that SAMD9L is a crucial component of a protein complex that facilitates the degradation of cytokine receptors through the homotypic fusion of endosomes. Collectively, our study suggests the contribution of haploinsufficiency of SAMD9L to the pathogenesis of myeloid diseases harboring -7/7q- through the prolonged activation of cytokine signals that results in the enhancement of stem cell self-renewal and/or delay in differentiation of early progenitors. Deletion of other haploinsufficient tumor-suppressor genes that reside in 7q would collaborate with the deficiency of SAMD9L for myeloid leukemogenesis. Disclosures:No relevant conflicts of interest to declare.

Endocytosis of the dermatan sulfate proteoglycan decorin utilizes multiple pathways and is modulated by epidermal growth factor receptor signaling

Human skin fibroblasts efficiently internalize the matrikine decorin by receptor-mediated endocytosis, however, very little is known about its intracellular trafficking routes up to lysosomal degradation. In an in vitro system measuring uptake and degradation of [ 35S]sulfate-labeled decorin, endocytosis was blocked by 46% when clathrin assembly/disassembly was inhibited using chlorpromazine. Pharmacological inhibition of EGF receptor signaling caused 34% reduction of decorin uptake, whereas inhibition of the IGF receptor had no effect. Using confocal immunofluorescence microscopy, we determined that only about 5–10% of internalized decorin colocalized with the EGFR. Thus, uptake depends on EGFR signaling rather than trafficking along the same pathway. Decorin passes through early endosomes towards trafficking to lysosomes, since more than 50% of decorin colocalized with EEA1. Moreover, inhibition of endosomal fusion by wortmannin caused a profound inhibition of decorin endocytosis. Overexpression of the clathrin-binding Hrs protein, which has previously been shown to inibit EGFR degradation blocked the degradation of decorin. Cholesterol depletion by filipin inhibited uptake of decorin by 34%, however, nearly no intracellular colocalization was found between decorin and caveolin-1. The combined use of filipin and chlorpromazine had an additive inhibitory effect on decorin endocytosis. Moreover, chlorpromazine diverted decorin from the chlorpromazine-sensitive pathway to an alternative uptake route. The CD44/hyaluronan pathway was excluded as an endocytic route for decorin. Our observations indicate that decorin is taken up by more than one endocytic pathway. Of note, lipid-raft-dependent EGFR signaling modulates decorin uptake, suggesting the presence of a potential feedback regulation mechanism for desensitization of signaling events mediated by decorin.

Inhibition of endosome fusion in primary hepatocytes prevents asialoglycoprotein degradation but not uptake of transferrin iron demonstrating that intracellular iron release occurs from early endosomes

A comparison of the effects of inhibitors of membrane fusion on the uptake of asialoglycoprotein and transferrin by primary rat hepatocytes was made. This showed that while high potassium medium inhibited the degradation but not the uptake of asialoorosomucoid, both transferrin endocytosis and iron delivery to the cells were unaffected. This difference between the two pathways was also observed with an inhibitor of phospholipase A2, bromophenacyl bromide. With the latter, it was found that the asialoglycoproteins failed to traverse from a low-density to a high-density intracellular compartment, implying a role for phospholipase A2 in the trafficking of asialoglycoprotein receptor but not that for transferrin or iron. This demonstrates that, after its release from transferrin, iron is transported to the cytoplasm directly from the early endosome without the need for fusion of the iron-containing vesicle with a lysosome.

Studies on the inhibition of endosome fusion by GTPgammaS-bound ARF.

Using a cell free assay, we have previously shown that ARF is not required for endosome fusion but that inhibition of fusion by GTPgammaS is dependent on a cytosolic pool of ARFs. Since ARF is proposed to function in intracellular membrane traffic by promoting vesicle biogenesis, and components of clathrin- and COP-coated vesicles have been localized on endosomal structures, we investigated whether ARF-mediated inhibition of early endosome fusion involves the recruitment or irreversible association of these proteins onto endosomal membranes. We now report that depletion of components of clathrin coated vesicles (clathrin, AP-1 and AP-2) or COPI vesicles (beta COP) does not affect the capacity of GTPgammaS-activated ARF to inhibit endosome fusion. Inhibition of fusion by activated ARF is also independent of endosomal acidification since assays performed in the presence of the vacuolar ATPase inhibitor bafilomycin A1 are equally sensitive to GTPgammaS-bound ARF. Finally, in contrast to reported effects on lysosomes, we demonstrate that ARF-GTPgammaS does not induce endosomal lysis. These combined data argue that sequestration of known coat proteins to membranes by activated ARF is not involved in the inhibition of early endosome fusion and that its capacity to inhibit fusion involves other specific interactions with the endosome surface. These results contrast with the mechanistic action of ARF on intra-Golgi transport and nuclear envelope assembly.

Involvement of the endosomal autoantigen EEA1 in homotypic fusion of early endosomes

In mammalian cells, fusion between early endocytic vesicles has been shown to require the ubiquitous intracellular fusion factors N-ethylmaleimide-sensitive factor (NSF) and α-SNAP, as well as a factor specific for early endosomes, the small GTPase Rab5 [1–3]. We have previously demonstrated an additional requirement for phosphatidylinositol 3-kinase (PI 3-kinase) activity [4]. The membrane association of early endosomal antigen 1 (EEA1), a specific marker of early endosomes [5,6], has recently been shown to be similarly dependent on PI 3-kinase activity [7], and we therefore postulated that it might be involved in endosome fusion. Here, we present evidence that EEA1 has an important role in determining the efficiency of endosome fusion in vitro. Both the carboxy-terminal domain of EEA1 (residues 1098–1411) and specific antibodies against EEA1 inhibited endosome fusion when included in an in vitro assay. Furthermore, depletion of EEA1, both from the membrane fraction used in the assay by washing with salt and from the cytosol using an EEA1-specific antibody, resulted in inhibition of endosome fusion. The involvement of EEA1 in endosome fusion accounts for the sensitivity of the endosome fusion assay to inhibitors of PI 3-kinase.

Inhibition of endosome fusion by wortmannin persists in the presence of activated Rab5.

Rab5-dependent endosome fusion is sensitive to the phosphoinositide 3-kinase inhibitor, wortmannin. It has been proposed that phosphoinositide 3-kinase activity may be required for activation of rab5 by influencing its nucleotide cycle such as to promote its active GTP state. In this report we demonstrate that endosome fusion remains sensitive to wortmannin despite preloading of endosomes with stimulatory levels of a GTPase-defective mutant rab5(Q79L) or of a xanthosine triphosphate-binding mutant, rab5(D136N), in the presence of the nonhydrolysable analogue XTPgammaS. These results suggest that activation of rab5 cannot be the principal function of the wortmannin-sensitive factor on the endosome fusion pathway. This result is extrapolated to all GTPases by demonstrating that endosome fusion remains wortmannin sensitive despite prior incubation with the nonhydrolysable nucleotide analogue GTPgammaS. Consistent with these results, direct measurement of clathrin-coated vesicle-stimulated nucleotide dissociation from exogenous rab5 was insensitive to the presence of wortmannin. A large excess of rab5(Q79L), beyond levels required for maximal stimulation of the fusion assay, afforded protection against wortmannin inhibition, and partial protection was also observed with an excess of wild-type rab5 independent of GTPgammaS.

Inhibition of endosome fusion by phospholipase A2 (PLA2) inhibitors points to a role for PLA2 in endocytosis.

Fusion of intracellular membrane-bound compartments is a common step in the transport of macromolecules along the endocytic and secretory pathways. A large number of factors active in the fusion process or its regulation have been identified; however, the actual sequence of events leading to membrane fusion is still unknown. In this study, we have assessed a possible role for PLA2 in endosome fusion by using an in vitro reconstitution assay and by examining endocytosis in intact cells. Several PLA2 inhibitors blocked endosome fusion in a broken-cell preparation. Inhibition was reversed by addition of arachidonic acid. At the electron microscope level, endosome clusters were observed even in the presence of inhibitors; however, actual fusion between endosomes was largely reduced. Fusion frequency increased upon the addition of arachidonic acid. A membrane-permeable PLA2 inhibitor blocked mixing of ligands internalized sequentially but did not affect internalization. The results indicate that vesicle fusion along the endocytic pathway requires a PLA2 activity. The effect of this activity would be, at least in part, mediated by arachidonic acid release.