RESUMO
Gp78 (also known as AMFR), an endoplasmic-reticulum (ER)-associated protein degradation (ERAD) E3 ubiquitin ligase, localizes to mitochondria-associated ER and targets the mitofusin (Mfn1 and Mfn2) mitochondrial fusion proteins for degradation. Gp78 is also the cell surface receptor for autocrine motility factor (AMF), which prevents Gp78-dependent mitofusin degradation. Gp78 ubiquitin ligase activity promotes ER-mitochondria association and ER-mitochondria Ca(2+) coupling, processes that are reversed by AMF. Electron microscopy of HT-1080 fibrosarcoma cancer cells identified both smooth ER (SER; â¼8â nm) and wider (â¼50-60â nm) rough ER (RER)-mitochondria contacts. Both short hairpin RNA (shRNA)-mediated knockdown of Gp78 (shGp78) and AMF treatment selectively reduced the extent of RER-mitochondria contacts without impacting on SER--mitochondria contacts. Concomitant small interfering RNA (siRNA)-mediated knockdown of Mfn1 increased SER-mitochondria contacts in both control and shGp78 cells, whereas knockdown of Mfn2 increased RER-mitochondria contacts selectively in shGp78 HT-1080 cells. The mitofusins therefore inhibit ER-mitochondria interaction. Regulation of close SER-mitochondria contacts by Mfn1 and of RER-mitochondria contacts by AMF-sensitive Gp78-mediated degradation of Mfn2 define new mechanisms that regulate ER-mitochondria interactions.
Assuntos
Retículo Endoplasmático Rugoso/genética , Retículo Endoplasmático Liso/genética , GTP Fosfo-Hidrolases/genética , Proteínas de Transporte da Membrana Mitocondrial/genética , Proteínas Mitocondriais/genética , Receptores do Fator Autócrino de Motilidade/genética , Animais , Células COS , Linhagem Celular , Chlorocebus aethiops , Retículo Endoplasmático Rugoso/metabolismo , Retículo Endoplasmático Liso/metabolismo , Degradação Associada com o Retículo Endoplasmático/fisiologia , Humanos , Mitocôndrias , Interferência de RNA , RNA Interferente PequenoRESUMO
The minute virus of mice, prototype strain (MVMp), is a non-enveloped, single-stranded DNA virus of the family Parvoviridae. Unlike other parvoviruses, the mechanism of cellular uptake of MVMp has not been studied in detail. We analyzed MVMp endocytosis in mouse LA9 fibroblasts and a tumor cell line derived from epithelial-mesenchymal transition through polyomavirus middle T antigen transformation in transgenic mice. By a combination of immunofluorescence and electron microscopy, we found that MVMp endocytosis occurs at the leading edge of migrating cells in proximity to focal adhesion sites. By using drug inhibitors of various endocytic pathways together with immunofluorescence microscopy and flow cytometry analysis, we discovered that MVMp can use a number of endocytic pathways, depending on the host cell type. At least three different mechanisms were identified: clathrin-, caveolin-, and clathrin-independent carrier-mediated endocytosis, with the latter occurring in transformed cells but not in LA9 fibroblasts.
Assuntos
Endocitose , Vírus Miúdo do Camundongo/fisiologia , Internalização do Vírus , Animais , Linhagem Celular , Células Epiteliais/virologia , Fibroblastos/virologia , Citometria de Fluxo , Camundongos , Camundongos Transgênicos , Microscopia Eletrônica , Microscopia de FluorescênciaRESUMO
Galectin-3 has previously been found to be required by the parvovirus minute virus of mice prototype strain (MVMp) for infection of mouse fibroblast cells. Since MVMp is an oncotropic virus, and galectin-3 is a multifunctional protein implicated in cancer metastasis, we hypothesized that galectin-3 and Mgat5, the Golgi enzyme that synthesizes high-affinity glycan ligands of galectin-3, might play a role in MVMp infection. Using siRNA-mediated knockdown of galectin-3 in mouse cells transformed with polyomavirus middle T antigen and Mgat5(-/-) mouse mammary tumor cells, we found that galectin-3 and Mgat5 are both necessary for efficient MVMp cell entry and infection, but not for cell binding. Moreover, we found that human cancer cells expressing higher levels of galectin-3 were more efficiently infected with MVMp than cell lines expressing lower galectin-3 levels. We conclude that galectin-3 and Mgat5 are involved in MVMp infection, and propose that galectin-3 is a determinant of MVMp oncotropism.
Assuntos
Galectina 3/metabolismo , Vírus Miúdo do Camundongo/fisiologia , Infecções por Parvoviridae/veterinária , Doenças dos Roedores/metabolismo , Animais , Linhagem Celular , Galectina 3/genética , Humanos , Camundongos , Vírus Miúdo do Camundongo/genética , N-Acetilglucosaminiltransferases/genética , N-Acetilglucosaminiltransferases/metabolismo , Infecções por Parvoviridae/genética , Infecções por Parvoviridae/metabolismo , Infecções por Parvoviridae/virologia , Doenças dos Roedores/genética , Doenças dos Roedores/virologiaRESUMO
The parvovirus minute virus of mice, prototype strain (MVMp), preferentially infects and kills cancer cells. This intrinsic MVMp oncotropism may depend in part on the early stages of MVMp infection. To test this hypothesis, we investigated the early events of MVMp infection in mouse LA9 fibroblasts and a highly invasive mouse mammary tumor cell line derived from polyomavirus middle T antigen-mediated transformation. Using a combination of fluorescence and electron microscopy, we found that various parameters of the cell migration process affect MVMp infection. We show that, after binding to the plasma membrane, MVMp particles rapidly cluster at the leading edge of migrating cells, which exhibit higher levels of MVMp uptake than non-motile cells. Moreover, promoting cell migration on a fibronectin matrix increased MVMp infection, and induction of epithelial-mesenchymal transition allowed MVMp replication in non-permissive epithelial cells. Hence, we propose that cell migration influences the early stages of MVMp infection.
