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1.
Int J Mol Sci ; 25(11)2024 May 23.
Article in English | MEDLINE | ID: mdl-38891874

ABSTRACT

Formin Homology Proteins (Formins) are a highly conserved family of cytoskeletal regulatory proteins that participate in a diverse range of cellular processes. FMNL2 is a member of the Diaphanous-Related Formin sub-group, and previous reports suggest FMNL2's role in filopodia assembly, force generation at lamellipodia, subcellular trafficking, cell-cell junction assembly, and focal adhesion formation. How FMNL2 is recruited to these sites of action is not well understood. To shed light on how FMNL2 activity is partitioned between subcellular locations, we used biotin proximity labeling and proteomic analysis to identify an FMNL2 interactome. The interactome identified known and new FMNL2 interacting proteins with functions related to previously described FMNL2 activities. In addition, our interactome predicts a novel connection between FMNL2 and extracellular vesicle assembly. We show directly that FMNL2 protein is present in exosomes.


Subject(s)
Formins , Formins/metabolism , Humans , Proteomics/methods , Exosomes/metabolism , Mass Spectrometry/methods , Protein Binding , HEK293 Cells , Protein Interaction Maps
2.
J Extracell Vesicles ; 11(10): e12220, 2022 10.
Article in English | MEDLINE | ID: mdl-36214496

ABSTRACT

One of the functions of small extracellular vesicles (sEVs) which has received the most attention is their capacity to deliver RNA into the cytoplasm of target cells. These studies have often been performed by transfecting RNAs into sEV-producing cells, to later purify and study sEV delivery of RNA. Transfection complexes and other delivery vehicles accumulate in late endosomes where sEV are formed and over 50% of transfection complexes or delivery vehicles administered to cells are released again to the extracellular space by exocytosis. This raises the possibility that transfection complexes could alter sEVs and contaminate sEV preparations. We found that widely used transfection reagents including RNAiMax and INTERFERin accumulated in late endosomes. These transfection complexes had a size similar to sEV and were purified by ultracentrifugation like sEV. Focusing on the lipid-based transfection reagent RNAiMax, we found that preparations of sEV from transfected cells contained lipids from transfection complexes and transfected siRNA was predominantly in particles with the density of transfection complexes, rather than sEV. This suggests that transfection complexes, such as lipid-based RNAiMax, may frequently contaminate sEV preparations and could account for some reports of sEV-mediated delivery of nucleic acids. Transfection of cells also impaired the capacity of sEVs to deliver stably-expressed siRNAs, suggesting that transfection of cells may alter sEVs and prevent the study of their endogenous capacity to deliver RNA to target cells.


Subject(s)
Extracellular Vesicles , Lipids , RNA, Small Interfering , Transfection , Ultracentrifugation
3.
Nat Biomed Eng ; 4(1): 52-68, 2020 01.
Article in English | MEDLINE | ID: mdl-31937944

ABSTRACT

A small percentage of the short interfering RNA (siRNA) delivered via passive lipid nanoparticles and other delivery vehicles reaches the cytoplasm of cells. The high doses of siRNA and delivery vehicle that are thus required to achieve therapeutic outcomes can lead to toxicity. Here, we show that the integration of siRNA sequences into a Dicer-independent RNA stem-loop based on pre-miR-451 microRNA-which is highly enriched in small extracellular vesicles secreted by many cell types-reduces the expression of the genes targeted by the siRNA in the liver, intestine and kidney glomeruli of mice at siRNA doses that are at least tenfold lower than the siRNA doses typically delivered via lipid nanoparticles. Small extracellular vesicles that efficiently package siRNA can significantly reduce its therapeutic dose.


Subject(s)
Extracellular Vesicles/metabolism , MicroRNAs/metabolism , RNA, Small Interfering/administration & dosage , RNA, Small Interfering/metabolism , Animals , Cell Line, Tumor , Drug Delivery Systems/methods , Gene Expression/drug effects , Humans , Mice , MicroRNAs/chemistry , Motor Neurons/drug effects , Nanoparticles/administration & dosage , RNA Interference , RNA, Small Interfering/chemistry
4.
Cell Rep ; 26(8): 2150-2165.e5, 2019 02 19.
Article in English | MEDLINE | ID: mdl-30784596

ABSTRACT

The autophagy pathway is an essential facet of the innate immune response, capable of rapidly targeting intracellular bacteria. However, the initial signaling regulating autophagy induction in response to pathogens remains largely unclear. Here, we report that AMPK, an upstream activator of the autophagy pathway, is stimulated upon detection of pathogenic bacteria, before bacterial invasion. Bacterial recognition occurs through the detection of outer membrane vesicles. We found that AMPK signaling relieves mTORC1-mediated repression of the autophagy pathway in response to infection, positioning the cell for a rapid induction of autophagy. Moreover, activation of AMPK and inhibition of mTORC1 in response to bacteria is not accompanied by an induction of bulk autophagy. However, AMPK signaling is required for the selective targeting of bacteria-containing vesicles by the autophagy pathway through the activation of pro-autophagic kinase complexes. These results demonstrate a key role for AMPK signaling in coordinating the rapid autophagic response to bacteria.


Subject(s)
Autophagy-Related Protein-1 Homolog/metabolism , Bacterial Outer Membrane/metabolism , Class III Phosphatidylinositol 3-Kinases/metabolism , Macroautophagy , Protein Kinases/metabolism , AMP-Activated Protein Kinase Kinases , Animals , Cells, Cultured , HCT116 Cells , HEK293 Cells , Host-Pathogen Interactions , Humans , MCF-7 Cells , Macrophages/metabolism , Macrophages/microbiology , Mechanistic Target of Rapamycin Complex 1/metabolism , Mice , Mice, Inbred NOD , Salmonella/pathogenicity
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