Identification of an FMNL2 Interactome by Quantitative Mass Spectrometry.

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.

Contaminating transfection complexes can masquerade as small extracellular vesicles and impair their delivery of RNA.

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.

AMPK Promotes Xenophagy through Priming of Autophagic Kinases upon Detection of Bacterial Outer Membrane Vesicles.

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.

Multivesicular bodies associate with components of miRNA effector complexes and modulate miRNA activity.

In animals, P-bodies or GW-bodies appear to cause the congregation of proteins involved in microRNA (miRNA)-mediated post-transcriptional silencing. The localization of P-bodies does not overlap with that of known organelles and are thus considered independent of lipid bilayers. Nonetheless, an miRNA effector protein, argonaute 2 (AGO2), was initially identified as membrane-associated, and some miRNAs have been found in secreted vesicles (exosomes) that derive from endo-lysosomal compartments called multivesicular bodies (MVBs). Proteins can be sorted in a ubiquitin-dependent manner into MVBs by three heteromeric subcomplexes, collectively termed ESCRT (endosomal sorting complex required for transport), to be further secreted in exosomes and/or degraded by the lysosome. Here we show that GW-bodies containing GW182 and AGO2, two main components of the RNA-induced silencing complex (RISC), are distinct from P-bodies due to their congregation with endosomes and MVBs. Moreover, miRNAs and miRNA-repressible mRNAs are enriched at these cellular membranes, suggesting that endosomes and/or MVBs are sites of miRNA-loaded RISC (miRISC) accumulation and, possibly, action. We further show that purified exosome-like vesicles secreted by MVBs are considerably enriched in GW182, but not P-body components, AGO2 or miRNA-repressible mRNA. Moreover, cells depleted of some ESCRT components show compromised miRNA-mediated gene silencing and over-accumulate GW182, which associates with ubiquitylated proteins. Therefore, GW182, possibly in association with a fraction of miRNA-loaded AGO2, is sorted into MVBs for secretion and/or lysosomal degradation. We propose that this process promotes continuous assembly or disassembly of membrane-associated miRISCs, which is possibly required for miRNA loading or target recognition and subsequent silencing.

CD8 alpha is expressed by human monocytes and enhances Fc gamma R-dependent responses.

BACKGROUND: CD8 alpha enhances the responses of antigen-specific CTL activated through TCR through binding MHC class I, favoring lipid raft partitioning of TCR, and inducing intracellular signaling. CD8 alpha is also found on dendritic cells and rat macrophages, but whether CD8 alpha enhances responses of a partner receptor, like TCR, to activate these cells is not known. TCR and FcR, use analogous or occasionally interchangeable signaling mechanisms suggesting the possibility that CD8 alpha co-activates FcR responses. Interestingly, CD8 alpha+ monocytes are often associated with rat models of disease involving immune-complex deposition and FcR-mediated pathology, such as arthritis, glomerulonephritis, ischaemia, and tumors. While rat macrophages have been shown to express CD8 alpha evidence for CD8 alpha expression by mouse or human monocytes or macrophages was incomplete. RESULTS: We detected CD8 alpha, but not CD8 beta on human monocytes and the monocytic cell line THP-1 by flow cytometry. Reactivity of anti-CD8 alpha mAb with monocytes is at least partly independent of FcR as anti-CD8 alpha mAb detect CD8 alpha by western blot and inhibit binding of MHC class I tetramers. CD8 alpha mRNA is also found in monocytes and THP-1 suggesting CD8 alpha is synthesized by monocytes and not acquired from other CD8 alpha+ cell types. Interestingly, CD8 alpha from monocytes and blood T cells presented distinguishable patterns by 2-D electrophoresis. Anti-CD8 alpha mAb alone did not activate monocyte TNF release. In comparison, TNF release by human monocytes stimulated in a FcR-dependent manner with immune-complexes was enhanced by inclusion of anti-CD8 alpha mAb in immune-complexes. CONCLUSION: Human monocytes express CD8 alpha. Co-engagement of CD8 alpha and FcR enhances monocyte TNF release, suggesting FcR may be a novel partner receptor for CD8 alpha on innate immune cells.