Minimal information for studies of extracellular vesicles (MISEV2023): From basic to advanced approaches

Extracellular vesicles (EVs), through their complex cargo, can reflect the state of their cell of origin and change the functions and phenotypes of other cells. These features indicate strong biomarker and therapeutic potential and have generated broad interest, as evidenced by the steady year-on-year increase in the numbers of scientific publications about EVs. Important advances have been made in EV metrology and in understanding and applying EV biology. However, hurdles remain to realising the potential of EVs in domains ranging from basic biology to clinical applications due to challenges in EV nomenclature, separation from non-vesicular extracellular particles, characterisation and functional studies. To address the challenges and opportunities in this rapidly evolving field, the International Society for Extracellular Vesicles (ISEV) updates its 'Minimal Information for Studies of Extracellular Vesicles', which was first published in 2014 and then in 2018 as MISEV2014 and MISEV2018, respectively. The goal of the current document, MISEV2023, is to provide researchers with an updated snapshot of available approaches and their advantages and limitations for production, separation and characterisation of EVs from multiple sources, including cell culture, body fluids and solid tissues. In addition to presenting the latest state of the art in basic principles of EV research, this document also covers advanced techniques and approaches that are currently expanding the boundaries of the field. MISEV2023 also includes new sections on EV release and uptake and a brief discussion of in vivo approaches to study EVs. Compiling feedback from ISEV expert task forces and more than 1000 researchers, this document conveys the current state of EV research to facilitate robust scientific discoveries and move the field forward even more rapidly

Protein Kinase A Activation Enhances β-Catenin Transcriptional Activity through Nuclear Localization to PML Bodies

<div><p>The Protein Kinase A (PKA) and Wnt signaling cascades are fundamental pathways involved in cellular development and maintenance. In the osteoblast lineage, these pathways have been demonstrated functionally to be essential for the production of mineralized bone. Evidence for PKA-Wnt crosstalk has been reported both during tumorigenesis and during organogenesis, and the nature of the interaction is thought to rely on tissue and cell context. In this manuscript, we analyzed bone tumors arising from mice with activated PKA caused by mutation of the PKA regulatory subunit Prkar1a. In primary cells from these tumors, we observed relocalization of β-catenin to intranuclear punctuate structures, which were identified as PML bodies. Cellular redistribution of β-catenin could be recapitulated by pharmacologic activation of PKA. Using 3T3-E1 pre-osteoblasts as a model system, we found that PKA phosphorylation sites on β-catenin were required for nuclear re-localization. Further, β-catenin's transport to the nucleus was accompanied by an increase in canonical Wnt-dependent transcription, which also required the PKA sites. PKA-Wnt crosstalk in the cells was bi-directional, including enhanced interactions between β-catenin and the cAMP-responsive element binding protein (CREB) and transcriptional crosstalk between the Wnt and PKA signaling pathways. Increases in canonical Wnt/β-catenin signaling were associated with a decrease in the activity of the non-canonical Wnt/Ror2 pathway, which has been shown to antagonize canonical Wnt signaling. Taken together, this study provides a new understanding of the complex regulation of the subcellular distribution of β-catenin and its differential protein-protein interaction that can be modulated by PKA signaling.</p></div

PKA activation promotes nuclear relocalization of phospho-β-catenin.

<p>A. Immunofluorescence for pS675-β-catenin (green), PML (red), with DAPI nuclear staining (blue) in MC3T3-E1 cells treated with vehicle (DMSO) or FSK. Note the nuclear accumulation of phospho-β-catenin in response to FSK. B. MC3T3-E1 cells were treated with vehicle or FSK and nuclear and cytosolic protein fractions prepared and blotted for the proteins shown. Specificity of the fractionation is demonstrated by blotting for Lamin A (nuclear marker) and α-tubulin (cytosolic marker). Note the enhanced phospho-β-catenin only in the nuclear fraction in response to FSK. 8 µg of nuclear and 20 µg of cytosolic protein were loaded per lane. C. Control or Prkar1a-knockdown MC3T3-E1 cells were studied by IF as in panel A. Scale bar for all images: 10 µm.</p

Distribution of TCF and CREB binding sites in genes with altered transcription in <i>Prkar1a^+/− bone tumors</i>^*.

<p>*Distribution of promoters with neither or both sites is NS. Distribution of Tcf sites vs. up- and down-regulated genes has p = 0.037 by Fisher's exact test. Distribution of CREB and TCF sites vs. up- and down-regulated genes shows p<0.0001 by Fisher's exact test.</p><p>Distribution of TCF and CREB binding sites in genes with altered transcription in <i>Prkar1a^+/− bone tumors</i>^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109523#nt101" target="_blank">*</a>}.</p

Mutation of phosphorylation sites affects nuclear localization of β-catenin.

<p>MC3T3-E1 cells were transfected with WT or mutated FLAG-tagged β-catenin constructs, treated with vehicle or forskolin for 6 h and then subjected to confocal microscopy with anti-FLAG antibodies. The bar graph shows the quantification of immunofluorescence data. 30-40 fields were counted for each transfection, yielding 70–100 transfected cells for each condition. Note that very little nuclear localization (<3%) occurs in the absence of FSK with any of the constructs used. Scale bars: 10 µm.</p

PKA activation enhances basal and stimulated Wnt/β-catenin -dependent transcriptional activity in MC3T3-E1 cells.

<p>A. Cells were transfected with a Wnt/β-catenin -reporter plasmid (TOPFlash) or the same plasmid with a mutation in the Wnt-responsive elements (FOPFlash). Luciferase assay was performed following treatment with vehicle, FSK, or Wnt3a (100 ng/ml) as indicated (* P<0.05, ** P<0.01). B. Luciferase assay was performed in cells with control or <i>Prkar1a</i> knockdown to measure the Wnt/β-catenin-reporter activity (** P<0.01). C. mRNA expression of Wnt/β-catenin target genes was determined using QPCR analysis. The expression of each was normalized to GAPDH expression (** P<0.01 versus DMSO treated cells). (D) Cells were transfected with TOPFlash or FOPFlash along with FLAG-tagged β-catenin or its PKA phosphorylation mutants, as indicated. The Wnt/β-catenin -reporter activity was measured by luciferase assay following stimulation of cells with forskolin. (** P<0.01 versus WT or single mutants, ∧ P<0.05 versus DMSO treated counterparts).</p

β-catenin forms punctate nuclear lesions in response to PKA activation in primary cultures.

<p>Primary osteoblasts from wild type (WT) bones or from bone tumors arising in <i>Prkar1a^+/−</i> mice were studied by immunofluorescence for β-catenin (green). For reference, cell nuclei were stained with DAPI. The left column shows β-catenin, only, whereas the right column shows merger of the β-catenin and DAPI stains. Top) WT osteoblasts. Middle) Tumor osteoblasts. Bottom) WT osteoblasts treated with forskolin (FSK). Note the punctate nuclear localization of β-catenin observed in Tumor cells or WT cells treated with FSK. Magnification: 400x.</p

PKA activation represses Wnt5a/Ror2 pathway.

<p>A. and B. mRNA expression of Wnt5a and Ror2 was determined using QPCR analysis in MC3T3-E1 cells treated with FSK (A) or with <i>Prkar1a</i> knockdown (B) (** P<0.01 versus DMSO or control shRNA treated cells). Error bars represent standard deviation. C. 20 ug of protein lysates from MC3T3-E1 cells were analyzed for Wnt5a/b by Western blotting. Actin was used as the internal control. This experiment was repeated at least twice with similar results, and a representative blot is shown.</p

Stimulation of PKA by FSK increases β-catenin phosphorylation and nuclear relocalization in MC3T3-E1 cells.

<p>A. Protein lysates were prepared from MC3T3-E1 cells treated with vehicle for Forskolin (FSK) for the times indicated and blotted with the antibodies shown. Note the increases in pS133-CREB, pS552- and pS675- β-catenin at both timepoints without changes in total protein levels. Actin is shown as a loading control. 20 µg of protein were loaded per lane. B. Immunofluorescence tracking of β-catenin after vehicle (DMSO) or FSK treatment in MC3T3-E1 cells. The bottom row shows a merged images from the panels above. Scale bar: 10 µm.</p