Biology subjects

Li Calzi, M.

Publications and source records attributed to Li Calzi, M..

3 recordsLinked to original sources

bioRxiv2025

Tetraspanin positivity as a function of extracellular vesicle size measured by a modified immuno-TEM protocol

The identification of surface markers that correlate with specific subpopulations of extracellular vesicles (EVs) is important for EV identification, classification, purification, sorting, and functional analysis. Tetraspanins such as CD9, CD63 and CD81 were once considered to be universal markers of exosomes: small EVs released into the extracellular space when late endosomes / multivesicular bodies fuse with the plasma membrane. In contrast, plasma membrane-derived ectosomes (also called microvesicles) have a different biogenesis, were often regarded as being larger than exosomes, and display a different surface proteome. However, recent studies have shown that tetraspanins such as CD9 and CD81 are highly enriched on ectosomes derived from various sources. Thus, it is currently unclear how tetraspanin content correlates with specific EV subpopulations. Here, we present a modified immuno-TEM protocol that can be easily applied to heterogeneous EV populations comprising both small and large EVs (and presumably also a collection of exosomes and ectosomes). In EVs purified from U-2 OS cells by size-exclusion chromatography, we show that the percentage of particles positive for CD9 and CD81 is significantly higher in the subpopulation of EVs [≤] 100 nm (i.e., small EVs). These results also explain discrepancies in the size distribution profiles that we obtained using the same EV preparations by alternative single-vesicle characterization platforms such as nano flow cytometry and SP-IRIS / ExoView. The latter, when used to capture tetraspanin-positive particles, returns a population that is relatively small in size.

molecular biology↗

bioRxiv2024

Ribonuclease activity undermines immune sensing of naked extracellular RNA

The plasma membrane and the membrane of endosomal vesicles are considered physical barriers preventing extracellular RNA uptake. While naked RNA can be spontaneously internalized by certain cells types, functional delivery of naked RNA into the cytosol has been rarely observed. Here we show that extracellular ribonucleases, mainly derived from cell culture supplements, have so far hindered the study of extracellular RNA functionality. In the presence of active ribonuclease inhibitors (RI), naked bacterial RNA is pro-inflammatory when spiked in the media of dendritic cells and macrophages. In murine cells, this response mainly depends on the action of endosomal Toll-like receptors. However, we also show that naked RNA can perform endosomal escape and engage with cytosolic RNA sensors and ribosomes. For example, naked mRNAs encoding reporter proteins can be spontaneously internalized and translated by a variety of cell types, in an RI-dependent manner. In vivo, RI co-injection enhances the activation induced by naked extracellular RNA on splenic lymphocytes and myeloid-derived leukocytes. Furthermore, naked extracellular RNA is inherently pro-inflammatory in ribonuclease-poor compartments such as the peritoneal cavity. Overall, these results demonstrate that naked RNA is bioactive and does not need encapsulation inside synthetic or biological lipid vesicles for functional uptake, making a case for nonvesicular extracellular RNA-mediated intercellular communication.

molecular biology↗

bioRxiv2022

Nicked tRNAs are stable reservoirs of tRNA halves in cells and biofluids

Nonvesicular extracellular RNAs (nv-exRNAs) constitute the majority of the extracellular RNAome, but little is known about their stability, function and potential use as disease biomarkers. Herein, we measured the stability of several naked RNAs when incubated in human serum, urine and cerebrospinal fluid (CSF). We identified extracellularly produced tRNA-derived small RNAs (tDRs) with half-lives of up to three hours in CSF. Contrary to widespread assumptions, these intrinsically stable small RNAs are full-length tRNAs containing broken phosphodiester bonds (i.e., nicked tRNAs). Standard molecular biology protocols, including phenol-based RNA extraction and heat, induce the artifactual denaturation of nicked tRNAs and the consequent in vitro production of tDRs. Broken bonds are roadblocks for reverse transcriptases, preventing amplification and/or sequencing of nicked tRNAs in their native state. To solve this, we performed enzymatic repair of nicked tRNAs purified under native conditions, harnessing the intrinsic activity of phage and bacterial tRNA repair systems. Enzymatic repair regenerated an RNase R-resistant tRNA-sized band in northern blot and enabled RT-PCR amplification of full-length tRNAs. We also separated nicked tRNAs from tDRs by chromatographic methods under native conditions, identifying nicked tRNAs inside stressed cells and in vesicle-depleted human biofluids. Dissociation of nicked tRNAs produces single-stranded tDRs that can be spontaneously taken up by human epithelial cells, positioning stable nv-exRNAs as potentially relevant players in intercellular communication pathways.

molecular biology↗