Specificities of exosome versus small ectosome secretion revealed by live intracellular tracking of CD63 and CD9.

Despite their roles in intercellular communications, the different populations of extracellular vesicles (EVs) and their secretion mechanisms are not fully characterized: how and to what extent EVs form as intraluminal vesicles of endocytic compartments (exosomes), or at the plasma membrane (PM) (ectosomes) remains unclear. Here we follow intracellular trafficking of the EV markers CD9 and CD63 from the endoplasmic reticulum to their residency compartment, respectively PM and late endosomes. We observe transient co-localization at both places, before they finally segregate. CD9 and a mutant CD63 stabilized at the PM are more abundantly released in EVs than CD63. Thus, in HeLa cells, ectosomes are more prominent than exosomes. By comparative proteomic analysis and differential response to neutralization of endosomal pH, we identify a few surface proteins likely specific of either exosomes (LAMP1) or ectosomes (BSG, SLC3A2). Our work sets the path for molecular and functional discrimination of exosomes and small ectosomes in any cell type

ImageJ macros for RUSH and colocalization analyses

rushAnalysis macros can be used to analyze movies of RUSH transfected cells, to quantify the fluorescence in the Golgi and in large compartments ("withLysosomes" macro) and the number of positive small compartments. Pearson_vs_time macro can be used to calculate Pearson's correlation coefficient between two channel over time for live imaging experiments. Part1, Part2 and Part3 are three macros to be used sequencially for calculation of Mander's co-localization coefficient of a double positive cell compartment for two proteins with a third protein

Different TCR-induced T lymphocyte responses are potentiated by stiffness with variable sensitivity

International audienceT cells are mechanosensitive but the effect of stiffness on their functions is still debated. We characterize herein how human primary CD4 + T cell functions are affected by stiffness within the physiological Young's modulus range of 0.5 kPa to 100 kPa. Stiffness modulates T lymphocyte migration and morphological changes induced by TCR/CD3 triggering. Stiffness also increases TCR-induced immune system, metabolism and cell-cycle-related genes. Yet, upon TCR/ CD3 stimulation, while cytokine production increases within a wide range of stiffness, from hundreds of Pa to hundreds of kPa, T cell metabolic properties and cell cycle progression are only increased by the highest stiffness tested (100 kPa). Finally, mechanical properties of adherent antigen-presenting cells modulate cytokine production by T cells. Together, these results reveal that T cells discriminate between the wide range of stiffness values found in the body and adapt their responses accordingly

Specificities of exosome versus small ectosome secretion revealed by live intracellular tracking of CD63 and CD9

Despite their roles in intercellular communications, the different populations of extracellular vesicles (EVs) and their secretion mechanisms are not fully characterized: how and to what extent EVs form as intraluminal vesicles of endocytic compartments (exosomes), or at the plasma membrane (PM) (ectosomes) remains unclear. Here we follow intracellular trafficking of the EV markers CD9 and CD63 from the endoplasmic reticulum to their residency compartment, respectively PM and late endosomes. We observe transient co-localization at both places, before they finally segregate. CD9 and a mutant CD63 stabilized at the PM are more abundantly released in EVs than CD63. Thus, in HeLa cells, ectosomes are more prominent than exosomes. By comparative proteomic analysis and differential response to neutralization of endosomal pH, we identify a few surface proteins likely specific of either exosomes (LAMP1) or ectosomes (BSG, SLC3A2). Our work sets the path for molecular and functional discrimination of exosomes and small ectosomes in any cell type

Compromised nuclear envelope integrity drives TREX1-dependent DNA damage and tumor cell invasion

International audienceWhile mutations leading to a compromised nuclear envelope cause diseases such as muscular dystrophies or accelerated aging, the consequences of mechanically induced nuclear envelope ruptures are less known. Here we show that nuclear envelope ruptures induce DNA damage which promotes senescence in non-transformed cells, and induces an invasive phenotype in human breast cancer cells. We find that the ER-associated exonuclease TREX1 translocates into the nucleus after nuclear envelope rupture and is required to induce DNA damage. Inside the mammary duct, cellular crowding leads to nuclear envelope ruptures which generate TREX1-dependent DNA damage, thereby driving the progression of in situ carcinoma to the invasive stage. DNA damage and nuclear envelope rupture markers were also enriched at the invasive edge of human tumors. We propose that DNA damage in mechanically challenged nuclei could affect the pathophysiology of crowded tissues by modulating proliferation and extracellular matrix degradation of normal and transformed cells

Definitions and treatment of oligometastatic oesophagogastric cancer according to multidisciplinary tumour boards in Europe

Background: Consensus about the definition and treatment of oligometastatic oesophagogastric cancer is lacking. Objective: To assess the definition and treatment of oligometastatic oesophagogastric cancer across multidisciplinary tumour boards (MDTs) in Europe. Material and methods: European expert centers (n Z 49) were requested to discuss 15 real-life cases in their MDT with at least a medical, surgical, and radiation oncologist present. The cases varied in terms of location and number of metastases, histology, timing of detection (i.e. synchronous versus metachronous), primary tumour treatment status, and response to systemic therapy. The primary outcome was the agreement in the definition of oligometastatic disease at diagnosis and after systemic therapy. The secondary outcome was the agreement in treatment strategies. Treatment strategies for oligometastatic disease were categorised into up -front local treatment (i.e. metastasectomy or stereotactic radiotherapy), systemic therapy followed by restaging to consider local treatment or systemic therapy alone. The agreement across MDTs was scored to be either absent/poor (= 75%). Results: A total of 47 MDTs across 16 countries fully discussed the cases (96%). Oligometastatic disease was considered in patients with 1-2 metastases in either the liver, lung, retroperitoneal lymph nodes, adrenal gland, soft tissue or bone (consensus). At follow-up, oligometastatic disease was considered after a median of 18 weeks of systemic therapy when no progression or progression in size only of the oligometastatic lesion(s) was seen (consensus). If at restaging after a median of 18 weeks of systemic therapy the number of lesions progressed, this was not considered as oligometastatic disease (fair agreement). There was no consensus on treatment strategies for oligometastatic disease. Conclusion: A broad consensus on definitions of oligometastatic oesophagogastric cancer was found among MDTs of oesophagogastric cancer expert centres in Europe. However, high practice variability in treatment strategies exists. (C) 2022 The Authors. Published by Elsevier Ltd