Giant organelle vesicles to uncover intracellular membrane mechanics and plasticity.

Tools for accessing and studying organelles remain underdeveloped. Here, we present a method by which giant organelle vesicles (GOVs) are generated by submitting cells to a hypotonic medium followed by plasma membrane breakage. By this means, GOVs ranging from 3 to over 10 µm become available for micromanipulation. GOVs are made from organelles such as the endoplasmic reticulum, endosomes, lysosomes and mitochondria, or in contact with one another such as giant mitochondria-associated ER membrane vesicles. We measure the mechanical properties of each organelle-derived GOV and find that they have distinct properties. In GOVs procured from Cos7 cells, for example, bending rigidities tend to increase from the endoplasmic reticulum to the plasma membrane. We also found that the mechanical properties of giant endoplasmic reticulum vesicles (GERVs) vary depending on their interactions with other organelles or the metabolic state of the cell. Lastly, we demonstrate GERVs' biochemical activity through their capacity to synthesize triglycerides and assemble lipid droplets. These findings underscore the potential of GOVs as valuable tools for studying the biophysics and biology of organelles.

Cholesterol esters form supercooled lipid droplets whose nucleation is facilitated by triacylglycerols.

Cellular cholesterol can be metabolized to its fatty acid esters, cholesteryl esters (CEs), to be stored in lipid droplets (LDs). With triacylglycerols (TGs), CEs represent the main neutral lipids in LDs. However, while TG melts at ~4 °C, CE melts at ~44 °C, raising the question of how CE-rich LDs form in cells. Here, we show that CE forms supercooled droplets when the CE concentration in LDs is above 20% to TG and, in particular, liquid-crystalline phases when the fraction of CEs is above 90% at 37 °C. In model bilayers, CEs condense and nucleate droplets when the CE/phospholipid ratio reaches over 10-15%. This concentration is reduced by TG pre-clusters in the membrane that thereby facilitate CE nucleation. Accordingly, blocking TG synthesis in cells is sufficient to strongly dampen CE LD nucleation. Finally, CE LDs emerged at seipins, which cluster and nucleate TG LDs in the ER. However, when TG synthesis is inhibited, similar numbers of LDs are generated in the presence and absence of seipin, suggesting that seipin controls CE LD formation via its TG clustering capacity. Our data point to a unique model whereby TG pre-clusters, favorable at seipins, catalyze the nucleation of CE LDs.

Triglyceride lipolysis triggers liquid crystalline phases in lipid droplets and alters the LD proteome.

Lipid droplets (LDs) are reservoirs for triglycerides (TGs) and sterol-esters (SEs), but how these lipids are organized within LDs and influence their proteome remain unclear. Using in situ cryo-electron tomography, we show that glucose restriction triggers lipid phase transitions within LDs generating liquid crystalline lattices inside them. Mechanistically this requires TG lipolysis, which decreases the LD's TG:SE ratio, promoting SE transition to a liquid crystalline phase. Molecular dynamics simulations reveal TG depletion promotes spontaneous TG and SE demixing in LDs, additionally altering the lipid packing of the PL monolayer surface. Fluorescence imaging and proteomics further reveal that liquid crystalline phases are associated with selective remodeling of the LD proteome. Some canonical LD proteins, including Erg6, relocalize to the ER network, whereas others remain LD-associated. Model peptide LiveDrop also redistributes from LDs to the ER, suggesting liquid crystalline phases influence ER-LD interorganelle transport. Our data suggests glucose restriction drives TG mobilization, which alters the phase properties of LD lipids and selectively remodels the LD proteome.

Assessment of a fully automated RPR assay (Mediace RPR) for serological diagnosis and follow-up of syphilis: a retrospective study.

OBJECTIVES: This study assessed the Mediace RPR assay, an automated RPR (aRPR), for syphilis diagnosis and serological follow-up. METHODS: Serums from patients positively screened for syphilis between January 2017 and December 2019 were retrospectively selected. A focus was performed on patients with a serological follow-up after treatment and/or a reinfection. Serums were tested by both manual (mRPR) and aRPR tests. Categorical and Quantitative Agreements (CA and QA), and serological follow-up conclusions were analyzed. RESULTS: 236 serums from 85 patients (99% of male, 66% of HIV-infected) were included. The overall QA was 54.2%. CA was low (79.7%) especially for samples with low RPR titers. No prozone effect was observed. Serological follow-up after treatment led to similar conclusions, although aRPR titers often decreased faster. Over 26 episodes of reinfection, 4 (15.4%) were misdiagnosed with the aRPR. CONCLUSIONS: While the Mediace aRPR presents the advantages of an automated test, its poor sensitivity in low titers may limit its use.

MOSPD2 is an endoplasmic reticulum-lipid droplet tether functioning in LD homeostasis.

Membrane contact sites between organelles are organized by protein bridges. Among the components of these contacts, the VAP family comprises ER-anchored proteins, such as MOSPD2, that function as major ER-organelle tethers. MOSPD2 distinguishes itself from the other members of the VAP family by the presence of a CRAL-TRIO domain. In this study, we show that MOSPD2 forms ER-lipid droplet (LD) contacts, thanks to its CRAL-TRIO domain. MOSPD2 ensures the attachment of the ER to LDs through a direct protein-membrane interaction. The attachment mechanism involves an amphipathic helix that has an affinity for lipid packing defects present at the surface of LDs. Remarkably, the absence of MOSPD2 markedly disturbs the assembly of lipid droplets. These data show that MOSPD2, in addition to being a general ER receptor for inter-organelle contacts, possesses an additional tethering activity and is specifically implicated in the biology of LDs via its CRAL-TRIO domain.

Foxp3+ Regulatory and Conventional CD4+ T Cells Display Similarly High Frequencies of Alloantigen-Reactive Cells.

Foxp3+ regulatory T cells (Tregs) play a major role in acquired immune tolerance to allogenic transplants. Their suppressive activity is thought to require T cell receptor (TCR)-driven antigen recognition; little, however, is known about the fraction of Tregs able to recognize alloantigens within this T cell subset primarily educated against self-antigens. Performing transfer experiments of Tregs or conventional T cells (Tconv) into both lymphoreplete and lymphopenic mice, we observed a similarly high proportion of cells signaling through their TCR and proliferating in allogenic hosts. Furthermore, using an in vivo proliferation assay with limited T cell numbers infused into lymphopenic mice, we found that the overall frequency of alloreactive Tregs was similar if not higher to that of alloreactive Tconv. Overall our study highlights a noticeably high level of alloreactive Foxp3+ regulatory T cells accounting for their predominant role in transplantation tolerance.

The Effect of Magnification Loupes on Spontaneous Posture Change of Dental Students During Preclinical Restorative Training.

Scientific evidence validating the beneficial effect of loupes in preventing musculoskeletal disorders is very scarce. The aim of this study was to assess the impact of dental loupes on dental students' posture during a preclinical restorative dentistry course. Using a randomized crossover design, this study was conducted at the School of Dentistry, University of Nantes, France, in 2017. Forty students in their second year of dental study were randomly divided into two groups of 20 each: group A used loupes, whereas group B did not. The week after, students reversed configurations (each subject served as his or her own treatment and control group). Students were video-recorded during cavity preparation. Trunk, head and neck, and upper arm positions were analyzed using continuous scores based on the modified Posture Assessment Instrument. Additionally, cavities were rated, and students completed a questionnaire on their perceptions of the loupes. On a scale on which lower scores indicated better posture, the results showed significantly higher posture ergonomic scores per minute for students without loupes (146.3±6.64 points/min) than with loupes (123.2±6.77 points/min; p<0.05). The majority of the students (32/39, 82%) showed improvements in ergonomic postures with the use of loupes. Trunk, head, and neck were positively impacted by the use of loupes, but not the upper arms. Cavity preparations were not improved by the use of loupes. The questionnaire revealed negative aspects (pain and difficulty adapting) but underlined the perceived positive impact on posture. This study documented the ergonomic advantages and challenges of introducing magnification near the beginning of the dental training program.

Intrinsic transgene immunogenicity gears CD8(+) T-cell priming after rAAV-mediated muscle gene transfer.

Antitransgene CD8(+) T-cell responses are an important hurdle after recombinant adeno-associated virus (rAAV) vector-mediated gene transfer. Indeed, depending on the mutational genotype of the host, transgene amino-acid sequences of foreign origin can elicit deleterious cellular and humoral responses. We compared here two different major histocompatibility complex (MHC) class I epitopes of an engineered ovalbumin transgene delivered in muscle tissue by rAAV1 vector and found very different strength of CD8 responses, muscle destruction being correlated with the course of the immunodominant response. We further demonstrate that robust CD8(+) T-cell priming can occur through the cross-presentation pathway but requires the presence of either a strong MHC class II epitope or antibodies to the transgene product. Finally, manipulating transgene subcellular localization, we found that provided we avoid transgene expression in antigen presenting cells, the poorly accessible cytosolic form of ovalbumin transgene lacking strong MHC II epitope, evades CD8(+) T-cell priming and remains permanently expressed in muscle with no immune cell infiltration. Our results demonstrate that the intrinsic immunogenicity of transgenes delivered with rAAV vector in muscle can be manipulated in a rational manner to avoid adverse immune responses.

Extrathymic induction of Foxp3⁺ regulatory T cells declines with age in a T-cell intrinsic manner.

Extrathymically induced Foxp3⁺ regulatory T (Treg) cells contribute to the pool of Treg cells and are implicated in the maintenance of immune tolerance at environmental interfaces. The impact of T-cell senescence on their generation and function is, however, poorly characterized. We report here that steady-state induction of Foxp3 is impaired in aged T cells in vivo. In vitro assays further revealed that this defective generation of Treg cells was independent from the strength of TCR stimulation and arose before T-cell proliferation. Importantly, they also revealed that this impairment of Foxp3 induction is unrelated to known age-related T-cell defects, such as IL-2 secretion impairment, accumulation of activated T-cell populations, or narrowing of the T-cell repertoire. Finally, a loss of extrathymic induction of Foxp3 and tolerance to minor-mismatched skin graft were observed in aged mice treated by nondepleting anti-CD4 antibody. The T-cell intrinsic impairment of Treg-cell generation revealed here highlights age as a key factor to be considered in immune tolerance induction.