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Mitochondrial fission involves the preconstriction of an organelle followed by scission by dynamin-related protein Drp1. Preconstriction is facilitated by actin and non-muscle myosin II through a mechanism that remains unclear, largely due to the unknown cytoskeletal ultrastructure at mitochondrial constrictions. Here, using platinum replica electron microscopy, we show that mitochondria in cells are embedded in an interstitial cytoskeletal network that contains abundant unbranched actin filaments. Both spontaneous and induced mitochondrial constrictions typically associate with a criss-cross array of long actin filaments that comprise part of this interstitial network. Non-muscle myosin II is found adjacent to mitochondria but is not specifically enriched at the constriction sites. During ionomycin-induced mitochondrial fission, F-actin clouds colocalize with mitochondrial constriction sites, whereas dynamic myosin II clouds are present in the vicinity of constrictions. We propose that myosin II promotes mitochondrial constriction by inducing stochastic deformations of the interstitial actin network, which applies pressure on the mitochondrial surface and thus initiates curvature-sensing mechanisms that complete mitochondrial constriction. Using platinum replica electron microscopy, Yang and Svitkina find how myosin II enhances actin-mediated mitochondrial constriction and fission.

In flowering plants, pollen germinates on the stigma and pollen tubes grow through the style to fertilize the ovules. Enzymatic production of reactive oxygen species (ROS) has been suggested to be involved in pollen tube tip growth. Here, we characterized the function and regulation of the NADPH oxidases RbohH and RbohJ (Respiratory burst oxidase homolog H and J) in pollen tubes in Arabidopsis thaliana. In the rbohH and rbohJ single mutants, pollen tube tip growth was comparable to that of the wild type; however, tip growth was severely impaired in the double mutant. In vivo imaging showed that ROS accumulation in the pollen tube was impaired in the double mutant. Both RbohH and RbohJ, which contain Ca²⁺ binding EF-hand motifs, possessed Ca²⁺-induced ROS-producing activity and localized at the plasma membrane of the pollen tube tip. Point mutations in the EF-hand motifs impaired Ca²⁺-induced ROS production and complementation of the double mutant phenotype. We also showed that a protein phosphatase inhibitor enhanced the Ca²⁺-induced ROS-producing activity of RbohH and RbohJ, suggesting their synergistic activation by protein phosphorylation and Ca²⁺. Our results suggest that ROS production by RbohH and RbohJ is essential for proper pollen tube tip growth, and furthermore, that Ca²⁺-induced ROS positive feedback regulation is conserved in the polarized cell growth to shape the long tubular cell.

Neutrophils are short-lived innate immune cells. These cells respond quickly to stimuli, and die within minutes to hours; the relevance of DNA transcription in dying neutrophils remains an enigma for several decades. Here we show that the transcriptional activity reflects the degree of DNA decondensation occurring in both NADPH oxidase 2 (Nox)-dependent and Nox-independent neutrophil extracellular trap (NET) formation or NETosis. Transcriptomics analyses show that transcription starts at multiple loci in all chromosomes earlier in the rapid Nox-independent NETosis (induced by calcium ionophore A23187) than Nox-dependent NETosis (induced by PMA). NETosis-specific kinase cascades differentially activate transcription of different sets of genes. Inhibitors of transcription, but not translation, suppress both types of NETosis. In particular, promoter melting step is important to drive NETosis (induced by PMA, E. coli LPS, A23187, Streptomyces conglobatus ionomycin). Extensive citrullination of histones in multiple loci occurs only during calcium-mediated NETosis, suggesting that citrullination of histone contributes to the rapid DNA decondensation seen in Nox-independent NETosis. Furthermore, blocking transcription suppresses both types of NETosis, without affecting the reactive oxygen species production that is necessary for antimicrobial functions. Therefore, we assign a new function for transcription in neutrophils: Transcriptional firing, regulated by NETosis-specific kinases, helps to drive NETosis.

Expression of programmed death ligand 1 (PD-L1) on the surface of tumor cells and its interaction with programmed cell death protein 1 (PD-1) on tumor-infiltrating lymphocytes suppress anti-tumor immunity. In breast tumors, PD-L1 expression levels are the highest in estrogen receptor-negative, progesterone receptor-negative, and human epidermal growth factor receptor 2-negative (triple-negative) cancers. In this study, we show that a portion of PD-L1 exogenously expressed in several triple-negative breast cancer cell lines, as well as endogenous PD-L1, is proteolytically cleaved by cell surface metalloproteases. The cleavage generates an ~ 37-kDa N-terminal PD-L1 fragment that is released to the media and a C-terminal PD-L1 fragment that remains associated with cells but is efficiently eliminated by lysosomal degradation. We identify ADAM10 and ADAM17, two closely related members of the ADAM family of cell surface metalloproteases, as enzymes mediating PD-L1 cleavage. Notably, treatment of cells with ionomycin, a calcium ionophore and a known activator of ADAM10, or with phorbol 12-myristate 13-acetate, an activator of ADAM17, dramatically increases the release of soluble PD-L1 to the media. We postulate that ADAM10 and/or ADAM17 may contribute to the regulation of the PD-L1/PD-1 pathway and, ultimately, to anti-tumor immunity in triple-negative breast cancer.

The development of latency reversing agents that potently reactivate HIV without inducing global T cell activation would benefit the field of HIV reservoir research and could pave the way to a functional cure. Here, we explore the reactivation capacity of a lipid nanoparticle containing Tat mRNA (Tat-LNP) in CD4 T cells from people living with HIV undergoing antiretroviral therapy (ART). When combined with panobinostat, Tat-LNP induces latency reversal in a significantly higher proportion of latently infected cells compared to PMA/ionomycin (≈ 4-fold higher). We demonstrate that Tat-LNP does not alter the transcriptome of CD4 T cells, enabling the characterization of latently infected cells in their near-native state. Upon latency reversal, we identify transcriptomic differences between infected cells carrying an inducible provirus and non-infected cells (e.g. LINC02964 , GZMA , CCL5 ). We confirm the transcriptomic differences at the protein level and provide evidence that the long non-coding RNA LINC02964 plays a role in active HIV infection. Furthermore, p24+ cells exhibit heightened PI3K/Akt signaling, along with downregulation of protein translation, suggesting that HIV-infected cells display distinct signatures facilitating their long-term persistence. Tat-LNP represents a valuable research tool for in vitro reservoir studies as it greatly facilitates the in-depth characterization of HIV reservoir cells’ transcriptome and proteome profiles. Reactivating latent HIV reservoirs could be beneficial towards a functional cure. Here, the authors show that Tat-LNP effectively reactivates HIV while preserving the cell transcriptome. Upon reactivation, p24+ cells exhibit distinct genes and pathways potentially contributing to their persistence.

Anti-citrullinated protein antibodies (ACPAs) are a hallmark of rheumatoid arthritis, but the sources of citrullinated antigens as well as which peptidylarginine deiminases (PADs) are required for their production remain incompletely defined. Here, we investigated if macrophage extracellular traps (METs) could be a source of citrullinated proteins bound by APCAs, and if their formation requires PAD2 or PAD4. Thioglycolate-induced peritoneal macrophages from wild-type, PAD2 , and PAD4 mice or human peripheral blood-derived M1 macrophages were activated with a variety of stimulants, then fixed and stained with DAPI and either anti-citrullinated histone H4 (citH4) antibody or sera from ACPA+ or ACPA- rheumatoid arthritis subjects. METs were visualized by immunofluorescence, confirmed to be extracellular using DNase, and quantified. We found that ionomycin and monosodium urate crystals reliably induced murine citH4+ METs, which were reduced in the absence of PAD2 and lost in the absence of PAD4. Also, IgG from ACPA+, but not ACPA-, rheumatoid arthritis sera bound to murine METs, and in the absence of PAD2 or PAD4, ACPA-bound METs were lost. Finally, ionomycin induced human METs that are citH4+ and ACPA-bound. Thus, METs may contribute to the pool of citrullinated antigens bound by ACPAs in a PAD2- and PAD4-dependent manner, providing new insights into the targets of immune tolerance loss in rheumatoid arthritis.

Neutrophils play a major role in tumor biology. Among other functions, neutrophils can release extracellular traps (NETs), mesh-like structures of decondensed chromatin fibers, in a process termed NETosis. Originally characterized as an antimicrobial mechanism, NETosis has been described in cancer, but cancer-related predisposition is not clear. In the current study, we investigated the predisposition of circulating neutrophils to release NETs in lung cancer and the impact of G-CSF on this function, comparing circulating neutrophils isolated from cancer patients to the LLC and AB12 mouse models. We find that neutrophils from both healthy donors and cancer patients display high NETotic potential, with 30–60% of cells undergoing NETosis upon PMA stimulation. In contrast, neutrophils isolated from tumor-bearing mice displayed only 4–5% NETotic cells, though significantly higher than naive controls (1–2%). Despite differential mechanisms of activation described, Ionomycin and PMA mainly triggered suicidal rather than vital NETosis. G-CSF secreting tumors did not increase NETotic rates in murine neutrophils, and direct G-CSF stimulation did not promote their NET release. In contrast, human neutrophils strongly responded to G-CSF stimulation resulting also in a higher response to PMA + G-CSF stimulation. Our data show clear differences in NETotic potentials between human and murine neutrophils. We do not find a predisposition of neutrophils to release NETs in lung cancer patients compared to healthy controls, whereas cancer may modulate neutrophils' NETotic potential in mice. G-CSF secreted from tumors differentially affects murine and human NETosis in cancer. These important differences should be considered in future studies of NETosis in cancer.

Dogs are becoming an important model for human cancers, and successfully troubleshooting issues with genetically modified T cell immunotherapy for round cell and solid neoplasms in dogs provides a unique opportunity to improve efficacy, safety, and affordability for humans as well. Unfortunately, T cell activation in dogs for optimal viral transduction has not been determined, restricting advancements in canine T cell immunotherapy. Two αCD3 and two αCD28 antibody clones for canine T cell stimulation have been described in the literature, but no studies have been undertaken to evaluate which αCD3/αCD28 combination is most effective, nor has anyone directly compared the efficacy of the two most popular antibody presentation strategies: antibody-coated plates and antibody-conjugated beads. In evaluating the effects of plate- or bead-bound αCD3 stimulation alone versus αCD3/αCD28 in combination, we tested 12 possible antibody stimulation strategies in addition to evaluating two largely unexplored mitogens in canine T cell transduction, phorbol myristate acetate (PMA) with ionomycin and concanavalin A (ConA). We investigated the impact of these stimulation strategies on canine T cell activation, expansion, and transduction. For stimulation strategies producing the best results, we also examined how each strategy affected the proportions of CD4/CD8 T cell subsets and regulatory T cell (T reg ) prevalence. We determined that, in general, plate-bound antibodies were far superior to bead-bound antibodies for canine T cell stimulation, and that plate-bound αCD3 clone CA17.6F9 in combination with αCD28 clone 5B8 or the mitogen PMA with ionomycin produced better activation and expansion profiles, better transduction, and more desirable T cell subsets that are more likely to improve patient outcomes in dogs suffering from round cell and solid tumors.

Egg activation refers to events required for transition of a gamete into an embryo, including establishment of the polyspermy block, completion of meiosis, entry into mitosis, selective recruitment and degradation of maternal mRNA and pronuclear development. Here we show that zinc fluxes accompany human egg activation. We monitored calcium and zinc dynamics in individual human eggs using selective fluorophores following activation with calcium-ionomycin, ionomycin, or hPLCζ cRNA microinjection. These egg activation methods, as expected, induced rises in intracellular calcium levels and also triggered the coordinated release of zinc into the extracellular space in a prominent “zinc spark.” The ability of the gamete to mount a zinc spark response was meiotic-stage dependent. Moreover, chelation of intracellular zinc alone was sufficient to induce cell cycle resumption and transition of a meiotic cell into a mitotic one. Together, these results demonstrate critical functions for zinc dynamics and establish the zinc spark as an extracellular marker of early human development.

ObjectivesMurine models of interleukin (IL)-23-driven spondyloarthritis (SpA) have demonstrated entheseal accumulation of γδT-cells which were responsible for the majority of local IL-17A production. However, IL-23 blockers are ineffective in axial inflammation in man. This study investigated γδT-cell subsets in the normal human enthesis to explore the biology of the IL-23/17 axis.MethodsHuman spinous processes entheseal soft tissue (EST) and peri-entheseal bone (PEB) were harvested during elective orthopaedic procedures. Entheseal γδT-cells were evaluated using immunohistochemistry and isolated and characterised using flow cytometry. RNA was isolated from γδT-cell subsets and analysed by qPCR. Entheseal γδT-cells were stimulated with phorbol 12-myristate 13-acetate (PMA) and ionomycin, anti-CD3/28 or IL-23 and IL-17A production was measured by high-sensitivity ELISA and qPCR.ResultsEntheseal γδT-cells were confirmed immunohistochemically with Vδ1 and Vδ2 subsets that are cytometrically defined. Transcript profiles of both cell populations suggested tissue residency and immunomodulatory status. Entheseal Vδ2 cells expressed high relative abundance of IL-23/17-associated transcripts including IL-23R, RORC and CCR6, whereas the Vδ1 subset almost completely lacked detectable IL-23R transcript. Following PMA stimulation IL-17A was detectable in both Vδ1 and Vδ2 subsets, and following CD3/CD28 stimulation both subsets showed IL-17A and IL-17F transcripts with neither transcript being detectable in the Vδ1 subset following IL-23 stimulation.ConclusionSpinal entheseal Vδ1 and Vδ2 subsets are tissue resident cells with inducible IL-17A production with evidence that the Vδ1 subset does so independently of IL-23R expression.