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The actin-rich cortex plays a fundamental role in many cellular processes. Its architecture and molecular composition vary across cell types and physiological states. The full complement of actin assembly factors driving cortex formation and how their activities are spatiotemporally regulated remain to be fully elucidated. Using Dictyostelium as a model for polarized and rapidly migrating cells, we show that GxcM, a RhoGEF localized specifically in the rear of migrating cells, functions together with F-BAR protein Fbp17, a small GTPase RacC, and the actin nucleation-promoting factor WASP to coordinately promote Arp2/3 complex-mediated cortical actin assembly. Overactivation of this signaling cascade leads to excessive actin polymerization in the rear cortex, whereas its disruption causes defects in cortical integrity and function. Therefore, apart from its well-defined role in the formation of the protrusions at the cell front, the Arp2/3 complex-based actin carries out a previously unappreciated function in building the rear cortical subcompartment in rapidly migrating cells.

ObjectiveTo explore the risk factors of capillary leakage syndrome in patients with acute organophosphorus pesticide poisoning (AOPP), in order to provide reference for clinical screening and intervention timing.MethodsThe clinical manifestations, auxiliary examination and prognosis of AOPP patients admitted in Fuyang People's Hospital from November 2020 to June 2022 were prospectively analyzed, and the patients were divided into non-CLS group and CLS group based on the presence or absence of capillary leak syndrome (CLS). Patients were admitted to hospital as the starting point, and death or discharge as the endpoint. Multiple Logistic regression analysis was used to explore the risk factors of AOPP complicated with CLS.ResultsA total of 38 AOPP patients were included, of which 17 (44.7%) were complicated with CLS. There were statistical differences in age (P=0.015), blood concentration of dichlorvos (P=0.001), blood concentration of dipterex(P < 0.001), creatinine (P < 0.001) and APACHEⅡ score (P < 0.001) between non-CLS group and CLS group. Multivariate Logistic regression analysis showed that blood concentration of dipterex ≥0.44 μg/mL and APACHEⅡ score≥10.5 were independent risk factors for AOPP complicated with CLS. The mortality rate of CLS group was higher than that of CLS group (P < 0.001).ConclusionsPatients with AOPP are more likely to be complicated by CLS, and the mortality rate is higher. High-risk patients can be identified early by dipterex blood concentration and APACHE Ⅱ score, and early intervention can be carried out to improve the prognosis of patients.

Abstract: Macropinocytosis, an evolutionarily conserved mechanism mediating nonspecific bulk uptake of extracellular fluid, has been ascribed diverse functions. How nascent macropinosomes mature after internalization remains largely unknown. By searching for proteins that localize on macropinosomes during the Rab5-to-Rab7 transition stage in Dictyostelium, we uncover a complex composed of two proteins, which we name PripA and TbcrA. We show that the Rab5-to-Rab7 conversion involves fusion of Rab5-marked early macropinosomes with Rab7-marked late macropinosomes. PripA links the two membrane compartments by interacting with PI(3,4)P2 and Rab7. In addition, PripA recruits TbcrA, which acts as a GAP, to turn off Rab5. Thus, the conversion to Rab7 is linked to inactivation of the upstream Rab5. Consistently, disruption of either pripA or tbcrA impairs Rab5 inactivation and macropinocytic cargo processing. Therefore, the PripA-TbcrA complex is the central component of a Rab GAP cascade that facilitates programmed Rab switch and efficient cargo trafficking during macropinosome maturation.

ABSTRACT: Macropinocytosis mediates non-selective bulk uptake of extracellular fluid. It is the major route by which axenic Dictyostelium cells obtain nutrients and has emerged as a nutrient-scavenging pathway in mammalian cells. How environmental and cellular nutrient status modulates macropinocytic activity is not well understood. By developing a high-content imaging-based genetic screen in Dictyostelium discoideum we identified Slc15A, an oligopeptide transporter located at the plasma membrane and early macropinosome, as a novel macropinocytosis regulator. We show that deletion of slc15A but not two other related slc15 genes, leads to reduced macropinocytosis, reduced cell growth and aberrantly increased autophagy in cells grown in nutrient-rich medium. Expression of Slc15A protein or supplying cells with free amino acids rescues these defects. In contrast, expression of transport-defective Slc15A or supplying cells with amino acids in their di-peptide forms fails to rescue these defects. Therefore, Slc15A modulates the level of macropinocytosis by maintaining the intracellular availability of key amino acids through extraction of oligopeptides from the early macropinocytic pathway. We propose that Slc15A constitutes part of a positive feedback mechanism coupling cellular nutrient status and macropinocytosis. This article has an associated First Person interview with the first authors of the paper.

Polarity, which refers to the molecular or structural asymmetry in cells, is essential for diverse cellular functions. Dictyostelium has proven to be a valuable system for dissecting the molecular mechanisms of cell polarity. Previous studies in Dictyostelium have revealed a range of signaling and cytoskeletal proteins that function at the leading edge to promote pseudopod extension and migration. In contrast, how proteins are localized to the trailing edge is not well understood. By screening for asymmetrically localized proteins, we identified a novel trailing-edge protein we named Teep1. We show that a charged surface formed by two pleckstrin homology (PH) domains in Teep1 is necessary and sufficient for targeting it to the rear of cells. Combining biochemical and imaging analyses, we demonstrate that Teep1 interacts preferentially with PI(4,5)P2 and PI(3,5)P2in vitro and simultaneous elimination of these lipid species in cells blocks the membrane association of Teep1. Furthermore, a leading-edge localized myotubularin phosphatase likely mediates the removal of PI(3,5)P2 from the front, as well as the formation of a back-to-front gradient of PI(3,5)P2. Together our data indicate that PI(4,5)P2 and PI(3,5)P2 on the plasma membrane jointly participate in shaping the back state of Dictyostelium cells.

Polarity is essential for diverse functions in many cell types. Establishing polarity requires targeting a network of specific signaling and cytoskeleton molecules to different subregions of the cell, yet the full complement of polarity regulators and how their activities are integrated over space and time to form morphologically and functionally distinct domains remain to be uncovered. Here, by using the model system Dictyostelium and exploiting the characteristic chemoattractant-stimulated translocation of polarly distributed molecules, we developed a proteomic screening approach, through which we identified a leucine-rich repeat domain–containing protein we named Leep1 as a novel polarity regulator. We combined imaging, biochemical, and phenotypic analyses to demonstrate that Leep1 localizes selectively at the leading edge of cells by binding to PIP3, where it modulates pseudopod and macropinocytic cup dynamics by negatively regulating the Scar/WAVE complex. The spatiotemporal coordination of PIP3 signaling, Leep1, and the Scar/WAVE complex provides a cellular mechanism for organizing protrusive structures at the leading edge.

Background: Data comparing the outcomes of loose versus rigorous validation strategies for pulmonary vein isolation (PVI) in patients with paroxysmal atrial fibrillation (PAF) are limited. We aimed to prospectively assess the effectiveness of loose versus rigorous validation for PVI in patients with PAF with a maintained sinus rhythm. Methods: Patients (n = 117) with PAF were randomized to receive either loose validation (n = 59) or rigorous validation (n = 58) after PVI. The presence of dormant conduction in loose validation was assessed only by adenosine administration followed by isoproterenol infusion. The complete absence of pulmonary vein (PV) potentials in rigorous validation was confirmed by the combination of the Lasso catheter with isoproterenol plus adenosine. Dormant conduction, revealed by validation after PVI, was ablated until all reconnections were eliminated. Results: The procedure time in the rigorous validation group was greater than that in the loose validation group (161.3 ± 52.7 min vs. 142.5 ± 37.6 min, p = 0.03, respectively). After successful PVI, the detection of dormant PV reconnections in the rigorous validation group was significantly greater than that in the loose validation group (69.0% vs. 37.3%, p = 0.001). However, after reisolation of the sites of dormant PV conduction, the postablation recurrence rates in 1.3 years were similar between the groups (79.2% vs. 83.6%, p = 0.67). Conclusion: Rigorous validation can reveal dormant conduction in more than two‐thirds of patients with PAF undergoing PVI. However, rigorous validation and additional ablation of the resulting connections do not improve long‐term outcomes when a protocol that includes electrophysiological confirmation and pharmacological validation is used. [ABSTRACT FROM AUTHOR]

Meiosis is a specialized cell division process that generates gametes for sexual reproduction. However, the factors and underlying mechanisms involving meiotic progression remain largely unknown, especially in humans. Here, it is first showed that HSF5 is associated with human spermatogenesis. Patients with a pathogenic variant of HSF5 are completely infertile. Testicular histologic findings in the patients reveal rare postmeiotic germ cells resulting from meiotic prophase I arrest. Hsf5 knockout (KO) mice confirms that the loss of HSF5 causes defects in meiotic recombination, crossover formation, sex chromosome synapsis, and sex chromosome inactivation (MSCI), which may contribute to spermatocyte arrest at the late pachytene stage. Importantly, spermatogenic arrest can be rescued by compensatory HSF5 adeno‐associated virus injection into KO mouse testes. Mechanistically, integrated analysis of RNA sequencing and chromatin immunoprecipitation sequencing data revealed that HSF5 predominantly binds to promoters of key genes involved in crossover formation (e.g., HFM1, MSH5 and MLH3), synapsis (e.g., SYCP1, SYCP2 and SYCE3), recombination (TEX15), and MSCI (MDC1) and further regulates their transcription during meiotic progression. Taken together, the study demonstrates that HSF5 modulates the transcriptome to ensure meiotic progression in humans and mice. These findings will aid in genetic diagnosis of and potential treatments for male infertility. [ABSTRACT FROM AUTHOR]