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Background: Egress of Plasmodium falciparum, from erythrocytes at the end of its asexual cycle and subsequent parasite invasion into new host cells, is responsible for parasite dissemination in the human body. The egress pathway is emerging as a coordinated multistep programme that extends in time for tens of minutes, ending with rapid parasite extrusion from erythrocytes. While the Ca2+ regulation of the invasion of P. falciparum in erythrocytes is well established, the role of Ca2+ in parasite egress is poorly understood. This study analysed the involvement of cytoplasmic free Ca2+ in infected erythrocytes during the multistep egress programme of malaria parasites. Methods: Live-cell fluorescence microscopy was used to image parasite egress from infected erythrocytes, assessing the effect of drugs modulating Ca2+ homeostasis on the egress programme. Results: A steady increase in cytoplasmic free Ca2+ is found to precede parasite egress. This increase is independent of extracellular Ca2+ for at least the last two hours of the cycle, but is dependent upon Ca2+ release from internal stores. Intracellular BAPTA chelation of Ca2+ within the last 45 minutes of the cycle inhibits egress prior to parasitophorous vacuole swelling and erythrocyte membrane poration, two characteristic morphological transformations preceding parasite egress. Inhibitors of the parasite endoplasmic reticulum (ER) Ca2+-ATPase accelerate parasite egress, indicating that Ca2+ stores within the ER are sufficient in supporting egress. Markedly accelerated egress of apparently viable parasites was achieved in mature schizonts using Ca2+ ionophore A23187. Ionophore treatment overcomes the BAPTA-induced block of parasite egress, confirming that free Ca2+ is essential in egress initiation. Ionophore treatment of immature schizonts had an adverse effect inducing parasitophorous vacuole swelling and killing the parasites within the host cell. Conclusions: The parasite egress programme requires intracellular free Ca2+ for egress initiation, vacuole swelling, and host cell cytoskeleton digestion. The evidence that parasitophorous vacuole swelling, a stage of unaffected egress, is dependent upon a rise in intracellular Ca2+ suggests a mechanism for ionophore-inducible egress and a new target for Ca2+ in the programme liberating parasites from the host cell. A regulatory pathway for egress that depends upon increases in intracellular free Ca2+ is proposed. [ABSTRACT FROM AUTHOR]

Investigates the consequences of having multiple fusion complexes on exocytotic granules. Different physiological responses to calcium; Sea urchin egg cortical granule; N-ethylmaleimide inhibition; Spare active fusion complexes of cortical granules; Random distribution of Poisson process; Effects of high calcium concentration.

Individuals infected with the SARS-CoV-2 Delta variant, lineage B.1.617.2, exhibit faster initial infection with a higher viral load than prior variants, and pseudotyped viral particles bearing the SARS-CoV-2 Delta variant spike protein induce a faster initial infection rate of target cells compared to those bearing other SARS-CoV-2 variant spikes. Here, we show that pseudotyped viral particles bearing the Delta variant spike form unique aggregates, as evidenced by negative stain and cryogenic electron microscopy (EM), flow cytometry, and nanoparticle tracking analysis. Viral particles pseudotyped with other SARS-CoV-2 spike variants do not show aggregation by any of these criteria. The contribution to infection kinetics of the Delta spike's unique property to aggregate is discussed with respect to recent evidence for collective infection by other viruses. Irrespective of this intriguing possibility, spike-dependent aggregation is a new functional parameter of spike-expressing viral particles to evaluate in future spike protein variants. [ABSTRACT FROM AUTHOR]

In adipose tissue, resistance to insulin's ability to increase glucose uptake can be induced by multiple factors, including obesity. Impaired insulin action may take place at different spatial loci at the cellular or subcellular level. To begin to understand the spatial response to insulin in human subcutaneous adipose tissue (hSAT), we developed a quantitative imaging method for activation of a major signaling node in the glucoregulatory insulin signaling pathway. After treatment with insulin or control media, biopsied tissues were immunostained for Akt phosphorylation at Thr-308/9 (pAkt) and then imaged by confocal fluorescence microscopy automated to collect a large grid of high resolution fields. In hSAT from 40 men and women with obesity, substantial heterogeneity of pAkt densities in adipocyte membranes were quantified in each image mosaic using a spatial unit of at least twice the size of the point spread function. Statistical analysis of the distribution of pAkt spatial units was best fit as the weighted sum of two separate distributions, corresponding to either a low or high pAkt density. A "high pAkt fraction" metric was calculated from the fraction of high pAkt distributed units over the total units. Importantly, upon insulin stimulation, tissues from the same biopsy showed either a minimal or a substantial change in the high pAkt fraction. Further supporting a two-state response to insulin stimulation, subjects with similar insulin sensitivity indices are also segregated into either of two clusters identified by the amount of membrane- localized pAkt. [ABSTRACT FROM AUTHOR]

Introduction/aims: Individuals with dysferlinopathies, a group of genetic muscle diseases, experience delay in the onset of muscle weakness. The cause of this delay and subsequent muscle wasting are unknown, and there are currently no clinical interventions to limit or prevent muscle weakness. To better understand molecular drivers of dysferlinopathies, age-dependent changes in the proteomic profile of skeletal muscle (SM) in wild-type (WT) and dysferlin-deficient mice were identified., Methods: Quadriceps were isolated from 6-, 18-, 42-, and 77-wk-old C57BL/6 (WT, Dysf +/+ ) and BLAJ (Dysf -/- ) mice (n = 3, 2 male/1 female or 1 male/2 female, 24 total). Whole-muscle proteomes were characterized using liquid chromatography-mass spectrometry with relative quantification using TMT10plex isobaric labeling. Principle component analysis was utilized to detect age-dependent proteomic differences over the lifespan of, and between, WT and dysferlin-deficient SM. The biological relevance of proteins with significant variation was established using Ingenuity Pathway Analysis., Results: Over 3200 proteins were identified between 6-, 18-, 42-, and 77-wk-old mice. In total, 46 proteins varied in aging WT SM (p < .01), while 365 varied in dysferlin-deficient SM. However, 569 proteins varied between aged-matched WT and dysferlin-deficient SM. Proteins with significant variation in expression across all comparisons followed distinct temporal trends., Discussion: Proteins involved in sarcolemma repair and regeneration underwent significant changes in SM over the lifespan of WT mice, while those associated with immune infiltration and inflammation were overly represented over the lifespan of dysferlin-deficient mice. The proteins identified herein are likely to contribute to our overall understanding of SM aging and dysferlinopathy disease progression., (© 2023 Wiley Periodicals LLC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.)

Introduction: Glioblastoma (GBM) is an aggressive primary brain cancer. Lack of effective therapy is related to its highly invasive nature. GBM invasion has been studied with reductionist systems that do not fully recapitulate the cytoarchitecture of the brain. We describe a human-derived brain organotypic model to study the migratory properties of GBM IDH-wild type ex vivo., Methods: Non-tumor brain samples were obtained from patients undergoing surgery (n = 7). Organotypic brain slices were prepared, and green fluorescent protein (GFP)-labeled primary human GBM IDH-wild type cells (GBM276, GBM612, GBM965) were placed on the organotypic slice. Migration was evaluated via microscopy and immunohistochemistry., Results: After placement, cells migrated towards blood vessels; initially migrating with limited directionality, sending processes in different directions, and increasing their speed upon contact with the vessel. Once merged, migration speed decreased and continued to decrease with time (p < 0.001). After perivascular localization, migration is limited along the blood vessels in both directions. The percentage of cells that contact blood vessels and then continue to migrate along the vessel was 92.5% (- 3.9/ + 2.9)% while the percentage of cells that migrate along the blood vessel and leave was 7.5% (- 2.9/ + 3.9) (95% CI, Clopper-Pearson (exact); n = 256 cells from six organotypic cultures); these percentages are significantly different from the random (50%) null hypothesis (z = 13.6; p < 10 -7 ). Further, cells increase their speed in response to a decrease in oxygen tension from atmospheric normoxia (20% O 2 ) to anoxia (1% O 2 ) (p = 0.033)., Conclusion: Human organotypic models can accurately study cell migration ex vivo. GBM IDH-wild type cells migrate toward the perivascular space in blood vessels and their migratory parameters change once they contact vascular structures and under hypoxic conditions. This model allows the evaluation of GBM invasion, considering the human brain microenvironment when cells are removed from their native niche after surgery., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Niemann-Pick, type C1 (NPC1) is a fatal, neurodegenerative disease, which belongs to the family of lysosomal diseases. In NPC1, endo/lysosomal accumulation of unesterified cholesterol and sphingolipids arise from improper intracellular trafficking resulting in multi-organ dysfunction. With the proximity between the brain and cerebrospinal fluid (CSF), performing differential proteomics provides a means to shed light to changes occurring in the brain. In this study, CSF samples obtained from NPC1 individuals and unaffected controls were used for protein biomarker identification. A subset of these individuals with NPC1 are being treated with miglustat, a glycosphingolipid synthesis inhibitor. Of the 300 identified proteins, 71 proteins were altered in individuals with NPC1 compared to controls including cathepsin D, and members of the complement family. Included are a report of 10 potential markers for monitoring therapeutic treatment. We observed that pro-neuropeptide Y (NPY) was significantly increased in NPC1 individuals relative to healthy controls; however, individuals treated with miglustat displayed levels comparable to healthy controls. In further investigation, NPY levels in a NPC1 mouse model corroborated our findings. We posit that NPY could be a potential therapeutic target for NPC1 due to its multiple roles in the central nervous system such as attenuating neuroinflammation and reducing excitotoxicity., (© 2022 The Authors. Proteomics published by Wiley-VCH GmbH.)

To infect, enveloped viruses employ spike protein, spearheaded by its amphipathic fusion peptide (FP), that upon activation extends out from the viral surface to embed into the target cellular membrane. Here we report that synthesized influenza virus FPs are membrane active, generating pores in giant unilamellar vesicles (GUV), and thus potentially explain both influenza virus' hemolytic activity and the liposome poration seen in cryo-electron tomography. Experimentally, FPs are heterogeneously distributed on the GUV at the time of poration. Consistent with this heterogeneous distribution, molecular dynamics (MD) simulations of asymmetric bilayers with different numbers of FPs in one leaflet show FP aggregation. At the center of FP aggregates, a profound change in the membrane structure results in thinning, higher water permeability, and curvature. Ultimately, a hybrid bilayer nanodomain forms with one lipidic leaflet and one peptidic leaflet. Membrane elastic theory predicts a reduced barrier to water pore formation when even a dimer of FPs thins the membrane as above, and the FPs of that dimer tilt, to continue the leaflet bending initiated by the hydrophobic mismatch between the FP dimer and the surrounding lipid., (© 2022. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Fluorescent proteins (FPs) have revolutionized cell biology by allowing genetic tagging of specific proteins inside living cells. In conjunction with Förster's resonance energy transfer (FRET) measurements, FP-tagged proteins can be used to study protein-protein interactions and estimate distances between tagged proteins. FRET is mediated by weak Coulombic dipole-dipole coupling of donor and acceptor fluorophores that behave independently, with energy hopping discretely and incoherently between fluorophores. Stronger dipole-dipole coupling can mediate excitonic coupling in which excitation energy is distributed near instantaneously between coherently interacting excited states that behave as a single quantum entity. The interpretation of FP energy transfer measurements to estimate separation often assumes that donors and acceptors are very weakly coupled and therefore use a FRET mechanism. This assumption is considered reasonable as close fluorophore proximity, typically associated with strong excitonic coupling, is limited by the FP β-barrel structure. Furthermore, physiological temperatures promote rapid vibrational dephasing associated with a rapid decoherence of fluorophore-excited states. Recently, FP dephasing times that are 50 times slower than traditional organic fluorophores have been measured, raising the possibility that evolution has shaped FPs to allow stronger than expected coupling under physiological conditions. In this study, we test if excitonic coupling between FPs is possible at physiological temperatures. FRET and excitonic coupling can be distinguished by monitoring spectral changes associated with fluorophore dimerization. The weak coupling mediating FRET should not cause a change in fluorophore absorption, whereas strong excitonic coupling causes Davydov splitting. Circular dichroism spectroscopy revealed Davydov splitting when the yellow FP Venus A206 dimerizes, and a novel approach combining photon antibunching and fluorescence correlation spectroscopy was used to confirm that the two fluorophores in a Venus A206 homodimer behave as a single-photon emitter. We conclude that excitonic coupling between Venus A206 fluorophores is possible at physiological temperatures., (Published by Elsevier Inc.)