Phagocytes engulf unwanted particles into phagosomes that then fuse with lysosomes to degrade the enclosed particles. Ultimately, phagosomes must be recycled to help recover membrane resources that were consumed during phagocytosis and phagosome maturation, a process referred to as “phagosome resolution.” Little is known about phagosome resolution, which may proceed through exocytosis or membrane fission. Here, we show that bacteria-containing phagolysosomes in macrophages undergo fragmentation through vesicle budding, tubulation, and constriction. Phagosome fragmentation requires cargo degradation, the actin and microtubule cytoskeletons, and clathrin. We provide evidence that lysosome reformation occurs during phagosome resolution since the majority of phagosome-derived vesicles displayed lysosomal properties. Importantly, we show that clathrin-dependent phagosome resolution is important to maintain the degradative capacity of macrophages challenged with two waves of phagocytosis. Overall, our work suggests that phagosome resolution contributes to lysosome recovery and to maintaining the degradative power of macrophages to handle multiple waves of phagocytosis.

Phagocytosis is an evolutionarily conserved process. In Protozoa, phagocytosis fulfills a feeding mechanism, while in Metazoa, phagocytosis diversified to play multiple organismal roles, including immune defence, tissue homeostasis, and remodeling. Accordingly, phagocytes display a high level of plasticity in their capacity to recognize, engulf, and process targets that differ in composition and morphology. Here, we review how phagocytosis adapts to its multiple roles and discuss in particular the effect of target morphology in phagocytic uptake and phagosome maturation.

Abbreviations: Arl 8: Arf-like GTPase 8, CLC-GFP: GFP-fusion of the clathrin-light chain; ConA: concanamycin A; DQ-BSA: dye-quenched bovine serum albumin; IKA: ikarugamycin; LAMP1: lysosomal-associated membrane protein 1; LAMP2: lysosomal-associated membrane protein 2; LB: Luria-Bertani medium; Lp: Legionella pneumophila; mTORC1: mechanistic target of rapamycin complex 1; PDV: phagosome-derived vesicle; PtdIns(3)P: phosphatidylinositol 3-phosphate; PtdIns(3,5)P 2 : phosphatidylinositol 3,5-biphosphate; PtdIns(4)P: phosphatidylinositol 4-phosphate; TFEB: transcription factor EB Summary Phagocytes engulf particles into phagolysosomes for degradation. However, the ultimate fate of phagolysosomes is undefined. Lancaster, Fountain et al. show that phagosomes undergo fragmentation to reform lysosomes in a clathrin-dependent manner. This process is necessary to maintain the degradative capacity of phagocytes during subsequent phagocytosis. AbstractDuring phagocytosis, phagocytes like macrophages engulf and sequester unwanted particles like bacteria into phagosomes. Phagosomes then fuse with lysosomes to mature into phagolysosomes, resulting in the degradation of the enclosed particle. Ultimately, phagosomes must be recycled to help recover membrane resources like lysosomes consumed during phagocytosis, a process referred to as phagosome resolution. Little is known about phagosome resolution, which may proceed through exocytosis or membrane fission. Here, we show that bacteria-containing phagolysosomes in macrophages undergo fragmentation through vesicle budding, tubulation, and constriction. Phagosome fragmentation required cargo degradation, the actin and microtubule 3 cytoskeletons, and clathrin. We provide evidence that lysosome reformation occurs during phagosome resolution since the majority of phagosome-derived vesicles displayed lysosomal properties. Importantly, we showed that the clathrin-dependent phagosome resolution is important to maintain the degradative capacity of macrophages challenged with two waves of phagocytosis. Overall, our work suggests that phagosome resolution contributes to lysosome recovery and to maintain the degradative power of macrophages to handle multiple waves of phagocytosis.

Bone loss is a major health problem that many aging individuals will face and thus research focusing on enhancing bone formation is of great importance. Cell biology or in vitro studies are particularly useful in exploring the exact effects a vitamin, supplement or drug has on particular processes within a certain cell type. Although there have been many cell biology articles focusing on the effects of vitamin D, K 1 or K 2 addition on bone formation in vitro, there has yet to be a consensus amongst the literature. The purpose of this article is to determine the effects of vitamin D, K 1 and K 2 supplementation on osteoblast maturation parameters through meta-analysis of past cell biology literature. A Hedges d effect size was calculated for each experiment extracted from past literature and the experiments were grouped by experiment and cell type. Homogeneity was assessed by the Cochran's Q test, while the effect sizes' departure from zero was assessed by a 95% confidence interval and a non-directional test. Supplementation with vitamin D, K 1 and K 2 , along with the combination of vitamin K 2 + 1,25-dihydroxyvitamin D, increased bone mineralization, while not consistently affecting all of the other parameters associated with bone formation. Vitamin K 2 and D addition had variable effects on bone formation using different cell types, which calls into question the suitability of particular cell lines as models for clinical trials. Therefore, the conditions and parameters in which bone formation is studied in vitro must be considered carefully before running a vitamin supplementation or drug-testing experiment.

Aluminum salts have been successfully utilized as adjuvants to enhance the immunogenicity of vaccine antigens since the 1930s. However, the cellular mechanisms behind the immune adjuvanticity effect of these materials in antigen‐presenting cells are poorly understood. In this study, we investigated the uptake and trafficking of aluminum oxy‐hydroxide (AlOOH), in RAW 264.7 murine and U‐937 human macrophages‐like cells. Furthermore, we determined the impact that the adsorption to AlOOH particulates has on the trafficking of a Bordetella pertussis vaccine candidate, the genetically detoxified pertussis toxin (gdPT). Our results indicate that macrophages internalize AlOOH by constitutive macropinocytosis assisted by the filopodial protrusions that capture the adjuvant particles. Moreover, we show that AlOOH has the capacity to nonspecifically adsorb IgG, engaging opsonic phagocytosis, which is a feature that may allow for more effective capture and uptake of adjuvant particles by antigen‐presenting cells (APCs) at the site of vaccine administration. We found that AlOOH traffics to endolysosomal compartments that hold degradative properties. Importantly, while we show that gdPT escapes degradative endolysosomes and traffics toward the retrograde pathway, as reported for the wild‐type pertussis toxin, the adsorption to AlOOH diverts gdPT to traffic to the adjuvant’s lysosome‐type compartments, which may be key for MHC‐II‐driven antigen presentation and activation of CD4+ T cell. Thus, our findings establish a direct link between antigen adsorption to AlOOH and the intracellular trafficking of antigens within antigen‐presenting cells and bring to light a new potential mechanism for aluminum adjuvancy. Moreover, the in‐vitro single‐cell approach described herein provides a general framework and tools for understanding critical attributes of other vaccine formulations.

Phagocytosis is the selective internalization of large particles and is critical for immunity, development and tissue homeostasis. The conversion of phagosomes into microbicidal compartments is a highly regulated process, which has been studied in great detail. Conversely, the fate of phagolysosomes, after their cargo is digested, is poorly understood. By utilizing fluorescent bacteria and labelling phagosomal markers, we followed mature phagolysosomes to their resolution. Despite previous assumptions, phagolysosomes are not exocytosed, but instead undergo a continuous dismembering process resulting in the shedding of myriads of vesicles containing degraded cargo. These phagosome‐derived vesicles resemble lysosomes, based on the acquisition of lysosomal markers, acidification and fusogenicity with newly formed phagosomes. Combining pharmacological and genetic strategies, we found that phagosomal fragmentation is dependent on cargo degradation, clathrin and the integrity of the actin and microtubule cytoskeleton. In addition, we provide evidence that phagosome‐derived vesicles are required to recycle lysosomal components allowing for the formation of new phagosomes and consequently maintaining the capacity of macrophages to ingest targets. Thus, we propose that phagolysosome resolution recycles cellular resources for the continuous generation of new phagosomes and the prevention of this phase will critically impair the immune and housekeeping functions of the phagocytes. Support or Funding Information Canadian Institutes of Health Research Canada Research Chairs Program Ryerson University Natural Sciences and Engineering Council of Canada Clathrin mediates phagosome resolution. A. Macrophages engulfed mRFP1‐expressing E. coli. At 1 h post‐phagocytosis, phagosomes are rod‐shaped, like E. coli and mRFP1 is mostly associated with phagosomes. After 4 h of phagosome maturation, phagosomes shrunk and mRFP1 is found in small vesicular compartments. Clathrin inhibitors, pitstop and ikarugamycin, impair phagosome fragmentation. B. Quantification of phagosome‐derived vesicles, where b is significantly different from other conditions.

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<p>Phagocytes engulf unwanted particles into phagosomes that then fuse with lysosomes to degrade the enclosed particles. Ultimately, phagosomes must be recycled to help recover membrane resources that were consumed during phagocytosis and phagosome maturation, a process referred to as “phagosome resolution.” Little is known about phagosome resolution, which may proceed through exocytosis or membrane fission. Here, we show that bacteria-containing phagolysosomes in macrophages undergo fragmentation through vesicle budding, tubulation, and constriction. Phagosome fragmentation requires cargo degradation, the actin and microtubule cytoskeletons, and clathrin. We provide evidence that lysosome reformation occurs during phagosome resolution since the majority of phagosome-derived vesicles displayed lysosomal properties. Importantly, we show that clathrin-dependent phagosome resolution is important to maintain the degradative capacity of macrophages challenged with two waves of phagocytosis. Overall, our work suggests that phagosome resolution contributes to lysosome recovery and to maintaining the degradative power of macrophages to handle multiple waves of phagocytosis. </p>