Phagocytosis: A (Sphingo)Lipid Story

Saharan, Ojal; Mehendale, Neelay; Kamat, Siddhesh S.

doi:10.1016/j.crchbi.2022.100030

Cited by 4 publications

(2 citation statements)

References 20 publications

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“…These span from the facilitation of receptor clustering and activation [ 7 , 8 ], to the initial membrane deformation of phagocytic cups [ 9 , 10 ], to the promotion of phago–lysosome fusion [ 11 , 12 , 13 ], and the inhibition of antigen presentation in dendritic cells [ 14 , 15 ]. However, compared to phosphoinositides, less is known about the dynamics, regulation, and roles of ceramide and its related lipids during this critical immune process [ 16 ].…”

Section: Introductionmentioning

confidence: 99%

Development of Genetically Encoded Fluorescent KSR1-Based Probes to Track Ceramides during Phagocytosis

Girik,

van Ek,

Dentand Quadri

et al. 2024

IJMS

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Ceramides regulate phagocytosis; however, their exact function remains poorly understood. Here, we sought (1) to develop genetically encoded fluorescent tools for imaging ceramides, and (2) to use them to examine ceramide dynamics during phagocytosis. Fourteen enhanced green fluorescent protein (EGFP) fusion constructs based on four known ceramide-binding domains were generated and screened. While most constructs localized to the nucleus or cytosol, three based on the CA3 ceramide-binding domain of kinase suppressor of ras 1 (KSR1) localized to the plasma membrane or autolysosomes. C-terminally tagged CA3 with a vector-based (C-KSR) or glycine-serine linker (C-KSR-GS) responded sensitively and similarly to ceramide depletion and accumulation using a panel of ceramide modifying drugs, whereas N-terminally tagged CA3 (N-KSR) responded differently to a subset of treatments. Lipidomic and liposome microarray analysis suggested that, instead, N-KSR may preferentially bind glucosyl-ceramide. Additionally, the three probes showed distinct dynamics during phagocytosis. Despite partial autolysosomal degradation, C-KSR and C-KSR-GS accumulated at the plasma membrane during phagocytosis, whereas N-KSR did not. Moreover, the weak recruitment of C-KSR-GS to the endoplasmic reticulum and phagosomes was enhanced through overexpression of the endoplasmic reticulum proteins stromal interaction molecule 1 (STIM1) and Sec22b, and was more salient in dendritic cells. The data suggest these novel probes can be used to analyze sphingolipid dynamics and function in living cells.

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Section: Introductionmentioning

confidence: 99%

Development of Genetically Encoded Fluorescent KSR1-Based Probes to Track Ceramides during Phagocytosis

Girik,

van Ek,

Dentand Quadri

et al. 2024

IJMS

View full text Add to dashboard Cite

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“…These span from facilitation of receptor clustering and activation (Abdel Shakor, Kwiatkowska, and Sobota 2004;Avota, Gulbins, and Schneider-Schaulies 2011), to initial membrane deformation of phagocytic cups (Magenau et al 2011;Niekamp et al 2021), to promotion of phago-lysosome fusion (Schramm et al 2008;Wu et al 2018;Hinkovska-Galcheva et al 2005), and inhibition of antigen presentation in dendritic cells (Sallusto et al 1996;Eva Fuglsang, Louise Boye, and Hanne Frokiaer 2017). However, compared to phosphoinositides, less is known about the dynamics, regulation and roles of Cer and its related lipids during this critical immune process (Saharan, Mehendale, and Kamat 2022).…”

Section: Introductionmentioning

confidence: 99%

Development of genetically-encoded fluorescent KSR1-based probes to track ceramides during phagocytosis

Girik,

van Ek,

Quadri

et al. 2023

Preprint

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Ceramides (Cer) and their derivatives have been suggested to regulate phagocytosis, however their exact function remains poorly understood. Genetically-encoded fluorescent biosensors have been immensely useful in deciphering the critical role of phagosomal phosphoinositide regulation, and thus we sought to develop similar tools to examine Cer dynamics during phagocytosis. EGFP fusion of several known Cer-binding domains were generated and expressed in cells. While most constructs localized to the nucleus or cytosol, the CA3 cysteine-rich domain of the pseudokinase KSR1 displayed additionally plasma membrane (PM) or endolysosomal localization, depending on the position of the fluorescent tag and linker. C-terminally tagged KSR1-CA3 (C-KSR) and a more stable version C-KSR-GS responded as expected to Cer depletion and accumulation using a panel of Cer modifying drugs. In contrast, N-terminally tagged N-KSR did not respond to Cer induction by palmitate or glucosylceramide (GlcCer) synthase inhibitor Genz-123346. Liposome microarray analysis suggested N-KSR binding to the Cer derivative GlcCer. Interestingly, the three probes showed distinct dynamics during phagocytosis in model phagocytic fibroblasts. Despite partial lysosomal degradation, C-KSR robustly but transiently re-localized to the PM during phagocytosis, whereas N-KSR re-localized from the PM to the cytoplasm at later timepoints. Moreover, occasional weak recruitment of C-KSR-GS to endoplasmic reticulum and phagosome was enhanced upon overexpression of the endoplasmic reticulum proteins STIM1 and Sec22b, and was more salient in dendritic cells. The data suggest these novel probes may be valuable new members of the growing molecular toolkit used to analyze sphingolipid dynamics and function in living cells.

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Mapping lipid pathways during phagocytosis

Saharan

Kamat

2023

Biochemical Society Transactions

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Phagocytosis is an evolutionarily conserved important immunological process in higher organisms, and acts as the first line of defense against invading pathogenic microbial infections. Additionally, this dynamic innate immune response is also critical for clearing apoptotic cells and/or tissues, is responsible for maintaining homeostasis and acts as a systemic regulator of critical physiological processes such as wound healing and tissue regeneration. Over the past two decades, numerous studies have shown that phagocytosis occurs in three spatiotemporally distinct steps, namely formation, maturation and resolution of the phagosome, and that, both the protein and lipid composition change as a function of the aforementioned steps during this immunological process. While significant knowledge is now available on the proteomic content of a phagosome during the different stages of phagocytosis, the lipidome however, remained lesser explored, until the past few years. In this review, we summarize recent efforts towards mapping the physiological roles and functions of three lipid classes, the phosphatidylinositols, cholesterol and sphingolipids during the various stages of phagocytosis, and discuss strategies evolved by microbes to hijack and/or disrupt these lipid pathways to evade the immune system. We conclude this review with some potential avenues that may be pursued towards mapping hitherto unknown lipid pathways during phagocytosis, and how this research might be beneficial in our ongoing battle to overcome pathogenic infections.

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Phagocytosis: A (Sphingo)Lipid Story

Cited by 4 publications

References 20 publications

Development of Genetically Encoded Fluorescent KSR1-Based Probes to Track Ceramides during Phagocytosis

Development of Genetically Encoded Fluorescent KSR1-Based Probes to Track Ceramides during Phagocytosis

Development of genetically-encoded fluorescent KSR1-based probes to track ceramides during phagocytosis

Mapping lipid pathways during phagocytosis

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