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

• Published in: International journal of molecular sciences Vol. 25; no. 5; p. 2996
• Main Authors: Girik, Vladimir, van Ek, Larissa, Dentand Quadri, Isabelle, Azam, Maral, Cruz Cobo, María, Mandavit, Marion, Riezman, Isabelle, Riezman, Howard, Gavin, Anne-Claude, Nunes-Hasler, Paula
• Format: Journal Article
• Language: English
• Published: Switzerland MDPI AG 01.03.2024; MDPI
• Subjects: Biosensors; C6-ceramide; Ceramides; Ceramides - metabolism; Dendritic cells; DNA microarrays; Fluorescence; Fluorescent Dyes; Glycine; kinase suppressor of ras 1 (KSR1); Kinases; Lipids; Localization; Mass spectrometry; Membranes; Phagocytosis; phosphatase 2A inhibitor 2 (I2PP2A); Plant lipids; protein kinase c zeta (PRKCZ); Protein Kinases; Proteins; Scientific imaging; SET nuclear proto-oncogene; Signal Transduction - physiology; sphingomyelinase; Canada; United Kingdom; Massachusetts; Germany; stromal interaction molecule 1 (STIM1); C6-ceramide; SET nuclear proto-oncogene; fumonisin B1; protein kinase c zeta (PRKCZ); sphingomyelinase; myriocin; phosphatase 2A inhibitor 2 (I2PP2A); Sec22b; lipidomics; kinase suppressor of ras 1 (KSR1)
• ISSN: 1422-0067; 1661-6596; 1422-0067
• DOI: 10.3390/ijms25052996

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.