Biology subjects

Nunes-Hasler, P.

Publications and source records attributed to Nunes-Hasler, P..

3 recordsLinked to original sources

bioRxiv2023

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

Ceramides regulate phagocytosis, however their exact function remains poorly understood. Here we sought 1) to develop genetically encoded fluorescent tools for imaging ceramide, and 2) to use them to examine ceramide dynamics during phagocytosis. Fourteen 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 KSR1 localized to plasma membrane or endolysosomes. 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 preferentially binds to glucosyl-ceramide. Additionally, the three probes showed distinct dynamics during phagocytosis. Despite partial lysosomal degradation, C-KSR robustly accumulated at the plasma membrane during phagocytosis, whereas N-KSR becomes cytoplasmic at later timepoints. Moreover, weak recruitment of C-KSR-GS to endoplasmic reticulum and phagosomes was enhanced by overexpression of the endoplasmic reticulum proteins 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.

cell biology↗

bioRxiv2022

The lipid transfer proteins Nir2 and Nir3 sustain phosphoinositide signaling and actin dynamics during phagocytosis

Changes in membrane phosphoinositides and local Ca2+ elevations at sites of particle capture coordinate the dynamic remodeling of the actin cytoskeleton during phagocytosis. Here, we show that the phosphatidylinositol (PI) transfer proteins PITPNM1 (Nir2) and PITPNM2 (Nir3) maintain PI(4,5)P2 homeostasis at phagocytic cups, thereby promoting actin contractility and the sealing of phagosomes. Nir3 and to a lesser extent Nir2 accumulated in ER cisternae juxtaposed to phagocytic cups when expressed in phagocytic mouse fibroblasts. CRISPR-Cas9 editing of Nir2 and Nir3 genes decreased plasma membrane PI(4,5)P2 levels, store-operated Ca2+ entry (SOCE), and receptor-mediated phagocytosis, stalling particle capture at cup stage. Re-expression of either Nir2 or Nir3 restored phagocytosis, but not SOCE, proportionally to the PM PI(4,5)P2 levels. Phagosomes forming in Nir2/3-edited cells had decreased overall PI(4,5)P2 levels but normal periphagosomal Ca2+ signals. Nir2/3 editing reduced the density of contractile actin rings at sites of particle capture, causing repetitive low-intensity contractile events indicative of abortive phagosome closure. We conclude that Nir-mediated lipid transfer maintains phosphoinositide homeostasis at phagocytic cups, thereby sustaining the signals that initiate the remodeling of the actin cytoskeleton during phagocytosis. Summary statementChanges in membrane phosphoinositides coordinate actin remodeling during phagocytosis, but whether lipid transport proteins contribute to this process is not known. Here, we show that the phosphatidylinositol transfer proteins Nir2 and Nir3 are recruited to phagocytic cups and drive the formation of contractile actin rings during particle engulfment. Using gene editing and re-expression, we show that Nir2 and Nir3 maintain PI(4,5)P2 signaling competence at phagocytic cups and promote the actin-dependent sealing of phagocytic vacuoles. These observations establish that lipid transport proteins maintain the phosphoinositide signals that drive the remodeling of the actin cytoskeleton during phagocytosis.

cell biology↗

bioRxiv2022

The SNARE Sec22b regulates phagosome maturation by promoting ORP8-mediated PI(4)P exchange at ER-phagosome contact sites.

The precise control of phagosome maturation is critical for innate and adaptive immunity, determining whether phagocytosed material is destroyed or used to present antigens. We observed previously that non-fusogenic contacts between the endoplasmic reticulum (ER) and phagosomes, called membrane contact sites (MCS), are tethered by the calcium regulator STIM1 and fine-tune phagosomal maturation. The secretory pathway SNARE protein Sec22b has been implicated in controlling phagocytosis, phagosome maturation and antigen presentation, though its effects are controversial, and its mechanism of action poorly understood. Recently, Sec22b was shown to tether MCS at the plasma membrane without mediating membrane fusion. Here, we show that Sec22b localizes to and regulates the frequency of ER-phagosome contacts independently of STIM proteins. Sec22b knockdown and overexpression of a an MCS-disrupting mutant Sec22b-P33 induced only mild or no effect on global and local calcium signalling. However, Sec22b knockdown altered phagosomal phospholipids including PI(3)P, PI(4)P and PS, but not PI(4,5)P2. Increased PI(4)P in shSec22b cells was rescued by re-expression of Sec22b or the artificial MCS tether MAPPER but not the P33 mutant. Moreover, Sec22b co-precipitated and was co-recruited to phagosomes with the PS/PI(4)P lipid exchange protein ORP8. Expression of wild-type, but not mutant ORP8, also rescued phagosomal PI(4)P. Concordantly, Sec22b, MAPPER and ORP8 but not P33 or the ORP8 mutant decreased phagolysosome fusion in shSec22b cells. These results clarify a novel mechanism through which Sec22b controls phagosome maturation and beg a reassessment of the relative contribution of Sec22b-mediated fusion versus tethering to phagosome biology.

cell biology↗