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The inhibition of Fcγ receptors (FcγR) is an attractive strategy for treating diseases driven by IgG immune complexes (IC). Previously, we demonstrated that an engineered tri-valent arrangement of IgG1 Fc domains (SIF1) potently inhibited FcγR activation by IC, whereas a penta-valent Fc molecule (PentX) activated FcγR, potentially mimicking ICs and leading to Syk phosphorylation. Thus, a precise balance exists between the number of engaged FcγRs for inhibition versus activation. Here, we demonstrate that Fc valency differentially controls FcγR activation and inhibition within distinct subcellular compartments. Large Fc multimer clusters consisting of 5-50 Fc domains predominately recruited Syk-mScarlet to patches on the plasma membrane, whereas PentX exclusively recruited Syk-mScarlet to endosomes in human monocytic cell line (THP-1 cells). In contrast, SIF1, similar to monomeric Fc, spent longer periods docked to FcγRs on the plasma membrane and did not accumulate and recruit Syk-mScarlet within large endosomes. Single particle tracking (SPT) of fluorescent engineered Fc molecules and Syk-mScarlet at the plasma membrane imaged by total internal reflection fluorescence microscopy (SPT-TIRF), revealed that Syk-mScarlet sampled the plasma membrane was not recruited to FcγR docked with any of the engineered Fc molecules at the plasma membrane. Furthermore, the motions of FcγRs docked with recombinant Fc (rFc), SIF1 or PentX, displayed similar motions with D ~ 0.15 μm2/s, indicating that SIF1 and PentX did not induce reorganization or microclustering of FcγRs beyond the ligating valency. Multicolor SPT-TIRF and brightness analysis of docked rFc, SIF1 and PentX also indicated that FcγRs were not pre-assembled into clusters. Taken together, activation on the plasma membrane requires assembly of more than 5 FcγRs. Unlike rFc or SIF1, PentX accumulated Syk-mScarlet on endosomes indicating that the threshold for FcγR activation on endosomes is lower than on the plasma membrane. We conclude that the inhibitory effects of SIF1 are mediated by stabilizing a ligated and inactive FcγR on the plasma membrane. Thus, FcγR inhibition can be achieved by low valency ligation with SIF1 that behaves similarly to FcγR docked with monomeric IgG.

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SUMMARYAs the principal internalization mechanism in mammalian cells, clathrin-mediated endocytosis (CME) is critical for cellular signal transduction, receptor recycling, and membrane homeostasis. Acute depletion of cholesterol disrupts CME, motivating analysis of CME dynamics in the context of disrupted cholesterol synthesis, sterol specificity, mechanisms involved, and relevance to disease pathology. Using genome-edited cell lines, we demonstrate that inhibition of post-squalene cholesterol biosynthesis as observed in inborn errors of cholesterol metabolism, results in striking immobilization of CME and impaired transferrin uptake. Imaging of membrane bending dynamics and CME pit ultrastructure revealed prolonged clathrin pit lifetimes and accumulation of shallow clathrin-coated structures that scaled with diminishing sterol abundance. Moreover, fibroblasts derived from Smith-Lemli-Opitz syndrome subjects displayed reduced CME function. We conclude that sterols lower the energetic costs of membrane bending during pit formation and vesicular scission during CME and suggest reduced CME contributes to cellular phenotypes observed within disorders of cholesterol metabolism.

Clathrin‐mediated endocytosis (CME) is a critical cellular pathway for regulation of intracellular signaling, vesicular recycling, and receptor sensitization. While CME is a molecularly complex process which was previously shown to be inhibited by cholesterol depletion, the precise impact of sterol metabolism on CME, the cellular mechanisms involved, and the relevance to disease pathology remain unclear. To determine the impact of sterol homeostasis on CME and possible roles for CME within diseases impacted by cholesterol metabolism, CRISPR/Cas9 gene editing was utilized to fluorescently label clathrin light chain A and dynamin 2 in both immortalized and induced pluripotent stem cell (iPSC) models. To model how cholesterol levels and disease‐relevant sterol changes observed in cholesterol biosynthetic disorders (characterized by the substitution of cellular cholesterol for sterol intermediates) impact endocytosis, CME was analyzed under varying biochemical states. Live cell imaging demonstrated clathrin immobilization at the cell membrane and functional CME impairment occurred following either cholesterol depletion or upon accumulation of sterol species incapable of supporting ordered lipid domains. Polarized total internal reflection fluorescence microscopy demonstrated prolonged clathrin lifetimes associated with endocytic pits with variable curvature in correlation with sterol abundance and support of phase separation. To address the impact of sterol‐mediated CME on neurodevelopment, human iPSCs were analyzed at various stages of differentiation for trafficking deficits following sterol depletion or cholesterol metabolism disruption. CME deficits in iPSCs preceded loss of pluripotency, morphological changes, or aberrant differentiation events observed following cholesterol depletion or sterol intermediate accumulation. CME deficits were also cell‐stage specific, CME activity corresponded to the rate of intracellular sterol metabolism, and organellar deficits with disease relevance resulted from CME deficiencies. Ongoing studies are investigating the consequences of sterol depletion and sterol substitution on developmental signaling pathways and cellular function to provide insight into the mechanistic requirements for sterol homeostasis in CME‐mediated cellular trafficking.

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