Macropinocytosis, or ?cell drinking,? is central to critical macrophage functions including wound healing, antigen presentation, and the resolution of inflammation. However, there are large gaps in the mechanistic understanding of this process. The long-term goal of this project is to identify the mediators and the cellular mechanisms of macropinocytosis. The overall objective is to investigate how the macrophage mannose receptor (MRC1), a cell surface carbohydrate receptor, mediates uptake of fluids and solutes from the cellular environment via macropinocytosis. The central hypothesis is that MRC1 promotes the uptake of branched sugars and other ligands by binding extracellular ligands and mediating their subsequent internalization on newly forming macropinosomes. This hypothesis stems from preliminary CRISPR/Cas9 whole genome screen data produced in the applicant's laboratory, demonstrating that the Mrc1 gene and molecules that regulate MRC1 protein abundance on the cell surface are key regulators of macropinocytosis. The hypothesis will be tested by pursuing two specific aims: 1) Determine how MRC1 promotes uptake of dextran in macrophages, and 2) Determine the mechanisms of MRC1 internalization from the cell surface in the presence or absence of dextran and other ligands. Under the first aim, a chemical conjugation technique established in the PI's lab will be used to prepare dextrans of different molecular weights, charges and fluorophore conjugations for evaluation of the chemical and physical parameters that modulate the uptake efficiency of dextrans by wildtype and MRC1-deficient macrophages.
The second aim will determine the route of MRC1 uptake into macropinosomes using an immunofluorescent staining/microscopy approach established by the applicant to image the movements of MRC1 during macropinocytosis. Specifically, co-localization of MRC1 with sites of actin polymerization, membrane protrusion, and 3'-phosphoinositide production will be measured. This approach is innovative because the hypothesis was generated from new mediators of macropinocytosis identified by a CRISPR/Cas9 whole genome screen. Furthermore, this strategy uses targeted gene disruptions in combination with new cellular fluorescent probes and live-cell microscopy techniques to interrogate the mechanisms of macropinocytosis. The proposed research is significant because it is expected to expand the understanding of machinery and molecular mechanisms of macropinocytosis. Ultimately, such knowledge has the potential to identify therapeutic targets for the modulation of macropinocytosis and to optimize the targeting of therapeutics to macrophages for treatment of cancer or immune diseases using MRC1 ligands.
The proposed research is relevant to public health because the discovery of mechanisms regulating macropinocytosis will increase the understanding of the pathogenesis of inflammatory and auto-immune diseases and cancer. Thus, the proposed research is relevant to NIH's mission, in that it will contribute to developing fundamental knowledge that will help reduce the burden of human disease.
