Document Type

Dissertation - Open Access

Award Date


Degree Name

Doctor of Philosophy (PhD)

Department / School

Chemistry and Biochemistry

First Advisor

Adam D. Hoppe


CRISPR/Cas9, Fc receptors Liposomes, Macropinocytosis, Nanoparticles, Traffick


Macrophages are tissue-resident phagocytes that play critical roles in immune response and tissue homeostasis. They have a tremendous capacity to internalize objects of various sizes ranging from nanoscale viral particles to micron sized bacteria and tumor cells. This phenomenon, termed phagocytosis for the uptake of large particles, or endocytosis for the uptake of small particles, is integral to the immune response in multicellular organisms. Macrophages express FcγRs, which are tyrosine kinase receptors that bind IgG on an opsonized target. Binding of IgG Fc domain to the extracellular domain of an FcγR triggers signaling cascades that coordinate internalization of the opsonized target, generation of reactive oxygen species and release of cytokines. FcγR mediated phagocytosis of large particles has been characterized in macrophages and dendritic cells, but an understanding of FcγR endocytosis of small particles is limited. Insight into FcγR endocytosis in macrophages will be useful in the design and targeting of therapeutic antibodies for treatment of cancers and viral infections. Here, ~100nm fluorescent PEGylated liposomes displaying surface biotin antigen and antibiotin IgG2a were used to investigate FcγR trafficking. Internalization of liposomes occurred by FcγR dependent endocytosis with no contributions from macropinocytosis. Rather, liposomes associated with clathrin on the plasma membrane and within the cytoplasm at early time points, demonstrating that the endocytic process involved the participation of clathrin. Internalized IgG2a-liposome complexes were trafficked to the limiting membrane of macropinosomes where IgG was segregated from cargo liposomes in a pH dependent manner. Contrary to trafficking of CSF-1 in macrophages, segregated IgG and liposomes were excluded from the lumen of macropinosomes and did not undergo immediate intraluminal budding, demonstrating that lysosomes did not immediately destroy the immune complex. Segregated IgG was recycled back to the cell surface where it was capable of phagocytosing new biotinylated SRBCs. Although macropinosomes and liposomes formed a multivesicular object, three-color live cell microscopy showed that fluid phase marker movement did not predict movement of liposomes to the lysosome. Lysosomes rapidly internalized luminal content of macropinosomes via piranhalysis (or squidlysis), but interacted with IgG-liposome complexes by kiss-and-run events resulting in early transition of fluid phase marker and delayed transition of liposome cargo into lysosomal compartments. Thus, we show that the macropinosome has a novel role in organizing antigen-antibody segregation in macrophages. The contributions of murine FcγRs (I, IIb, and III) and FcRγ to endocytosis of IgG2aliposomes were delineated using single and multiple receptor knockouts in macrophages. Knockouts were created by means of synthetic gRNAs targeting FcγRs and FcRγ in FLM cells harvested from CRISPR/Cas9 transgenic mice. Analysis of particle uptake showed that FcγRs drive differential internalization of IgG2a-liposomes with binding and uptake of particles heavily dependent on high affinity FcγR I and FcRγ. Knockout of three receptors namely FcγR I, IIb, and III abolished binding and uptake of particles. Analysis of fluid phase uptake in knockout lines showed that FcγR I, III, IIb&III, FcRγ, and TKO were defective for macropinosome formation, whilst FcγR IIb knockouts produced more than twice the number of macropinosomes compared to parental cells. Together, these demonstrate that activating FcγRs may have a novel role in driving constitutive macropinosome formation attenuated by inhibitory FcγR IIb.

Library of Congress Subject Headings

Fc receptors.


Includes bibliographical references



Number of Pages



South Dakota State University



Rights Statement

In Copyright