Document Type

Thesis - Open Access

Award Date

2017

Degree Name

Master of Science (MS)

Department / School

Biology and Microbiology

First Advisor

Natalie Thiex

Keywords

Clathrin, CRISPR/Cas9, lysosome, Macrophages, Macropinosome maturation, Vesicular trafficking

Abstract

Macrophages nonspecifically take up extracellular fluids, solutes and macromolecules by macropinocytosis. Understanding the mechanisms of macropinosome maturation will inform the study of lipid uptake, viral entry, antigen processing and presentation, as well as regulation of cell growth. Colony stimulating factor-1 receptor (CSF-1R) is internalized by small vesicle endocytosis, trafficked to nascent macropinosomes and degraded. These CSF-1R positive macropinosomes mature through a sequence similar to endosomes, progressing from EEA1 and Rab5 to Rab7 positive vesicles before fusing with lysosomes. Here we report the assembly of clathrin on internalized macropinosomes shown both by live-cell microscopy of cells expressing clathrin light chain-yellow fluorescent protein (CLTA-YFP) and by immuno-staining of endogenous clathrin heavy chain (CHC). Partial depletion of the clathrin heavy chain by siRNA prevented macropinosome-lysosome fusion and impaired degradation of the CSF- 1R, with only minimal effects on the delivery of the CSF-1R to the macropinosome. Expression of fluorescent protein fusions demonstrates that clathrin assembled on macropinosomes co-localizes with dynamin and possibly the clathrin adaptor, clathrinassembly lymphoid myeloid leukaemia protein (CALM). These data indicate a novel role for clathrin in mediating macropinosome maturation, cargo trafficking and macropinosomes-lysosome fusion in macrophages. We hypothesize that clathrin is involved in an outward budding event during the Rab5 to Rab7 stage of macropinosome maturation. Protein and membrane sorting at this time may predispose the macropinosome to further maturation processes such as attachment to motor proteins and lysosome fusion.

Library of Congress Subject Headings

Macrophages.
Bone marrow.
Endocytosis.
Lysosomes.

Description

Includes bibliographical references

Format

application/pdf

Number of Pages

94

Publisher

South Dakota State University

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Rights Statement

In Copyright