Visualizing the Early Stages of Phagocytosis

Ali Rashidfarrokhi; Veronica Richina; Fikadu G. Tafesse

doi:10.3791/54646

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Method Article

Visualizing the Early Stages of Phagocytosis

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DOI:

10.3791/54646

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February 3rd, 2017

Ali Rashidfarrokhi^*² , Veronica Richina^*² , Fikadu G. Tafesse¹^,²

¹Department of Molecular Microbiology & Immunology, Oregon Health & Science University, ²Ragon Institute of MGH, MIT and Harvard

^* These authors contributed equally

Summary

Here we describe a microscope-based technique to visualize and quantify the early cascades of events during phagocytosis of pathogens such as the fungi Candida albicans and particulates that are larger than 0.5 µm including zymosan and IgG-coated beads.

Abstract

The mammalian body is equipped with various layers of mechanisms that help to defend itself from pathogen invasions. Professional phagocytes of the immune system — such as neutrophils, dendritic cells, and macrophages — retain the innate ability to detect and clear such invading pathogens through phagocytosis¹. Phagocytosis involves choreographed events of membrane reorganization and actin remodeling at the cell surface²^,³. Phagocytes successfully internalize and eradicate foreign molecules only when all stages of phagocytosis are fulfilled. These steps include recognition and binding of the pathogen by pattern recognition receptors (PRRs) residing at the cell surface, formation of phagocytic cup through actin-enriched membranous protrusions (pseudopods) to surround the particulate, and scission of the phagosome followed by phagolysosome maturation that results in the killing of the pathogen³^,⁴.

Imaging and quantification of various stages of phagocytosis is instrumental for elucidating the molecular mechanisms of this cellular process. The present manuscript reports methods to study the different phases of phagocytosis. We describe a microscope-based approach to visualize and quantify the binding, phagocytic cup formation, and the internalization of particulate by phagocytes. As phagocytosis occurs when innate receptors on phagocytic cells encounter ligands on a target particle bigger than 0.5 µm, the assays we present here comprise the use of pathogenic fungi Candida albicans and other particulates such as zymosan and IgG-coated beads.

Introduction

Despite the continuous exposure to pathogens such as bacteria, viruses and fungi, our body is well equipped with immune mechanism that provide protection against infection. The innate immune system is the first line of defense against invading pathogens and relies mainly on phagocytic cells that recognize and internalize foreign targets.

Phagocytosis is an evolutionarily conserved cellular process that encompasses the engulfment of unwanted particulates greater than 0.5 µm. Phagocytic cells express a wide range of immune receptors (also known as pattern recognition receptors, PRRs) at the cell surface that enable them to recognize path....