Two-photon excitation imaging of exocytosis and endocytosis and determination of their spatial organization

Abstract

Two-photon excitation imaging is the least invasive optical approach to study living tissues. We have established two-photon extracellular polar-tracer (TEP) imaging with which it is possible to visualize and quantify all exocytic events in the plane of focus within secretory tissues. This technology also enables estimate of the precise diameters of vesicles independently of the spatial resolution of the optical microscope, and determination of the fusion pore dynamics at nanometer resolution using TEP-imaging based quantification (TEPIQ). TEP imaging has been applied to representative secretory glands, e.g., exocrine pancreas, endocrine pancreas, adrenal medulla and a pheochromocytoma cell line (PC12), and has revealed unexpected diversity in the spatial organization of exocytosis and endocytosis crucial for the physiology and pathology of secretory tissues and neurons. TEP imaging and TEPIQ analysis are powerful tools for elucidating the molecular and cellular mechanisms of exocytosis and certain related diseases, such as diabetes mellitus, and the development of new therapeutic agents and diagnostic tools.

Introduction

Exocytic secretion is one of the fundamental cellular mechanisms for the delivery of biosynthetic materials contained in cytosolic vesicles to the extracellular space [1], [2]. Exocytosis involves the transport of secretory vesicles to target regions in the plasma membrane, the docking of vesicles to the plasma membrane and fusion of the two biological membranes, which may be followed by compound exocytosis on the fused vesicles and/or endocytosis. Exocytosis is regulated by cytosolic Ca²⁺, cAMP and GTPase in endocrine cells, exocrine cells, hematopoietic cells and neurons [3], [4]. Secretory substances are contained in large dense-core vesicles with diameters of 150–2000 nm in many secretory cells, while neurotransmitters such as glutamate and γ-aminobutyric acid (GABA) are contained in small vesicles with diameters of 30–90 nm. It has been clarified that every type of cell possesses Ca²⁺-dependent exocytosis of small vesicles and lysosomes, which may be utilized for housekeeping purposes such as membrane repair [5], [6], [7], [8]. Small synaptic vesicle exocytosis might have evolved from such housekeeping cellular mechanisms [9]. Thus, even single cells normally have multiple types of secretory vesicles with distinct diameters. The excess membrane inserted by exocytosis must be taken up by endocytic mechanisms [10], which also involve multiple types of vesicles with distinct diameters.

The key molecules involved in exocytosis have been identified over the last 15 years [11], [12], [13], [14]. The fundamental mechanisms of these complex processes, however, are still largely unknown. This situation is in striking contrast with ion-channel research, where the functions of interest are readily reduced to single ion-channel molecules, and can be precisely measured with electrophysiological techniques. In contrast, a single exocytic event involves multiple copies of distinct proteins, including SNAREs, synaptotagmins and myriad membrane lipid molecules. To make the situation worse, direct measurement of the individual steps of exocytosis itself is still a considerable challenge. For instance, a core step of exocytosis, membrane fusion, is triggered by hemifusion of two biological membranes and opening of a nanometer fusion pore that connects the extracellular space and vesicles. An ideal methodology would be able to track the sizes and histories of vesicles, reveal the dynamics of fusion pore formation and fates of Ω-shaped profile of fused vesicles, and follow endocytosis.

Several aspects of the research into exocytosis and endocytosis have tight links to clinical, pharmaceutical and industrial activities. It is known that exocytosis is impaired in some diseases, for example, insulin secretion in diabetes mellitus [15], zymogen granule exocytosis in pancreatitis [16], [17], and, very likely, synaptic vesicle exocytosis in mental disorders. Measurement of exocytosis in native and regenerated tissues is key to the development of new drugs and therapy. Exocytosis is an advanced form of drug delivery in biological systems, and understanding its mechanisms undoubtedly would help develop new approaches for drug delivery. In this review, we introduce two-photon excitation imaging of exocytosis as a powerful tool to achieve these goals [18], [19], [20], [21], [22], [23], [24], [25], [26], [27], and explain the importance of developing user-friendly two-photon microscope systems. We first introduce classical approaches to the study of exocytosis, and then explain the advantages of two-photon excitation imaging approaches.