Secretion and membrane fusion are fundamental cellular processes regulating endoplasmic reticulum (ER)-Golgi and Golgi-Golgi transport, plasma membrane recycling, cell division, sexual reproduction, acid secretion, histamine release, and the release of enzymes, hormones, and neurotransmitters, to name just a few. It is therefore no surprise that defects in secretion and membrane fusion lead to diabetes, Alzheimer’s, Parkinson’s, and a host of diseases. In view of this, there has been significant effort during the past half century to understand the molecular machinery and mechanism of secretion and membrane fusion in cells. Only in the last decade, studies using atomic force microscopy (AFM) and conventional biochemical, electrophysiological, and imaging approaches have provided a molecular understanding of these processes in cells (1–21). With these findings (1–21) made primarily in the author’s laboratory, a new understanding of cell secretion has emerged. These studies further demonstrate secretory vesicles to transiently dock and fuse at the cell plasma membrane, which has been confirmed by a number of laboratories (22–27).
Throughout history, the development of new imaging tools has provided new insights into our perceptions of the living world and profoundly impacted human health. The invention of the light microscope, almost 300 years ago, was the first catalyst, propelling us into an era of modern biology and medicine. Using the light microscope, a giant step into the gates of modern biology and medicine was made with the discovery of the unit of life, the cell. The structure and morphology of normal and diseased cells and of disease-causing microorganisms were revealed for the first time using the light microscope. Then in 1938, with the birth of the electron microscope (EM), dawned a new era. Through the mid 1940s and 1950s, a number of subcellular organelles were discovered and their functions determined using the EM. Viruses, the new life forms, were identified and observed for the first time and implicated in diseases ranging from the common cold to autoimmune disease (AIDS). Despite the capability of the EM to image biological samples at near-nanometer resolution, sample processing resulting in morphological alterations remained a major concern. Then, in the mid 1980s, scanning probe microscopy evolved (1, 28), further extending our perception of the living world to the near-atomic realm. One such scanning probe microscope, the AFM, has helped overcome both limitations of light and electron microscopy, enabling determination of the structure and dynamics of single biomolecules and live cells in three dimensions, at near-angstrom resolution. This unique capability of the AFM in combination with conventional tools and approaches has provided an understanding of cellular secretion (1–21) and membrane fusion (9, 17, 18, 29) at the molecular level.
The resolving power of the light microscope is dependent on the wavelength of the light used and, hence, 250–300 nm in lateral and much less in depth resolution can at best be achieved using light for imaging. The porosome or fusion pore in live secretory cells are cup-shaped structures measuring 100–180 nm at its opening and 15–35 nm in relative depth in the exocrine pancreas and just 10 nm at the presynaptic membrane of nerve terminals. As a result, it had evaded visual detection until its discovery using the AFM (3–8, 15). The development of the AFM (28) has enabled the imaging of live cells in physiological buffer at nanometer to subnanometer resolution. In AFM, a probe tip microfabricated from silicon or silicon nitride and mounted on a cantilever spring is used to scan the surface of the sample at a constant force. Either the probe or the sample can be precisely moved in a raster pattern using an x-y-z piezo tube to scan the surface of the sample. The deflection of the cantilever measured optically is used to generate an isoforce relief of the sample (30). Force is thus used by the AFM to image surface profiles of objects, such as live cells and subcellular structures, submerged in physiological buffer solutions, at ultrahigh resolution and in real time (3–8).