Fracture faces of frozen membranes: 50th anniversary

摘要

In 1961, the development of an improved freeze-etching (FE) procedure to prepare rapidly frozen biological cells or tissues for electron microscopy raised two important questions. How does a frozen cell membrane fracture? What do the extensive face views of the cell’s membranes exposed by the fracture process of FE tell us about the overall structure of biological membranes? I discovered that all frozen membranes tend to split along weakly bonded lipid bilayers. Consequently, the fracture process exposes internal membrane faces rather than either of the membrane’s two external surfaces. During etching, when ice is allowed to sublime after fracturing, limited regions of the actual membrane surfaces are revealed. Examination of the fractured faces and etched surfaces provided strong evidence that biological membranes are organized as lipid bilayers with some proteins on the surface and other proteins extending through the bilayer. Membrane splitting made it possible for electron microscopy to show the relative proportion of a membrane’s area that exists in either of these two organizational modes. Odd as it may seem, tedium paved the way to the astonishing discovery that the membranes of a frozen living cell can easily be cleft into two sheets. In 1961, shortly after starting a two-year National Science Foundation Postdoctoral Fellowship with Fritz Ruch in Frey-Wyssling’s department at the ETH in Switzerland, I realized that the motivating hypothesis for my project was misguided. Ruch convinced me to endure a few months carefully proving and explaining why prior investigators’ qualitative data were misleading, but it was dull research ( Branton and Ruch, 1964 ). The tedium motivated me to find a project that would open new insights rather than simply falsify a hypothesis. I was intrigued by the freeze-etching (FE) methods being developed in the department by Hans Moor. Initiated by Russel Steere, FE was a method of preparing biological material for the electron microscope without chemical fixation, dehydration, and embedding ( Steere, 1957 ). Steere’s idea was to rapidly freeze a living specimen in a tiny block of ice, plane away overlying ice with a scalpel to expose the material of interest, etch away (sublime) a small amount of water from the exposed frozen material in a vacuum, and then make a replica of the exposed, etched surface material that could be viewed in an electron microscope. Studies of baker’s yeast ( Saccharomyces cerevisiae ) by Moor and his colleagues showed FE to be the first really productive alternative to the usual fixation–dehydration–embedding routines and associated artifacts ( Moor and Muhlethaler, 1963 ). The “plane away overlying ice” step envisaged by Steere often fractured the frozen material in a plane that followed any membrane’s contours for many microns. This resulted in surprisingly expansive three-dimensional views of many cellular membranes. Such in-plane views had never been seen in thin sections of biological material. Only later, after the correct locus and physical basis of the fracture process were established, would the signal importance of these views become fully apparent. My initial work focused on easily grown onion root-tip cells whose freeze-etched cellular features could be compared with similar cells that had been visualized following classical methods. At first, freeze-etched root tips seemed to present only three-dimensional versions of the usual structures seen in fixed, sectioned root tips. But unfamiliar 7–10 nm diameter particles were observed on most of the membrane faces. Moor had observed similar particles on yeast endoplasmic membranes and assumed they must be ribosomes, even though they were much smaller than any known ribosomes ( Moor and Muhlethaler, 1963 ). Although Moor and his colleagues insisted that the similar particles in my root-tip cells must also be ribosomes, I was reluctant to use this designation. Thus, in my first paper reporting freeze-etch results, written jointly with Moor ( Branton and Moor, 1964 ), we compromised and concluded that “further studies … will establish a definitive correlation between surfaces seen in freeze-etched preparations and structures visualized by other techniques.” At issue was not only the nature of particles seen on the freeze-etched membrane faces but also the identity of the membrane faces on which the particles were found. Newly appointed as an assistant professor at University of California, Berkeley, I undertook a thorough reexamination of the replicas made during my postdoctoral research. Doing so revealed a previously overlooked feature: a small ridge at the base of every exposed membrane face. When observed in views that extended from a face-on view into a cross-section of the same membrane, the edge of the face-on view and the small ridge at the base of the face-on view merged and became continuous with, and indistinguishable from, the two ridges that together represented the FE appearance o