PERSPECTIVES fractionation of liver homogenates. The TIMELINE emphasis was on the quantitative monitoring of the distribution of the chemical con- stituents of the cell, rather than organelle George Emil Palade: charismatic purity5–8. Trial and error must have been the norm, and cold-room stamina a prerequisite, virtuoso of cell biology but this early period ultimately established the procedures that allowed organelles to remain intact without agglutination or lysis. Many Alan M. Tartakoff obstacles confronted these investigators, including a “…biochemical Zeitgeist that par- George Palade has created, shared and on the state of knowledge at that time in his ticles were a nuisance and stood in the way passed on a multidisciplinary view of the Nobel lecture,“…biologists [had been] in the of purification of … enzymes.”9 Whereas functional organization, biogenesis and same situation as astronomers and astro- biochemistry was developing rapidly, the dynamics of organelles. His open- physicists, who were permitted to see the understanding of the compartmentalization mindedness and tenacity, along with his objects of their interest, but not to touch of subcellular activities and the significance of rigour and sense of intellectual elegance, them; the cell was as distant from us as the organelles was still in its infancy. have been remarkable. This focus on the stars and galaxies were from them. More dra- Claude returned to his native Belgium in logic of organelles defined a crucial turning matic and frustrating was that we knew that 1949, but not before he and his colleagues point in biomedical science. The following the instrument at our disposal, the micro- had systematized the use of differential sedi- article sketches Palade’s research, as part scope … had … reached, irremediably, the mentation to isolate a comprehensive set of of a larger community that flourished after theoretical limits of its resolving power.”4 fractions from tissue homogenates using the Second World War. Claude had begun his investigations of hypertonic sucrose (in place of saline or organelle isolation by sedimenting intact cells, water) as a homogenization medium. These George Emil Palade (FIG.1) was born into an the organelles of which became beautifully fractions comprised heavy particles, a large- academic family in Moldavia, Romania. He stratified, without the rupture of the plasma particle fraction that was enriched in mito- received his medical training at the School of membrane (see TIMELINE). As a result of his chondria and lysosomes, a light fraction that Medicine in Bucharest and carried out his efforts to isolate the Rous sarcoma virus, he was enriched in microsomes — membrane first research as part of a doctoral thesis on then — in conjunction with the Canadian- vesicles which were so small that they could the kidneys of dolphins, which were obtained born developmental biologist Keith Porter not be seen with the microscopes that were with the help of Black-Sea fishermen. He then (FIG. 1), George Hogeboom and Walter available at the time — and a soluble fraction. served on the faculty of the Institute of Schneider — undertook the development of These fractions were studied both biochemi- Anatomy at the University of Bucharest until procedures for the systematic subcellular cally and by using the electron microscope — having survived the Nazi threat — in 1946, at the age of 34, he moved with his wife and two small children to New York City 1-3. New methods and approaches Palade spent his first few months in the United States with Robert Chambers at New York University, where he studied cellular membranes. After seeing the first of Albert Claude’s electron micrographs, however, Palade joined Claude (FIG. 1) — initially as a volunteer — in the Department of Pathology Figure 1 | Portraits. From left to right: George Palade (reproduced with permission from REF. 1 © The and Bacteriology at the Rockefeller Institute, Rockefeller University Press), Albert Claude (reproduced with permission from REF. 21 © (2002) The where the young doctor Claude had arrived Rockefeller University Press), Keith Porter (photograph courtesy of Blair Bowers) and Christian de Duve from Belgium in 1929. Claude commented (© The Nobel Foundation). NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 3 | NOVEMBER 2002 | 871 PERSPECTIVES Timeline | Microscopy and subcellular fractionation ×270 visualization Magnifying of bacteria (van Acceptance of the Abbé’s treatise Apochromatic Description of Successful glasses ×5–10 Leeuwenhoek) cell theory on optics objectives ergastoplasm (Garnier) osmium fixation ~1500 ~1650 1683 1823 ~1850 1869 1876 1878 1886 1896 1897–1898 1898 1927 Magnifying glasses Achromatic lenses Miescher’s attempts Oil immersion First edition of The Cell Description of the ×30 visualization of (resolution: 1 micron) at isolation of nuclei lenses in Development and Golgi complex (Golgi) plant cell walls in Heredity24 (Wilson) cork (Hooke) (EM), which scanned the stratified particulate 1953,“…what we were doing was trying to of the Rockefeller Institute.”11 “The new field contents of every pellet from the bottom to separate populations that … might at best be had virtually no tradition; everybody working the top10. And yet, as Christian de Duve (FIG. 1) only partly separable from each other. In in it came from some other province in nat- pointed out, before the introduction of den- addition, we were using a poorly discriminat- ural sciences … Added to all this excitement sity gradients and swinging bucket rotors in ing method for this purpose.”8 was a pervading free spirit — often irreverent, A few years earlier, in the period but always helpful, because it acted as an anti- 1945–1947, Porter, Claude, Ernest Fullam and dote against imagined grandeur. Keith Porter Edward Pickels had examined intact thinly was responsible for a good part of that spread cells using the earliest of the EMs, and spirit…”11. When Porter later left Rockefeller had already distinguished a lace-like structure University for Harvard University, and as a that later became known as the endoplasmic fond reminder of his contributions, the reticulum (ER)11,12. Some of the remaining Palade laboratory for many years displayed a technical hurdles were resolved by the intro- prominent photo of Porter with the caption duction of buffered osmium tetroxide for fixa- ‘Our Father Who Art at Harvard.’ tion (by Palade, in 1952), plastic (such as methacrylate) as an embedding medium (by Vesicular transport Newman, Borysko and Swerdlow, in 1949), In 1953, Palade described a ‘small particulate glass knives (by Latta and Hartmann, in 1950) component of the cytoplasm’ — which later and ingenious microtomes that produced thin became known as ribosomes. By 1956, the sections (by Joseph Blum and Porter), which painstaking microscopic and biochemical became commercially available in 1953. characterization of microsomal fractions was Our present knowledge of the fundamen- published by Palade and Philip Siekevitz, who tal properties of the cell is, largely, a function had joined the group in 1955. This work veri- of the early solutions to these methodological fied the hypothesis that the main component problems. Considering the novelty of the of the fraction was derived from fragmented procedures that were developed and the ER13. With the procedures established for sepa- refinements that were made over the years rating fractions enriched in microsomes from that followed, it is a great tribute to these those enriched in secretion granules, Siekevitz investigators that the quality of their EM and Palade then initiated biochemical investi- images and subcellular fractionation in many gations of macromolecular transport along the cases match present standards. Figure 2 | Hepatocyte membrane differentiation. This electron micrograph was published as part of a Summing up this period — when many of study of membrane induction during hepatocyte the leading cell biologists of the future passed differentiation. Note the rough-surfaced cisternae of through the laboratory5,7,11 — Palade wrote “These concepts have the endoplasmic reticulum (ER), which include some that “After so many years, it is difficult to become so central to our areas without attached ribosomes (long arrows). recapture in words the atmosphere of intense Also note the relative scarcity of ribosomes in activity, remarkable achievements, great thinking that the grazing sections of the ER (short arrows). A few cisternal granules (cg) are present within the ER. excitement, and unlimited optimism that pre- intellectual advance that Microbodies (mb) and mitochondria (m) are also vailed in the laboratory, which otherwise they represent has gone included. Reproduced with permission from REF.22 looked like an unattractive dungeon sunk in © (2002) The Rockefeller University Press. the third basement of one of the old buildings almost unrecognized.” 872 | NOVEMBER 2002 | VOLUME 3 www.nature.com/reviews/molcellbio PERSPECTIVES Production of commercial Introduction of Introduction of Isolation of microsomes phase microscopes that methacrylate for buffered Satisfactory routine using centrifuges capable were based on Zernike’s embedment (Newman, osmium fixative plastic resins for of 18,000 x g (Claude) model (Nobel prize 1953) Borysko and Swerdlow) (Palade) embedment (Luft) 1934 1938 1939 1941 1945 1949 1950 1952 1953 1961 1963 Attempts at isolation of Production of Examination of thinly- Glass knives for thin Commercial swinging bucket Introduction of mitochondria (Bensley)