Cell fractionation has enjoyed widespread use among cell biologists for half a century. It continues to be a fruitful, if not essential, approach in the reductionistic efforts to define the composition and functions of the multiple compartments in eukaryotic cells. It also provides the essential ingredients for the increasing number of cell-free assays now being used in test-tube reconstructions of complex cellular events involving intercompartmental interactions. Much of the knowledge regarding the composition and function of cell organelles has resulted from fractionation of mammalian tissues, where cells are both abundant and highly differentiated (and thus organelle-rich). However, with new techniques in molecular biology and widened interest in combining studies of intact cells and functional reconstitution, interest in fractionation has spread to cultured cell lines and genetically tractable lower eukaryotic cells.
The goals of cell fractionation often differ depending on the nature of the experiments being conducted. In preparative procedures, in which the intent is to isolate quantities of a particular cell organelle for further study or for subfractionation, the emphasis is on purity and (secondarily) on yield. In analytical experiments, in which the intent is not isolation of organelles but evaluation of associations of selected macromolecules with particular organelles, the emphasis is on using one-step procedures that result in different distributions of various organelles (as defined by marker activities) rather than on separating organelles outright. Finally, in preparing organelles for cell-free reconstitution, the goal is to maintain them in a functional state. The investigator generally has developed a specific assay for intercompartmental interaction that does not rely on organelle purity, so the extent of contamination by irrelevant organelles is less important.
The separation of distinct organelles during cell fractionation results from their differing physical properties-size (and shape), buoyant density, and surface charge density-which reflect their differing compositions. Particular fractionation techniques capitalize on one or more of these properties. For example, gel filtration separates on the basis of size, centrifugation separates on the basis of size and density, and electrophoresis separates on the basis of surface charge density. As knowledge of the specific composition of particular organelles has developed, it has become possible to apply newer techniques such as affinity chromatography and selective density-shift perturbation. Whichever fractionation method is used, the procedures may have to be modified to adapt them to individual needs. Even isolation of a particular kind of organelle from different tissue or cell sources may necessitate adjustment of fractionation conditions. This also means that in addition to isolating the organelle of interest, one should also plan on confirming the identity of what has been isolated.
Centrifugation is the most widely used procedure in cell fractionation. It is the only approach commonly used to separate crude tissue homogenates (often having quite large volumes) into subfractions as starting material for more refined purification procedures. Further, the technology available, using rotors with a variety of geometries and diverse media that enable separation according to size, density, or both, now routinely permits refined separations on volumes ranging from submilliliter to several liters. No other technology is this versatile. Gel filtration is limited by the pore sizes of available resins, such that only regularly shaped vesicles with diameters <100 to 200 nm can be purified away from larger or irregularly shaped organelles. Use of electrophoresis for organelle purification, especially at the preparative level, is relatively recent, and, as with gel filtration, its success relies on generating starting material by centrifugation. Although it shows promise for purifying selected organelles (e.g., endosomes) that have been difficult to obtain otherwise, surface charge densities on most organelles may not be different enough (or able to be manipulated sufficiently) to make this a versatile procedure.
Figure 1 The separation of distinct organelles during cell fractionation by centrifugation