1.5: How We Know the Functions of Cellular Organelles and Structures- Cell Fractionation

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We can see and describe cell parts in the light or electron microscope, but we could not definitively know their function until it became possible to release them from cells and separate them from one another. This became possible with the advent of differential centrifugation. Under centrifugal force generated by a spinning centrifuge, subcellular structures separate by differences in mass. Structures that are more massive reach the bottom of the centrifuge tube before less massive ones. A cell fractionation scheme is illustrated below. Biochemical analysis of the isolated cell fractions can reveal what different organelles and cellular substructures do.

107 Dissecting the Cell; a Cell Fractionation Scheme

Cell fractionation separates cells into their constituent parts. The first step of a cell fractionation is to break open the cells and release their contents. This can be done by physical means such as grinding in a mortar and pestle, tissue grinder or similar device, exposure to ultrasound or high pressure, or exposure to enzymes or other chemicals that can selectively degrade the plasma membrane. The next step is to isolate the subcellular organelles and particles from the cytoplasm (i.e., cytosol) by differential centrifugation. As noted, centrifugation of broken cells at progressively higher centrifugal force separates particulate cell components based on their mass. At the end of this process, a researcher will have isolated mitochondria, chloroplasts, nuclei, ribosomes etc. After re-suspension, each pellet can be re-suspended and prepared for microscopy.

Below are electron micrographs of several isolated subcellular fractions.

These structures can be tentatively identified by microscopy based on their dimensions and appearance. Molecular analyses and biochemical tests on the cell fractions then help to confirm these identities.

108 Isolated Nuclei 109 Isolated RER 110 Isolated Golgi vesicles 111 Lysosomes & Peroxisomes

112 Isolated Mitochondria 113 Isolated Chloroplasts 114 Isolated Plasma Membrane

Can you tell what organelles have been purified in each of these fractions based on the electron micrographs alone? Consider the structures on the left as an example. These were found in a low speed centrifugal pellet, implying that they are large structures. They look a bit like nuclei, which are also the largest structures in a eukaryotic cell. What biochemical or functional tests might you do to confirm that the four structures shown from left to right are isolated nuclei, rough endoplasmic reticulum, Golgi vesicles and mitochondria? Physical separation combined with biochemical-molecular analysis of subcellular structures has revealed their basic functions and continue to reveal previously un-noticed structures and functions in cells.

All of cell and molecular biology is devoted to understanding how prokaryotic and eukaryotic cells (and organisms) use their common structural and biochemical inheritance to meet very different survival strategies. As you progress in your studies, watch for experiments in which cell parts are separated and reassembled, or reconstituted. Reconstitution is one of the recurring experimental themes involving the functional analysis of cell components. Look for this theme as you continue your studies. Look also for another theme, namely how evolution can account for the common biochemistry and genetics of life…, and its structural diversity!