1.5: How we Know Functions of Organelles and Cell Structures- Cell Fractionation
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\(\newcommand{\avec}{\mathbf a}\) \(\newcommand{\bvec}{\mathbf b}\) \(\newcommand{\cvec}{\mathbf c}\) \(\newcommand{\dvec}{\mathbf d}\) \(\newcommand{\dtil}{\widetilde{\mathbf d}}\) \(\newcommand{\evec}{\mathbf e}\) \(\newcommand{\fvec}{\mathbf f}\) \(\newcommand{\nvec}{\mathbf n}\) \(\newcommand{\pvec}{\mathbf p}\) \(\newcommand{\qvec}{\mathbf q}\) \(\newcommand{\svec}{\mathbf s}\) \(\newcommand{\tvec}{\mathbf t}\) \(\newcommand{\uvec}{\mathbf u}\) \(\newcommand{\vvec}{\mathbf v}\) \(\newcommand{\wvec}{\mathbf w}\) \(\newcommand{\xvec}{\mathbf x}\) \(\newcommand{\yvec}{\mathbf y}\) \(\newcommand{\zvec}{\mathbf z}\) \(\newcommand{\rvec}{\mathbf r}\) \(\newcommand{\mvec}{\mathbf m}\) \(\newcommand{\zerovec}{\mathbf 0}\) \(\newcommand{\onevec}{\mathbf 1}\) \(\newcommand{\real}{\mathbb R}\) \(\newcommand{\twovec}[2]{\left[\begin{array}{r}#1 \\ #2 \end{array}\right]}\) \(\newcommand{\ctwovec}[2]{\left[\begin{array}{c}#1 \\ #2 \end{array}\right]}\) \(\newcommand{\threevec}[3]{\left[\begin{array}{r}#1 \\ #2 \\ #3 \end{array}\right]}\) \(\newcommand{\cthreevec}[3]{\left[\begin{array}{c}#1 \\ #2 \\ #3 \end{array}\right]}\) \(\newcommand{\fourvec}[4]{\left[\begin{array}{r}#1 \\ #2 \\ #3 \\ #4 \end{array}\right]}\) \(\newcommand{\cfourvec}[4]{\left[\begin{array}{c}#1 \\ #2 \\ #3 \\ #4 \end{array}\right]}\) \(\newcommand{\fivevec}[5]{\left[\begin{array}{r}#1 \\ #2 \\ #3 \\ #4 \\ #5 \\ \end{array}\right]}\) \(\newcommand{\cfivevec}[5]{\left[\begin{array}{c}#1 \\ #2 \\ #3 \\ #4 \\ #5 \\ \end{array}\right]}\) \(\newcommand{\mattwo}[4]{\left[\begin{array}{rr}#1 \amp #2 \\ #3 \amp #4 \\ \end{array}\right]}\) \(\newcommand{\laspan}[1]{\text{Span}\{#1\}}\) \(\newcommand{\bcal}{\cal B}\) \(\newcommand{\ccal}{\cal C}\) \(\newcommand{\scal}{\cal S}\) \(\newcommand{\wcal}{\cal W}\) \(\newcommand{\ecal}{\cal E}\) \(\newcommand{\coords}[2]{\left\{#1\right\}_{#2}}\) \(\newcommand{\gray}[1]{\color{gray}{#1}}\) \(\newcommand{\lgray}[1]{\color{lightgray}{#1}}\) \(\newcommand{\rank}{\operatorname{rank}}\) \(\newcommand{\row}{\text{Row}}\) \(\newcommand{\col}{\text{Col}}\) \(\renewcommand{\row}{\text{Row}}\) \(\newcommand{\nul}{\text{Nul}}\) \(\newcommand{\var}{\text{Var}}\) \(\newcommand{\corr}{\text{corr}}\) \(\newcommand{\len}[1]{\left|#1\right|}\) \(\newcommand{\bbar}{\overline{\bvec}}\) \(\newcommand{\bhat}{\widehat{\bvec}}\) \(\newcommand{\bperp}{\bvec^\perp}\) \(\newcommand{\xhat}{\widehat{\xvec}}\) \(\newcommand{\vhat}{\widehat{\vvec}}\) \(\newcommand{\uhat}{\widehat{\uvec}}\) \(\newcommand{\what}{\widehat{\wvec}}\) \(\newcommand{\Sighat}{\widehat{\Sigma}}\) \(\newcommand{\lt}{<}\) \(\newcommand{\gt}{>}\) \(\newcommand{\amp}{&}\) \(\definecolor{fillinmathshade}{gray}{0.9}\)We could 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 in a spinning centrifuge rotor, 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 in Fig 1.25. Biochemical analysis of the isolated cell fractions can reveal what different organelles and cellular substructures do.
107-2 Dissecting the Cell-a Cell Fractionation Scheme
The cell fractionation scheme pictured in Figure 1.25 does not include sucrose density gradient centrifugation. Offer an explanation.
Cell fractionation separates cells into their constituent parts. The first step 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. The 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 ribosomes, mitochondria, chloroplasts, nuclei,and other subcellular structures. After re-suspension, each pellet can be prepared for microscopy. Micrographs of some isolated subcellular fractions are shown in Figure 1.26.
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.
114-2 Isolated Membranes Form Vesicles
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 in fact, the largest structures in a eukaryotic cell)—and indeed that’s what they are!
Physical separation and the biochemical and molecular analysis of subcellular structures have revealed their basic functions and continue to reveal previously un-noticed structures and functions in cells. What biochemical tests might you do to confirm the identities of the structures shown? At this point you may realize that all 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 keep studying, watch for experiments in which cell parts are separated and reconstituted. Reconstitution is a recurring experimental theme in the functional analysis of cell parts. Also look for another, even bigger theme: how evolution accounts for the common biochemistry and genetics of life—and its structural diversity!