.2 A FEW APPROACHES TO THE STUDY OF ENDOMEMBRANES cell. These subjects will bedt
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.2 A FEW APPROACHES TO THE STUDY OF ENDOMEMBRANES cell. These subjects will bedtokcetal s and aperWeyydaring and dissecting the machinery that en- to the appropriate sites in the en containing radioactivity are revealed under the microscope by containing silver grains in the overlying emalsion (Figure 8.3) be discussed in detail in the following l elements, which play in the following chapter. We proteins and cytoskeletal elements thesized, Palade and Jamieson incubated slices of pancreatic amino acids for a To determine the sites where secretory proteins are syn trsport vesicles and other en tissue in a solution containing radioactive amino wnll begin the study of endomembranes by discuasinga ted brief period of time. During this period, labeled amino acids were taken up by the living cells and incorporated into the di- by discussing a few of tissues were quickly fixed, and the locations of proteins synthesized during the brief incubation with la- that had been beled amino acids were determined ing this approach, the REVIEW contrast the biosyathetic pathway with thetbe the site of synthesis of secretory proteins (Figure 8.3a). To determine the intracellular path followed by secretory from their site of synthesis to their site of discharge, and Jamieson carried out an additional experiment. After incubating the tissue for a brief period in radioactive amino acids, they washed the tissue free of excess isotope and transferred the tissue to a medium containing only unlabeled amino acids. An experiment of this type is called a puse-chase The pube refers to the brief incubation with radioactivity during h labeled amino acids are incorporated into protein. The chase refers to the period when the tissue is exposed to the un- Early studies with the electron microscope provided biologists labeled medium, a period during which additional proteins are with a detailed portrait of the structure of cells but gave them synthesized using nonradioactive amino acids. The longer the they were chase, the farther the radioactive proteins manufactured observing. Determining the functions of cytoplasmic or during the pulse will have traveled from their site of synthe- ganelles required the development of new techniques and the sis within the cell. Using this approach, one can ideally fol- 2. How are particular proteins targeted to particular suboel 8.2 A FEW APPROACHES TO THE STUDY OF ENDOMEMBRANES little insight into the functions of the execution of innovative experiments. The experimental ap-low the movements of newly synthesized molecules by proaches described in the following sections have proven par-observing a wave of radioactive material moving through the ticularly useful in providing the foundation of knowledge on which current research on cytoplasmic organelles is based cytoplasmic organelles of cells from one location to the next until the process is complete. The results of these experiments- which first defined the biosynthetic (or secretory) pathway and tied a number of seemingly separate membranous com- partments into an integrated functional unit -are summa- Among the hundreds of different cells in the body, the acinar rized in Figure 8.36-d cells of the pancreas have a brane system. These cells function primarily in the and secretion of digestive enzymes. After secretion from the pancreas, these small intestine, where thcy degrade ingested food matter. The autoradiographic experiments described in the previous Where within the pancreatic acinar cells are the secretory teins synthesized, and how do they reach the surface of the ferent cells that have been fixed at various times after intro- cells where they are discharged? These questions are inher- duction of a radioactive label. An alternative technology ently difficult to answer because all of the steps in the process allows researchers to follow the dynamic movements of spe- of secretion occur simultaneously within the cell. To follow cific proteins with their own eyes as they occur within a single the steps of a single cycle from start to finish, that is, from the living cell. This technology utilizes a gene isolated from a jel- synthesis of a secretory protein to its discharge from the cell, lyfish that encodes a small protein, called the green luores James Jamieson and George Palade of Rockefeller University cent protein (GFP), which emits a green fluorescent light. In utilized the technique of autoradiography Insights Gained from the Use of the Green Fluorescent Protein section require investigators to examine thin sections of dif enzymes are shipped through ducts to the this approach, DNA encoding GFP is fused to DNA encod- As discussed in Chapter 18, autoradiography provides a ing the protein to be studied and the resulting chimeric means to visualize biochemical processes by allowing an in- (ie., composite) DNA is introduced into cells that can be ob- vestigator to determine the location of radioactively labeled served under the microscope. Once inside a cell, the chimeric materials within a cell In this technique, tissue sections conDNA eapreses a chimeric protein consisting of GFP fused to taining radioactive isotopes are covered with a thin layer of the end of the protein to be studied. In most cases, the pres photographic emulsion, which is exposed by radiation ema ence of GFP joined to the end of a nating from radioisotopes within the tissue. Sites in the cells effect on the movement or function of that n has little or noExplanation / Answer
1. In case of a longer chase the radioactive proteins that got manufactured during the brief incubation travels from their site of synthesis within the cell which eases the tracking of the movements of newly synthesized molecules which can be seen through a wave of radioactive material traversing through the cytoplasmic organelles of cells from one location to another until the entire process is complete.
2. Quick fixation of tissues and determination of the locations of proteins through autoradiography.
3. Screening of mutant cells is important to recognize genes whose encoded proteins is involved in the secretory pathway of vesicle buds from the ER traversing to golgi complex where fusion takes place with golgi cisternae. In case of a defective gene product, the mutant cells accumulate an excess number of unfused vesicles.
4. As GFP, i.e green fluorescent protein can track the movement of proteins within a living cell.
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