.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 ager Weyy i daring radioactivity are revealed under the microscope by to the appropriate sites in the silver grains in the overlying emalsion (Figure 8.3) To determine the sites where secretory proteins are syn l elements, which play thesized, Palade and Jamicson incubated slices of pancreatic o transport vesicles and other en- tissuc in a solution containing radioactive amino wll begin the study of endomembranes by discusinga ted in the following chapter. We by discussing a few of brief period of time. During this period, labeled amino acids were taken up by the living cells and incorporated into the di- tissues were quickly fixed, and the locations of proteins synthesized during the brief incubation with la- REVIEW 1. Compaand contrast the biosynthetic pathway with the 2. How are particular proteins targeted to particular suboel- beled amino acids were determined ing this approach, the to be 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 labeled amino acids are incorporated into protein. The 8.2 A FEW APPROACHES TO THE STUDY OF ENDOMEMBRANES chase refers to the period when the tissue is eoxposed 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- 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 ytoplasmic 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- little insight into the functions of the which current research on cytoplasmic organelles is based partments into an integrated functional unit -are summa- Among the hundreds of different cells in the body, the acinar cells ofthe pancreas have particularly extensive endomem brane system. These cells function and secretion of digestive enzymes. After secretion from the rized in Figure 8.36-d Insights Gained from the Use of the Green Fluorescent Protein ynthesis I enzymes are shipped through ducts to the small intestine, where they degrade ingested food matter. The autoradiographic experiments described in the previous Where within the pancreatic acinar cells are the secretory section require investigators to examine thin sections of dif 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 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- (ic., 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 materialis within a cell In this technique, tissue sections conDNA eapresses 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 protcin. n has little or noExplanation / Answer
1. Autoradiography is the technique to study the distribution of radioactively labeled substances with radioisotope in a living system. The radioactively active materiasl are localized within a particular cell, tissue, organelles or even biomolecules. This method can also be used for the quantitative estimation of substances using densitometer. The first autoradiograph was obtained in 1867 using uranium salts. Now many biological molecules can be labeled with radioisotopes not only the radioactively elements like uranium, thorium has opened up new area of investigations in biological systems.
In autoradiography a radioactive substance is put in direct contact with photographic emulsion of gelatin substances and silver halide crystals. This is kept in dark for several days during which silver halide crystals are converted to metallic silver giving a dark coloured band on the autoradiograph. The autoradiograph can be observed with the help of electron microscope, digital scanners etc.
The silver grains observed on the radiographs is produced by several ionization events. The film is preflashed with uniform primes due to which each grain of silver become reduced. This increases the sensitivity of the film and the response produced is directly proportional to the amount of radiation in the sample.
In pulse-chase experiment a group of cells is first exposed to a labeled compound called as pulse and then compound is allowed to go through the metabolic processes and synthesise the product which is studied.
In the given example in the first case where the cell is labeled for 3 mins and then fixed immediately the the black silver grains on the radiograph will appear over the endoplasmic reticulum in the radiograph.
In the second case where the cell is labeled for 3 mins and chased for 40 mins the black silver grain will appear in the Golgi bodies and surrounding vesicles in the radiograph.
2. The biochemical analysis requires disruption of the cell the gentle fractionation method is used to separate the various cell components preserving their individual structures and functions.
In general fractionation method the cells are subjected to ultrasonic vibration and then kept in blender which breaks many membranes of the cell into fragments. These fragments again reseal to form closed vesicles. The suspension of cells containing the organelles such as nuclei, mitochondria, Golgi bodies form a thick slurry. If proper homogenization method is chosen these components can be separated which retain their original biochemical properties.
The cell fractionation is done by preparative ultracentrifuge in which cells are separated by size an density. The larger units experience large centrifugal force.
3. The eukaryotic cells have ER and the ER membrane constitutes more than half of the total membrane of a living cell. The structure of ER is like netlike labyrinth of branching tubules and flattened sacs spanning the cytoplasm. The ER occupies about 10% of total cell volume and formas a continuous sheet enclosing an internal space by the ER membrane.
The ER has the main role in protein and lipid synthesis. Its membrane is the site of production of all trans membrane proteins and lipids like lysosomes, Golgi complex, endosomes. The ER membrane helps in the synthesis of mitochondrial and peroxisomal membranes. All the proteins of the Golgi bodies, lysosomose and ER are first of all delivered to the lumen of ER.
The Flourescent micrographs of the ER in which part of the ER netweork is cultured and stained with an antibody that binds to the protein in the ER. This micrograph can be viewed under Electron microscope which gives the idea of proteins present in the lumen of ER.
4. The Green Fluorescent Protein (GFP) technology are now used to study the dynamics of the cell. Photobleaching studies using GFP-tagged organelle markers is used to study the protein diffusional mobility and provide the knowledge of mechanism of protein retention within the cellular compartments like ER.
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