Biology 2180 Laboratory # 5 Name Plant Cell Fractionation In this lab, you will work with plant tissue to learn about cell fractionation. Cell Fractionation is the process that isolates different components of the cell so that their biochemistry and structure can be studied separate from the rest of the cell. You will use a blender to disrupt corn and spinach cells and a centrifuge to isolate their mitochondria and chloroplasts respectively. Cell Fractionation part 1: Homogenization Tissues or cells grown in culture can be homogenized a number of different ways. As shown on page 160 of your textbook, breakage can be accomplished using high frequency sound, mild detergents, mechanical shearing or by forcing the material through a tissue sieve. Plant cells are particularly difficult to disrupt due to the strength of their cell walls. Some protocols disrupt plant cells by using enzymes to degrade the cell wall then by using a mild detergent to break open the plasma membrane. You will use the blades of a blender to homogenize plant tissues and to break open the cells to release the organelles. The main problem with the blender approach, is that you never know how much processing will give the highest organelle yield. If you don t run the blender long enough, not many of the cells will be disrupted and you won t release enough of the organelles. If you run the blender too long, you will not only break open the cells but you will also destroy a large number of the organelles, again producing a low yield. To minimize this problem, people generally will use the blender for differing amounts of time. They may first run the blender 15 seconds then pour of one third of the sample. Next, they will run it for another 15 seconds then pour off another third of the original sample. Finally, the remaining third will get another 15 seconds. By subjecting the sample to three different amounts of processing, the researcher can get a reasonable yield each time the isolation is performed. Another major concern when trying to isolate cellular organelles, is that they may not be stable outside of the cell. When removed from the protective intracellular environment, many organelles will fall apart if steps are not taken to support them. In your isolation, the homogenization buffer will help alleviate this problem. The buffer contains 10 % Sucrose which makes the solution somewhat viscous and simulates the cytoplasm of the cell. The buffer also contains Tris which buffers the ph at about 7.4, again simulating the intracellular ph of the cell. Lastly, the buffer contains EDTA which removes magnesium from the solution and inactivates degradative enzymes that might disrupt the organelles. Together, these homogenization buffer components stabilize the organelles so they can be isolated by the next step. Cell Fractionation part 2: Centrifugation Once the tissue or cells have been disrupted and a homogenate has been prepared, the cellular compartments can be separated from each other by a process called Differential Centrifugation. As shown in your textbook on pages 160 and 161, the homogenate is poured into test tubes and subjected to increased amounts of gravitational force by rotating the sample at high speed in a centrifuge. In the centrifuge, cell parts are fractionated based on their size and density. For the most part, larger/denser structures sediment faster and at
lower gravitational force. At relatively low speed, unbroken cells, tissue fragments and even nuclei form a pellet at the bottom of the test tube. The smaller cell parts will remain in the liquid which is called the supernatant. This supernatant can then be centrifuged for a longer time and at a higher gravitational force to pellet various cellular organelles and compartments. Mitochondria and Chloroplasts The two intracellular organelles that are involved in helping eukaryotic cells manage their energy transformations, mitochondria and chloroplasts, have a lot in common. The size and shape of both organelles is about the same as bacteria. They each have a double membrane structure with the inner membrane folded into specific configurations that are critical to the organelle function. These organelles also have genetic systems that are independent of the nuclear system, including their own DNA molecules, ribosomes and transfer RNAs. Given these similarities, Lynn Margulis proposed in 1968 that the mitochondria in eukaryotic cells and the chloroplasts in plant cells emerged by way of an endosymbiotic mechanism. This was the idea that: (i) a prokaryote evolved the ability to engulf other organisms; (ii) some of the latter were not digested but lived as endosymbionts (endo = inside, symbiosis = living together); (iii) some of the organelles of eukaryotes evolved from these endosymbionts, losing most of their genes - some to the host nucleus (the origin of which was not explained). Specifically, it was proposed that mitochondria represent a distant relative of proteobacteria and that chloroplasts represent a distant relative of cyanobacteria or blue-green algae. Evidently, the symbiosis has been maintained to present day because the organelle/bacteria benefits by way of their continued existence and the eukaryotic cell benefits from the energy transforming capabilities of the organelles (respiration in the mitochondria and photosynthesis in the chloroplast.) While the endosymbiotic hypothesis was not well recieved when it was first proposed, it has over time gained acceptance by a majority of contemporary biologists. Helping this concept gain acceptance was the subsequent advent of DNA cloning and sequencing technology. Currently, nucleotide sequences have been determined for hundreds of partial mitochondria and chloroplast genomes and many have been completely deciphered. The complete nucleotide sequence is known for the mitochondrial genomes of over 40 animals including human, mouse, fruit fly and sea anemone. Additionally, several chloroplast genomes have been completely sequenced including rice and arabidopsis. Molecular evolutionary comparisons based on these known sequences clearly place the genomes of both organelles in the eubacterial kingdom. Further, estimates of the amount of time since divergence range from 1 to 1.5 billion years ago for mitochondria and 500 to 900 million years ago for chloroplasts.
Transmission EM view of a Mitochondria Mitochondria are sometimes called the cells' power sources because they are the site where most of the cells ATP is generated during respiration. They are distinct organelles with two membranes. Usually they are rod-shaped, however they can be round. The outer membrane limits the organelle. The inner membrane is thrown into folds or shelves that project inward. These folds are called cristae. This electron micrograph taken from Fawcett, A Textbook of Histology, Chapman and Hall, 12th edition, 1994. Transmission Electron Micrograph of a Chloroplast
This is a chloroplast, the plant organelle where photosynthesis occurs, with a double surrounding membrane. The fluid inside this double-membrane organelle is called the stroma. The stroma is, according to the endosymbiotic hypothesis, the cytoplasm of the prokaryotic endosymbiont. As one would expect, it has a nucleoid region to house the circular DNA. It also holds 70S (prokaryotic-type) ribosomes. The stroma is also the site of the Calvin Cycle Reactions. The Calvin Cycle is the series of enzyme-catalyzed chemical reactions that use ATP and NADPH from the Light Reactions to produce carbohydrates and other compounds from carbon dioxide. Floating in the stroma are tiny membrane sacs called thylakoids. The sacs are stacked in groups called a grana. There are many grana in each chloroplast. These thylakoid membranes are the site of the photosynthetic light reactions. The thylakoids have intrinsic and extrinsic proteins, some with special prosthetic groups, allowing for electrons to be moved from protein complex to protein complex. These proteins constitute an electron transport system sometimes known as the Z-scheme. How do these organelles replicate? A shown below, mitochondria replicate much like bacterial cells. When they get too large, they undergo fission. This involves a furrowing of the inner and then the outer membrane as if someone was pinching the mitochondrion. Then the two daughter mitochondria split. Of course, the mitochondria must first replicate their DNA. An electron micrograph depicting the furrowing process is shown in these figures. This TEM was taken from Fawcett, A Textbook of Histology, Chapman and Hall, 12th edition, 1994
Isolation of plant mitochondria and chloroplasts * Keep all reagents as cold as possible. * Perform the protocol separately for Maize (mitochondria) and Spinach (chloroplasts) 1) Cut the plant material into small pieces with a razor blade and place 50 grams into the cold blender cup. 2) Rinse the material with cold water. 3) Add to the blender cup 200 ml ice-cold 10% Sucrose TE. 4) Blend for 15 seconds and pour one third of the mixture into a cold beaker covered with cheesecloth. 5) Repeat this processing two more times until all of the material has been poured through the cheesecloth. 6) Allow the cell lysate to drain through the cheesecloth then remove the cheesecloth from the beaker and set aside the filtrate material. (Don t throw it away because you need to look at it in the microscope). 7) Allow the lysate to settle a few minutes to help eliminate the large chunks of tissue. 8) Pipette from the top of the lysate 15 ml into a conical centrifuge tube that is cold. 9) Centrifuge 10 minutes in the tabletop at 800 RPM. 10) Decant the supernatant into a fresh tube and repeat the above centrifugation. 11) Again decant the supernatant into a fresh cold tube and this time, centrifuge 15 minutes at the 2000 RPM. 12) Discard the supernatant and allow the tube to drain upside-down on a towel for 1 minute. 13) Re-suspend the mitochondria in 0.5 ml of cold 10% Sucrose TE and pipette up and down to mix.
14) 14) Examine all cell fractions with the light microscope and record below what you see at the end of steps 6 (cheesecloth), 9 (pellet) and 13 (pellet). Step 6 Maize Spinach Step 9 Step 1 Questions 1) What was the purpose for including each of the following in our plant organelle isolation procedure? sucrose EDTA Tris Cheesecloth Blender
2) Why did we use the blender three times when isolating each plant organelle? 3) Why did we use the centrifuge twice when isolating each plant organelle? 4) Do you think these organelles could function on their own, outside of the cell? Why? 5) Can you think of any other cellular organelles that may have arisen by an endosymbiotic mechanism? Web Page References http://www.jccc.net/~pdecell/photosyn/chlorpla.html http://cellbio.utmb.edu/cellbio/mitoch2.htm http://www.cytochemistry.net/cell-biology/mitoch1.htm http://koning.ecsu.ctstateu.edu/cell/chloroplast.html