Isolation of Cell Organelles - Subcellular Fractionation

In this article, we are going to see how to isolate different organelles? First of all, I would like to ask, why is there a need to isolate organelles of the cell? I would argue that there is electron microscopy to see all the minute details of the cell. Then why to isolate different organelles?
The answer is, yes, definitely, the electron microscopy can be used for detailed visualization of the cell; however, it cannot determine the functions of the cell. Here comes the need to isolate the components of the cell - to study about its functions and hence, to do so, it is necessary to isolate them in intact form which can be used for biochemical studies. Hence, this can be accomplished using the technique, differential centrifugation followed by density-gradient centrifugation.

This method (differential centrifugation) is based on the principle that the cell components separate on the basis of the size and density. Following are the steps to separate cell components:

Diagrammatic Representation of Subcellular Fractionation

• Disruption of plasma membrane/ Cell lysis: The first step in the process of differential centrifugation is the disruption of the plasma membrane or cell lysis in such a way that there is no harm to internal components of the cell i.e.; all the organelles remain intact. This can be done by several methods, like sonication (subjecting the cells to high-frequency sound); grinding in a homogenizer etc. This process is carried out in a buffer solution to maintain the osmotic environment to keep the components intact. This results in a solution containing broken cells, small fragments of plasma membrane, endoplasmic reticulum while it leaves the organelles of the cell (like mitochondria, nuclei, lysosomes etc.) intact. This solution is called a lysate or homogenate.
  
  
• Ultracentrifugation:  The lysate is subjected to series of centrifugations which will fractionate into several components in an 'ultracentrifuge' which will rotate the samples at very high speeds. This will produce very high force and these forces will enable the components of the cell to settle down and form a pellet. This process of sedimentation will depend on the size and the density of the components with largest and heaviest components sedimenting first. The homogenate is first subjected to low speeds of around 800g for 10 minutes which will sediment unbroken cells and the largest cell organelle, nucleus. Thus, an enriched pellet of nucleus is obtained while supernatant (the remaining solution) contains other cellular components.
  The supernatant is further subjected to higher speeds (10,000-20,000g for 10 minutes) to sediment mitochondria, lysosomes, peroxisomes. The supernatant is again centrifuged to further high speeds (this time at 100,000 for 1 hour) resulting in fragments of plasma membrane and endoplasmic reticulum in the pellet.
  The final centrifugation step is done by further spinning it at very high speed which results in the ribosome sediments. The supernatant left is just cytosol.

The fractions which are obtained here, are enriched in corresponding organelle components, still, these fractions are not pure. So, to obtain pure components, further, density-gradient centrifugations have to be performed. In this process, the components are separated by sedimentation by a gradient of dense substance like sucrose. So, here, the sample is layered at the top of a gradient (sucrose). There is sedimentation of particles according to their sizes through gradient at different rates and discrete layers are formed. Thus, there is collection of each fraction which contains organelles of similar size (like mitochondria, lysosomes etc.)

Cell Organelles Dicoverers

We have seen in the previous posts about all the organelles in an animal cell with their structure and functions. But have we wondered who discovered them? So, I just want to make it easy for you all by jotting down the organelles and their discoverers over here.

The Organelles of the Cell

1. Cell Membrane - Robert Hooke in 1665 first view the cells under the microscope and hence, he is credited with the discovery of cell membrane.
2. Centriole - Discovered by Edouard Van Beneden in 1883 and was described and coined by Theodor Boveri in 1888.
3. Centrosome - Discovered by Edouard Van Beneden in 1883 and was described and coined by Theodor Boveri in 1888.
4. Cytoskeleton - Nikolai K Koltsov in 1903 proposed that the shape of the cell is determined by the tubular network, the cytoskelton. However, the word was introduced and coined by Paul Wintrebert in 1931 (in French, cytosquelette).
   i. Microtubules - De Robertis and Franchi disovered microtubules in 1953 in nerve cell and later Sabatani, Bansch, Barnette in 1963 explained the structure of microtubule.
   ii. Microfilament/Actin Filaments - Edward David Korn discovered microfilament in 1968 in Acanthamoeba castellanii.
   iii. Intermediate Filaments - The groep of Howard Holtzer in 1968 discovered intermediate filaments.
5. Cytosol - Discovered in 1835and no single scientist can be accredited to its discovery.
6. Golgi Apparatus -Camillo Golgi identified it in 1897 and named after him in 1898.
7. Lysosomes - Discovered by Christian de Duve in 1949 (or 1950s).
8. Mitochondria - Albert von Kolliker studied mitochondria in muscle cell in 1857; Richard Altmann first recognized them as cell organelle in 1894; Term "mitochondria' coined by Carl Benda in 1898.
9. Nucleus - Robert Brown discovered cell nucleus in 1833.
10. Ribosomes -Discovered by George Palade in 1955.
11. Endoplasmic Reticulum - Albert Claude in Belgium and Keith Porter at Rockfeller Institue in 1945.
12. Vacuole - Antony van Leeuwenhoek is credited with discovery of vacuole when he was studying bacteria in 1676.
13. Plastids: 'Plastid' term was coined by Ernst Haeckel in 1866. however, in 1883, Andreas Schimper was the first to give the clear definition of plastid and he also explained the relationship between the different types. 
14. Nucleolus: The  nucleolus was first discovered in eel cells in 1774 by Felice Fontana.
15. Chromosomes: The chromosomes were first discovered by Karl Wilhelm von Nageli in 1842.

If any references are needed, feel free to contact me.!