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.)

Centrosome and Centriole

Centrosome is another organelle in all eukaryotic cells. It is present in the cytoplasm of the cell usually close to the nucleus.  It has a key role in organization of microtubule in all animal cells and hence also referred to as the Microtubule Organizing Center (MTOC).

Simple Structure of a Centriole

The centrosome is generally composed of two centrioles (centriole figure on the right) which are at right angles or perpendicular to one another as can be seen in the figure below. They are surrounded by amorphous mass of protein called the  Pericentriolar Material (abbreviated as PCM).

Diagram showing Two Centrioles
Perpendicular to One Another

 This PCM contain proteins which are responsible for microtubule nucleation and anchoring. So, the two centrioles (perpendicular to one another) along with PCM constitutes the centrosome.
You might be thinking, what are centrioles? The centriole is cylindrical shaped structure composed of nine triplets of microtubule. This can be clearly seen in the cross section of centriole (diagram below).

Cross Section of Centriole
showing Nine Triplets of Microtubule

Centrosomes play a key role in the process of mitosis. As we all know, during the interphase stage of mitosis, the nuclear membrane breaks down, the microtubules interacts with the chromosomes to build the mitotic spindle. Each daughter cell receives only one centrosome containing two centrioles. This is because of the simple reason that centrosome is copied only once per cell cycle. How? The reason is simple just as how some other organelles divide in the cell during mitosis. The centrosome replicates in the S phase. Now, there are two centrosomes. Next, during prophase, each of the centrosome migrate to the opposite pole of the cell. The mitotic spindle forms between both of them. Upon dividing, each daughter cell inherits one centrosome (containing two centrioles).

Centrioles are a very important part of centrosomes, which are involved in organizing microtubules in the cytoplasm.The position of the centriole determines the position of the nucleus and plays a crucial role in the spatial arrangement of the cell. Experiments have shown that centrioles are not required for the progression of mitosis. If the centrioles are not present, the microtubules of the spindle are focused by motors which allows them to form the bipolar spindle. So, it has been shown that many cells can completely undergo interphase without centrioles.

Vacuole

A vacuole is a membrane bound organelle. Lets understand its structure and functions.

Structure:

Simple Diagram of a Vacuole

Do you know that there is a special name given to the membrane surrounding this vacuole? It is called the tonoplast. You might think that being a membrane-bound organelle, it might have a specific shape or size? The answer however, is no. There is no specific shape or size. It varies depending on the needs of the cell. For example, in a plant cell, there is generally one large vacuole at the center. In animals, there are small vacuoles but multiple in number. What does this vacuole contains? This vacuole does not take anything from the cell nor do it gives anything to the cell. It only stores nutrients for the cell. These are water-filled organelles containing various inorganic and organic molecules. It is also known to have contain wastes of the cell. Vacuoles can be central vacuole or food vacuole. The central vacuole is present only in plant cells which stores cell sap for the plant. It also allows for cell growth by absorbing water. Food vacuole is created by the process of endocytosis and is generally useful in storing food which is absorbed by the organism.

Why are these vacuoles present in the cell? What role do they play? Lets have a look at their functions.

Functions: 

The inside of the vacuoles contain water because of which there is pressure inside the cell called the turgor pressure (in plant cells) which gives structural support to the plants.It contains various inorganic and organic molecules of the cell which also includes enzymes in the solution. It stores wastes and exports these unwanted products from the cell. It also plays an important role in intracellular digestion and the release of cellular waste products.

Cytoskeleton

The cytoskeleton is another most important structure present in all eukaryotic cells. What do you mean by cytoskeleton? 'Cyto' means 'cell' and 'skeleton' means 'supporting structure/frame'. Thus, as the name suggests, cytoskeleton helps in maintaining the shape of the cell. However, its primary importance/function is cell motility.

Structure:

Cytoskeleton is the dynamic structure that fills the spaces in the cytoplasm of the cell. Cytoskeleton is a complex and organized network of three filaments as - microtubules, microfilaments (actin filaments) and intermediate filaments. What are the differences between them? Lets try to understand.

Three Different Components of Cytoskeleton

a. Microtubules: Microtubules are hollow cylindrical structures which are around 20-25 nm in diameter. They are composed of further subunits of protein called tubulin and further differentiated into alpha and beta tubulin.

b. Microfilaments/ Actin filaments: Microfilaments as the name suggests are thread like filaments or fibers of around 3-6nm in diameter. They are composed of subunits of protein called actin. Do you know that actin is the most abundant protein present in the cell? Microfilaments are also known to associate with another protein called myosin and this is responsible for muscle contractions.

c. Intermediate filaments: These filaments are around 10nm in diameter. They are heterogeneous constituents of the cell. The intermediate filaments include proteins like vimentin, lamin (these give structural support to the cell), keratin (protein which is found in skin, nail, hair etc.)

Functions:

Microtubules act as scaffold and determines the shape of the cell. They also from spindle fibers for separating chromosomes during the process of mitosis (cell division). They are also involved in the synthesis of cell walls in plants.

Microfilaments are for muscle contraction. They are responsible for movement of cells, for eg.; gliding.

Intermediate filaments provide tensile strength for the cell. They act as structural components and also help in anchoring organelles. They also are involved in cell-cell junctions.

Lysosomes - Suicidal Bags

Lysosomes are another organelle which is mostly found in animal cells; very rarely in plant cells. It is also called 'suicidal bags' because they are responsible for digestion of cell's bio-molecules, old cell parts and micro-organisms.

Structure:

Structure of Lysosome

Lysosomes are sac-like organelles which are bound by a membrane. The lysosomes arise from Golgi apparatus by budding off from its membrane. The interior of lysosomes is acidic is nature with pH around 4-4.5. This acidic environment is maintained in the interior by proton pumps. The interior contains a variety of enzymes (acid hydrolases). These hydrolytic enzymes degrade proteins, nucleic acids, carbohydrates and lipids. These enzymes cannot work outside the lysosomes as the pH of the cell is slightly alkaline or neutral. So, if there is lysosomal rupture or leakage, this acid-dependent activity protects the cell from self-degradation.

Functions:

Diagramtic Representation of Process of Degradation by Lysosomes

The main function is the break down and removing of old parts of the cell or microorganism. The lysosomes fuses with membrane bound vesicle that arises from any of these pathways - endocytosis, phagocytosis or autophagocytosis. These vesicles are referred to as endosomes, phagosomes and autophagosomes respectively. These endosomes fuses with lysosomes (primary lysosomes) and forms secondary lysosomes (sometimes referred to as endolysosomes). The bio-molecules are further broken down into smaller forms like amino acids, monosaccharides, nucleotides and fatty acids which are then recycled in the cell.

Golgi Apparatus

Golgi apparatus, also called 'Golgi Complex' or simply 'Golgi' is the stack of flattened sacs which are bound by a single membrane also known as cisternae. They are important for packaging and transporting the molecules from one compartment/organelle of the cell to another for secretion from the cell.

Structure: 

As already mentioned, Golgi is the stack of flattened sacs. Golgi has four main regions as: cis-golgi, endo-golgi, trans-golgi and median-golgi networks. The vesicles are pinched off from the endoplasmic reticulum containing proteins and other biomolecules; fuses with the networks. The vesicles are gradually modified here and are further moved to other stacks and finally to trans golgi network. Here, they are packaged and are finally sent to their destination or released from golgi and moved to cell membrane for exocytosis.

Functions:

Golgi is basically involved in modifying and packaging of biomolecules for secretion from the cell by exocytosis.

It is also involved in the transport of lipids around the cell. It acts as a post office where it packages and labels different items which then sends them to different parts of the cell. 

The enzymes in the cisternae modifies the proteins by adding a phosphate group (phosphorylation) or carbohydrate chain (glycosylation) to the protein. These modifications sometimes forms the signal sequence which helps in determining the destination.

The golgi also play a role in apoptosis.

Ribosomes - Protein Assemblers of the Cell

Ribosomes are tiny organelles which are present in large numbers in both prokaryotic and eukaryotic cells (described here). They are important site for protein synthesis.

Structure:

Structure of Prokaryotic Ribosome

The ribosomes are designated as per their rate of sedimentation as 70S for bacterial ribosomes and 80S for eukaryotic ribosomes (where S stands for rate of sedimentation).Both prokaryotic and eukaryotic ribosomes are composed of two different subunits. these subunits comprises of proteins and rRNAs. Both of the ribosomes have similar structure; however, they differ in some details like different number of proteins and types of rRNAs.

The prokaryotic 70S ribosome is made up of large subunit as 50S and small subunit as 30S.

The small subunit 30S consists of 21 proteins and 16S rRNA whereas the large subunit is consists of 34 proteins and 23S and 5S rRNAs.

Structure of Eukaryotic Ribosome

Eukaryotic 80S ribosomes in eukaryotes is made up of  60S as large subunit and 40S as small subunit. the large subunit, 60S is made up of 28S rRNA, 5.8S rRNA, 5S rRNA and approximately 45 proteins. Whereas the small subunit, 40S is comprises of 18S rRNA and approximately 30 proteins.

One of the striking feature of ribosome is that they can be formed in vitro by self assembly of their RNA and protein constituents. This was first described by Masayasu Nomura when he purified the constituents of ribosome and mixed them in appropriate conditions and the functional ribosome was re-formed.

Functions:

Translation of mRNA by a Ribosome into a Peptide Chain.

As already mentioned, the ribosome are the site for protein synthesis; which is the process of translating mRNA into proteins. The mRNA comprises a series of codons (triplets of nucleotides) that will ultimately form the amino acids to make the protein.
The ribosome uses this mRNA as template and translates each codon by pairing it with appropriate amino acid which is provided by aminoacyl-tRNA (aminoacyl-tRNA contains a complementary anti-codon). The ribosome contains three RNA binding sites as A, P and E. The A site binds an aminoacyl-tRNA; the P site binds a peptidyl-tRNA and E site binds a free tRNA before it exits the ribosome as can be seen in the diagram.
 

Endoplasmic Reticulum

Endoplasmic Reticulum (abbreviated as ER) is a complex network and is composed of sacs and tubules. It is highly twisted.

Structure:

Diagram of RER

ER can be differentiated into three varieties. They are:

1. Rough Endoplasmic Reticulum (RER):

The membrane of this type of ER is in the form of sheets (sacs). They are present near the nucleus and is continuous with the nuclear outer membrane. The surface of the membrane of RER is studded with ribosomes (another type of organelle, described here) which are an important site for protein synthesis. The appearance of the membrane is rough as can be seen in the diagram and hence, is called “RER”

Diagram of SER

2. Smooth Endoplasmic Reticulum (SER):

The membrane of this type of ER is mostly in the form of tubules; sometimes it may branch and form reticulate kind of network. They lack the ribosomes; thereby gives the appearance as smooth and hence the name Smooth ER. Also, SER contains an enzyme glucose 6-phosphatase, which is involved in gluconeogenesis.

3. Sarcoplasmic Reticulum (SR):

this is a type of SER, which is present in smooth and striated muscle. The only difference between SER and SR is the variety of proteins present in each.

Functions: 
The main and foremost function of RER is the synthesis and manufacture of proteins.

SER is involved in lipid synthesis and steroid synthesis. It is also involved in drug detoxification and steroid metabolism.

SR is present in muscle cells where it regulates calcium ion concentrations.

Mitochondria - Power house of the Cell

The mitochondria is another very important organelle in the cell and is called the power house of the cell. It is so because mitochondria is capable of producing the energy.

Structure: 

Mitochondria is a membrane enclosed organelle. It is double layered i.e.; it has two membranes  as outer membrane and inner membrane.
Between these two membranes is the space called intermembrane space.
The outer membrane covers the organelle.
The inner membrane folds upon itself many-a-times to form convolutions which are called cristae. This folding basically increases the surface are; the more area, the more work can be done and the more ATP (energy) we will get. The inner membrane also contains various types of proteins with different functions.
The space enclosed within the inner membrane is matrix. This matrix plays an important role in the synthesis of ATP as it contains various enzymes required for this. It also contains mitochondrial ribosomes, mitochondrial DNA.

Functions: 
As already mentioned above, this is the most important organelle for providing us the energy in the form of ATP. This is because the enzymes for the important cycles involved in the production of ATP are present in the mitochondria.
Also, mitochondria is a storage site for calcium ions. MItochondria is also the site for apoptosis or programmed cell-death.

Nucleus - Brain of the Cell

Nucleus is also called the “brain of the cell”. Why so? Because it contains the genetic material and is responsible for a large number of functions. Nucleus is spheroid and is most prominent part occupying around 10% of the total cell volume.

Structure:
Inside the nucleus is present a structure called nucleolus which consists of rRNA and proteins but no DNA. It is the site of assembly of ribosomes which are important for the process of protein synthesis. Nucleolus disappears when the cell is dividing and reappears after the cell is formed. 

Then, there is chromatin consisting of long strands of DNA associated with proteins. When the cell is in resting stage, the chromatin is relaxed and when the cell is going to divide, chromatin condenses and forms what is known as  “chromosome” as can be seen in the figure.

Structure of Nucleus

Nucleus is surrounded by nuclear membrane/envelope which keeps the nucleolus and chromatin inside the nucleus. Nuclear membrane is double-layered. the outer layer is connected with another organelle as endoplasmic reticulum. The space between both the layers is  fluid-filled space called perinuclear space. 

Now, if the nucleus is membrane bound, then how do DNA, proteins or macromolecules pass through? For this, there are several opening in the nuclear membrane called the nuclear pores which are the sites for exchange of macromolecules.

Functions:
It stores the genetic information in the form of “chromatin”. The gene expression takes place in nucleus which includes transcription where DNA is translated to mRNA. This mRNA is then transported to cytoplasm as ribosomes (which are present outside the nucleus, described here) are required for translation.
So, basically, nucleus says,  ”Hy Dude! I contain your genetic information and will form proteins for you whenever necessary”

Cell Membrane - Protector of the Cell

Simple diagram showing
lipid bilayer and a protein

Cell membrane is the outermost covering of the cell. The thickness varies from 0.1uM to several microns. Almost all the membranes have a typical fluid mosaic model. According to this model, the membrane is composed primarily of lipids and proteins. The lipids vary from 20 to 80 percent with the remainder being proteins. Why proteins and lipids? The lipids give membrane its flexibility while proteins are responsible for maintaining the environment of the cell and also helps in transportation of molecules.

Structure:
A. Lipid bilayer - The cell membrane has two layers (hence called bilayer). Various components of lipids are: 

i. Phospholipids - It is the major component of cell membrane. Forms a lipid bilayer in which the hydrophilic (water-loving) heads are arranged to face the extracellular fluid while the hydrophobic (water-repelling) tails face the cytosolic fluid. This layer is semi-permeable i.e.; it allows only certain molecules to pass through

ii. Glycolipids: Are present on cell membrane surfaces and they have a carbohydrate sugar chain attached to them. This helps in recognizing other cells of the body. 

iii. Cholesterol: Another lipid component of cell membrane. Helps to stiffen the membrane. However, cholesterol is not found in the cell membrane of the plants.

Fluid Mosaic Model of Cell Membrane

 
B. Proteins - There are several types of proteins present in the cell membrane. Some of them are: 

i. Structural proteins - These gives support and shape to the cell.
ii. Receptor proteins - They help cells communicate with their external environment through the use of hormones, neurotransmitters and other signaling molecules.
iii. Transport proteins (globular proteins) - These proteins transport molecules across cell membranes through facilitated diffusion.
iv. Glycoproteins - They have a carbohydrate chain attached to them. They are embedded in the cell membrane and help in cell to cell communications and also helps transport of molecule across the membrane.
v. Channel Proteins: These proteins allow molecules of certain size to pass through the membrane.

Functions:
a. Maintains cell shape - The cell membrane encloses the cell and defines it. Also, it maintains physical integrity of the cell. It forms a barrier between the interior an exterior of the cell thereby protecting the organelles from the outside environment.

 
b. Selectively permeable - The cell membrane has selective permeability meaning it allows certain molecules to pass through thereby regulating entry and exit of molecules. It just acts like a guard saying “Hey Dude! You are not permitted inside unless you have a license!”.

Diagram showing Passive Transport - Simple and Facilitated Diffusion

 
c. Passive transport - It involves moving molecules through membranes without the expenditure of energy. the movement is down the gradient. It involves the process of diffusion.

i. Simple Diffusion - It is a form of passive transport where only some substances like small ions     and molecules like carbon dioxide, oxygen and water can easily move across the plasma membrane. 

ii. Facilitated Diffusion - In this process, the molecules move across the membrane but with the help of membrane transport proteins which temporarily binds the molecule which is to be moved. Again, as no energy (ATP) is required, it is a passive process.

Diagram showing Active Transport

 
d. Active Transport - It involves moving the substances across the membrane against concentration gradient. Energy (ATP) is required for this process. Generally requires two carrier proteins - one to recognize the substance to be carried and one to release ATP to provide energy for protein carriers (pumps).

 
e. Exocytosis - Certain substances can be transported out of the cell by fusing vesicles with the membrane by the process called exocytosis.

Diagram showing the process of Exocytosis

f. Endocytosis - This is exactly opposite of exocytosis where the substances are moved into the cell via fusing with the cell membrane. Endocytosis involves pinocytosis (internalizing liquid substances), phagocytosis (internalizing solid particles) and receptor-mediated endocytosis

 
g. Markers and Signaling: Surface protein markers are embedded in the cell membrane that identify the cell, enabling nearby cells to communicate with each other.

Cell Organization

So, in the last post, we discussed about the cell size and shape. Now, whether this cell is a part of an organism or an organism by itself, it has several components in common. Depending on this, the cells can fall into two categories as Prokaryotic and Eukaryotic Cells. Is it difficult to remember these words? Make it easy by knowing the meanings of these words. So, Pro means 'before' and karyotic comes from Greek word (karyos) meaning 'in a kernel', which refers to the nucleus of the cell. So, 'prokaryotic' means “before nucleus” and “eu” as in eukaryotic cell means “true” thereby 'eukaryotic' meaning “True nucleus”.

So, is there only the difference of nucleus being there or not in these two types of cells? No. There are many other differences as follows.. 

Lets start with Prokaryotes. Prokaryotes are unicellular and simplest kind of cells to evolve. The two forms of prokaryotic cells are bacteria and archaebacteria. The size of prokaryotic cell ranges from 0.0001 to 0.003 mm. They lack a nucleus (description of nucleus) and surrounding nuclear membrane. Apart from nucleus, it also lacks several organelles like mitochondria, endoplasmic reticulum, chloroplasts (in case of plants and some algae), golgi apparatus. You might wonder how do prokaryotes manage without these organelles and  what about their functions? Interestingly, the functions are taken up by prokaryotic plasma membrane. The prokaryotic cells can be distinguished into three regions as:

Diagram of a Prokaryotic Cell

1. Outside: Flagella and Pili - These are the proteins which are attached to the cell surface and used for movement and/or communication amongst themselves.
2. Cell Envelope: Consisting of cell wall, plasma membrane, and some have capsule which separates them from the exterior region and helps in intake of nutrients from the outside world.
3. Inside: Cytoplasmic Region - Consisting of the genetic material; the genome (DNA), ribosomes and various other inclusions. Also, there is an additional extra-chromosomal body present in bacteria as plasmids which confer additional functions like antibiotic resistance etc. As mentioned, prokaryotes do not have nucleus, instead they have nucleoid (suffix -oid meaning 'similar') because it is almost at the same place where DNA is present. Remember that nucleoid has no physical boundaries, it is just an imaginary structure.

Now, coming to the structure of eukaryotic cell. Eukaryotes include fungi, plants, animals and also some unicellular organisms (like yeast). As against prokaryotes, the eukaryotes have a well defined nucleus with nuclear membrane and also various membrane bound organelles like mitochondria, golgi apparatus etc. Other features are as follows:

Diagram of a Eukaryotic Cell
(Note: this is the diagram of an animal cell with no cell wall)

a. Cell/plasma membrane - Just like prokaryotes, all eukaryotic cells have an outer cell membrane. Cell walls may (in plants) or may not (animals) be present.
b. The cytoplasm has various membrane bound organelles. Inside the nucleus, there is more organized DNA (in the form of chromosome) which is associated with proteins called histones.
c. Many of the cells have cilia which helps in locomotion and movement and also functions as “antennae” for various cell signalling pathways.
d. Some cells do have flagella but with a more complex structure than prokaryotes.

Below is the table form of the above mentioned differences between prokaryotes and eukaryotes.
Don’t hesitate to give more differences if you know.! :)

Table 1: Differences between Prokaryotes and Eukaryotes.

Sr. No.	Characteristics	Prokaryotes	Eukaryotes
1.	Nucleus	Absent	Present
2.	Cellularity	Unicellular	Unicellular or multicellular
3.	Membrane bound organelles	Absent	Present
4.	Genetic material	Unorganized in nucleoid	Organized in nucleus as chromosomes
5.	Size of the cell	Very small (<5uM)	Relatively large (>10uM)
6.	Cell wall	Present	May or may not be present
7.	DNA	Circular without proteins	Linear with proteins
8.	Ribosomes	Smaller	Larger and more complex
9.	Cytoskeleton	Absent	Present
10,	Cell division	By binary fission	Generally by mitosis or meiosis
11	Reproduction	Always asexual	Sexual or asexual

Next, we will discuss the organelles in detail one by one with their functions.