PI3K Pathway

Phosphoinositide 3-Kinase enzyme

It is a signal transduction system that connects oncogenes and multiple receptor classes to many cellular functions such as cell survival, proliferation and differentiation. The key enzyme family in this pathway is PI3Ks (Phosphoinositide 3-Kinases) which transducer signals from various growth factors and cytokines into intracellular messages by generating phospholipids. These phospholipids activate the serine- threonine protein kinase AKT (also known as protein kinase B (PKB)) and other downstream effector pathways.

PIP2 (PtdIns (4,5) P₂) à PIP3 (PtdsIns (3,4,5) P₃)

The enzyme family is classified into three classes based on the structural characteristics and substrate specificity:

Class I enzyme: à These enzymes are activated directly by the cell surface receptors. They are further classified into two sub classes. i.e. class IA and class IB.

Class IA enzyme à they are heterodimers consisting of a P110 catalytic subunit and p85 regulatory subunit.

            p85 regulatory subunit mediates receptor binding, activation and localization of enzyme.

This subunit directly interacts with tyrosine phosphate motif of activated receptor (eg: platelet growth factor receptor) or to adapter proteins associated with receptor (Eg: IRS1)

Activated P110 catalytic subunit generates phosphoinositide 3,4,5 triphosphate which further activates multiple downstream signalling pathway.

Class IB enzyme à It’s also a heterodimer with p110γ catalytic subunit and p101 regulatory subunit. Apart from this they also contain some adapter proteins such as p84, p87.

P110γ subunit is activated by GPCRs through interaction of regulatory subunit with Gβγ subunit of trimeric G proteins. P110γ is expressed in leukocytes but also found in heart, pancreases, liver, and skeletal muscles.

Class II enzyme: à

It consists of only a single catalytic subunit. Phosphatidylinositol 4-phosphate (PtdsIns4P) is used as the substrate.

It is found in three isoforms: a) PI3KC2α b) PI3KC2β c) PI3KC2γ

And these can be activated by receptor tyrosine kinases, cytokine receptors, and integrins.

But still their specific role in cellular function remains unclear.

Class III enzyme: à

 It consists of a single catalytic subunit VPS34 (Homologue of the yeast vacuolar protein sorting associated protein 34). It’s also known as PIK3C3 which only producesPtdIns3P, which is an important regulator of membrane trafficking.

The subunit function as a nutrient regulated lipid kinase that mediates signalling throught mTOR (mammalian target of rapamycin).

The negative regulator of this PI3K pathway is a tumor suppressor protein called PTEN (Phosphotase and Tensin homologue). PIP3 (PtdsIns (3,4,5) P₃) is the key second messenger that drives several downstream signalling cascades that regulate cellular processes. The cellular levels of PIP3 are tightly regulated by the opposing activity of PTEN. PTEN functionally antagonizes PI3K activity through its intrinsic lipid phosphatase activity that reduces the cellular pool of PIP3 by converting PIP3 back to PtdIns (4,5) P₂.

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Phage Vector

Bacteriophages

1) Viruses specifically infect bacteria.

2) Phages have simple structure: Genetic material (DNA or RNA) is encapsulated in a protective 

proteineous coat called caspid.

3) It replicates within the bacteria and lysis them.

Bacteriophage λ in Gene cloning

* It’s an E.Coli virus that can carry an insert of a range between 15-20 kb

* After Bacteriophage λ infects the E.Coli, two things may happen:

a) Lytic cycle: where the phage replicates within cell and finally lysis the cell inorder to release 100 of phage particles and this occurs in 20 minutes.

b) Lysogenic cycle: the phage DNA can be incorporated into the bacterial genome as a prophage and maintained as a benign guest through successive cell divisions.

But under environmental circumstances, the integrated phage DNA is excised out and therefore the phage enters the lytic cycle.

* The length of the Bacteriophage λ is 50kb and integration-excision (I/E) events occurs at 20 kb respectively.

* This 20 kb I/E region is replaced with 20 kb of cloned DNA.  

Bacteriophage as Vector in gene cloning

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Ultraviolet Mutagenesis

Ultraviolet light (UV)

UV light, an electromagnetic radiation with a wavelength range between 400 nm to 10 nm, i.e. shorter and longer than visible light and X-ray.

Natural source of UV light is sun and its artificial emitted by electric arcs or by mercury lamps.

It has a strong genotoxic effects to produce DNA damage, induce mutations and causes tumor development. It also said to be the main cause of skin cancer.

Purines and pyrimidine absorbs UV at 260 nm wavelength, which makes a perfect source for identifying nucleic acids in Electrophoresis. 

There three bands of UV classified based on their wavelength: UVA àUVA1 (340-400 nm); UVA2 (320-340 nm), UVB à (290-400nm).  UVB is has the strong carcinogenic effect on skin. And UVA has some role in skin carcinogenesis.

UV Induced DNA lesions

A lesion in the DNA means the weak hydrogen bonds in the base pair denatures, which leads to instability in the DNA structure. It can be an abnormal base in the DNA or damage to the sugar-phosphate backbone.

UV induces DNA lesions by the two ways:

By producing photoproducts

UV photons are easily absorbed by the DNA molecules and an excited state is produced which allows rearrangement of electrons resulting in the formation of photoproducts. cyclobutane pyrimidine dimers (CPDs) and pyrimidine(6-4)pyrimidone (64PPs) are the photoproducts formed by the UVB radiation. These photoproducts are found to be the cause of UV-specific mutations.

By producing ROS

And UV can also induce oxidative stress in the irradiated cells through the production of reactive oxygen species (ROS) by activating small molecules such as riboflavin, tryptophan and porphyrin. This will further activate the cellular oxygen.

DNA exposed to these ROS will produce oxidative base damage such as 8-hydroxyguanine (8OH-G) and thym­ine glycol in DNA or can make strand breaks.  ROS also attack cellular nucleotide pools, producing oxidized nucle­otides such as 8-hydroxydeoxygunanosine-triphosphate (8OH-dGTP), which can still be used as nucleotide precursors for DNA synthesis.

UV induced mutation

UV photons are absorbed by the DNA molecular and therefore pyrimidine dimers (Thymine-thymine dimers) are formed. During this moment, the DNA repair mechanism works and correct the dimer units. But in some cases mutation occurs, which is basically meant as DNA repair error.

Two types of mutations are induced by UV:

1) Base substitution of Cytosine à Thymine at dipyrimidine site.

2) Tandem base substitution of cytosine-cytosine à thymine-thymine.

These two types of mutations are best known to be UV signatures, because detection of these mutations suggests the past exposure to UV.

UV mutation by Deamination

Cytosine containing CPDs are quite unstable and they are easily deaminated to uracil. So, the uracil containing CPD are the causative agent for DNA damage.

In Deamination process, an amino group is converted to a keto group in cytosine and adenine. In that case, cytosine is converted to uracil and adenine is converted to hypoxanthine. So this will alter the base-pairing specificities of these two bases during replication. i.e Normally Cytosine pairing with thymine, but after deaminated to uracil, it will now pair with adenine. Then further replication process will replace the uracil to thymine. The same case goes for adenine which gets converted to hypoxanthine, which has affinity for cytosine.

Translesion DNA synthesis (TLS)

Actually, these photoproducts such as CPDs, 64PPs and dewar isomer block the DNA synthesis by preventing the replicative DNA polymerase from passing them when they reside on a template strand during DNA replication. Nucleotide excision repair which is a DNA repair mechanism tries to excise these photolesions, but sometimes failure in the repair before replication fork passing would lead to stall and collapse of the fork at the damaged site. Thus it leads to DNA double strand break and cell death eventually.

Inorder to overcome these conditions, Translesion DNA synthesis process will overcome this replication blocks and save the cell from death. In this process, TLS polymerase enzyme restarts the DNA syn­thesis been stalled at obstructive, damaged bases on a template strand. It is meant to be error prone and it introduces mutation in the genome at high frequency.

But somehow DNA polymerase (pol) η suppresses efficiently the induction of mutations after UV irradiation by performing an error-free TLS opposite CPDs using the base-pairing ability still remaining for CPDs.

Patient with pol η are prone to this UV radiation and they show high photocarcinogenic sensitivity in skin regions exposed to sunlight. Therefore pol η is consider as a suppressor for CPD mediated mutation. But unintentionally, it will produce the UV signature mutation produced by the deamination process.

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Cloning Vector : Vehicle for transport foreign DNA into Host cell.

A DNA molecule that can carry inserted DNA and can be perpetuated in host cell is called cloning vector.

A Vector for cloning should

a) have origin of replication which makes them replicate inside an appropriate host, even though a foreign DNA is present.  

b) have a site for inserting the target gene.

c) have restriction enzyme site for cutting and inserting the target gene.

d) have a selectable marker for identify the cloning containing the targeting gene.

e) be small with an ideal size of less than 10 kb. (large size tends to break during purification process)

There four major types of vectors:

1) Plasmids

2) bacteriophages and other viruses

3) cosmids

4) Artificial chromosomes

(Note is incomplete. Will be updated soon. )

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Ti plasmid as vector for plants

• It is circular and not always
• It’s contains 56%GC content out of which 81% encodes for a protein.
• It has various types of opines

1. Octopine
2. Nopaline
3. Succinamopine
4. Leucinopine

• It is found in Agrobacterium tumefacien and Agrobacterium rhizogenes.
• There is no pseudogene.
• It works only in dicotyledon plants.
• The Ti plasmid is lost when Agrobacterium is grown above 28°C. Such cured bacteria do not induce crown galls, i.e. they become avirulent.

Genes in the virulence region are grouped into the operons vir ABCDEFG, which code for the enzymes responsible for mediating transduction of T-DNA to plant cells.

1. virA codes for a receptor which reacts to the presence of phenolic compounds such as acetosyringone, syringealdehyde oracetovanillone which leak out of damaged plant tissues.
2. virB encodes proteins which produce a pore/pilus-like structure.
3. virC binds the overdrive sequence.
4. virD1 and virD2 produce endonucleases which target the direct repeat borders of the T-DNA segment, beginning with the right border.
5. virG activates vir-gene expression after binding to a consensus sequence, once it has been phosphorylated by virA.

When Agrobacterium infects plants, a region of the Ti plasmid called the T-DNA is taken up by the plant cell and incorporated into one of its chromosomes.

The genes in the T-DNA are referred to as phyto-oncogenes because they induce neoplastic, or tumor-producing, growth. To use the Ti plasmid as a vector for introducing new genes into plants, it is necessary to disarm the plasmid so that it does not cause tumors. The task is accomplished by deleting the genes in the T-DNA that encode the enzymes controlling auxin and cytokinin synthesis. Genes for antibiotic resistance are normally used for this purpose.

A cloned gene can then be inserted into the T-DNA of the engineered Ti plasmid, and the plasmid can be used to infect cultured cells, leaf discs, or root slices. The infected cells are placed on a culture medium that contains auxin and cytokinin (to induce growth) and the antibiotic. Only the transformed cells can grow in the presence of the antibiotic, because they have received the T-DNA containing not only the foreign gene but also the gene for antibiotic resistance.

To obtain a plant containing the foreign gene, it is necessary to regenerate whole plants from the cultured, transformed cells. Fortunately, methods for accomplishing this regeneration have been developed for many plants, although not for all important crop species yet. The regeneration involves adjusting the ratio of cytokinin to auxin to stimulate both shoot and root formation.

Although the transformation of cereal crops with Agrobacterium and their regeneration from cultured cells has been difficult, some remarkable successes have resulted from this approach. Investigators have introduced numerous foreign genes into plants such as tobacco, including soybean storage protein genes. Genes for such desirable characteristics as disease resistance, herbicide resistance, and salt tolerance have been transferred to crop plants by these techniques, and commercial crops now are being grown with these genetically engineered strains.

Arabidopsis can also be transformed directly by exposing whole plants to a solution containing Agrobacterium that is carrying engineered or wild-type Ti plasmids. The plants must be treated in such a way to allow the Agrobacterium to enter tissue, either by applying a vacuum or by treating with detergents. The Agrobacterium penetrates the floral tissue and transforms the developing ovules. Isolation of seeds from these Agrobacterium-exposed plants yields up to 2% of the seeds that are transformed with the T-DNA. This approach is very useful for molecular genetic studies, such as for characterizing DNA sequences involved in the control of gene expression, or constructing large libraries of insertional mutants.

Gene transfer using Ti plasmids

In the Ti plasmid itself, the T-DNA is flanked by 25 bp imperfect direct repeats known as border sequences, which are conserved between Octopine and nopaline plasmids. The border sequences are not transferred intact to the plant genome, but they are involved in the transfer process. The analysis of junction regions isolated from plant genomic DNA has shown that the integrated T-DNA end-points lie internal to the border sequences.

The right junction is rather precise, but the left junction can vary by about 100 nucleotides. Deletion of the right border repeat abolishes T-DNA transfer, but the left-hand border surprisingly appears to be non-essential. Experiments in which the right border repeat alone has been used have shown that an enhancer, sometimes called the overdrive sequence, located external to the repeat is also required for high-efficiency transfer.

Two of these genes, virA and virG, are constitutively expressed at a low level and control the plant-induced activation of the other vir genes. VirA is a kinase that spans the inner bacterial membrane and acts as the receptor for certain phenolic molecules that are released by wounded plant cells.

Acetosyringone, a phenolic compound has been the most widely used in the laboratory to induce vir gene expression

But acetosyringone do not attract bacteria to wounded plant cells. Rather, the bacteria appear to respond to simple molecules, such as sugars and amino acids, and the vir genes are induced after attachment

Activated VirA transphosphorylates the VirG protein, which is a transcriptional activator of the other vir genes.

The induction of vir gene expression results in the synthesis of proteins that form a conjugative pilus through which the T-DNA is transferred to the plant cell. The components of the pilus are encoded by genes in the virB operon.

DNA transfer itself is initiated by an endonuclease formed by the products of thevirD1 and virD2 genes. This introduces either single-strand nicks or a double-strand break at the 25 bp borders of the T-DNA, a process enhanced by the VirC1 and VirC2 proteins, which recognize and bind to the overdrive enhancer element. The VirD2 protein remains covalently attached to the processed T-DNA.

T strands are coated with VirE2, a single-stranded DNA-binding protein. The whole complex, sometimes dubbed the firecracker complex because of its proposed shape, is then transferred through the pilus and into the plant cell. It has been proposed that the VirD2 protein protects the T-DNA against nucleases, targets the DNA to the plant-cell nucleus and integrates it into the plant genome. The protein has two distinct nuclear localization signals, with the C terminal signal thought to play the major role in targeting the T-DNA.

It has been observed that the nucleus of wounded plant cells often becomes associated with the cytosolic membrane close to the wound site, suggesting that the T-DNA could be transferred directly to the nucleus without extensive exposure to the cytosol.

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Plasmid

1) Self- replicating, double stranded, circular DNA molecule.

2) It’s maintained as an independent extrachromosomal entity in bacteria.

3) There are different types of plasmid that has been present in certain for survival and diversification:

• F plasmid à Involves in conjugation for transferring information to another bacteria. 

* Its total length may be 100 kb.
* It contains genes responsible for cell attachment and plasmid transfer between specific bacterial strains during conjugation.
* It has an insertion sequences that assist plasmid integration into the host cell chromosome.
(Small Picture of F plasmid.)

• R plasmid à resistance to antibiotic.

• Degradative plasmid à carry specific genes for the utilization of unusual metabolites.

• Cryptic plasmid à no functional coding genes. 

• Col plasmids à colicin production. Bacteriocin gene produces cloacins which kills bacteriophages. 

4) High copy number plasmids (relaxed plasmids) à 10-100 copies of plasmids per host cell.
5) Low copy number plasmids (Stringent plasmids) à 1-4 copies of plasmids per host cell.
6) Host range of plasmid is determined by its ori region. And there are two host range of plasmid i.e. Restricted where the plasmid is restricted to replicate in certain bacteria only (eg: Plasmid Col E1 replicates only in enteric bacteria like E.Coli or salmonella) and Broad host range where the plasmid can replicate any group of bacterium (RP4 plasmid can replicate in most of gram negative bacteria).

7) An episome is a plasmid that can exist either with or without being integrated into the host’s chromosome.

Plasmid exists in three forms: covalently closed circles (CCC) DNA, open circles (OC) DNA, linear DNA.

Plasmids look like a coiled rubber band and . Topoisomerase, an enzyme that regulates the winding (over or under) of the DNA. It relaxes the supercoiled plasmid and brings CCC form. But if a Endonuclease (Enzyme cleaving the phosphodiester bond of DNA) attacks this uncoiled CCC, then a nick is formed in one of the strands of plasmid DNA. So therefore it forms a open circular DNA. The nick can be cured by another enzyme called DNA ligase which joins both the ends of the DNA strand. This enzyme is one of the important enzyme for joining the fragmented DNA with the plasmid. 

Now, DNA gyrase is type of Topoisomerase II which provide negative supercoiling of DNA by cutting the double strands and then joining them together by twisting the ends. 

In certain bacteria like streptomyces sp or Borrelia burgdorferi, plasmids are found in linear form. To protect the ends from nuclease digestion, there are two general mechanism involved:
a) terminal contains a hairpin loop structure (Borrelia).
b) terminal are protected by covalent attachment of protein (Streptomyces sp.)

Certain traits exhibited by the plasmid carrying genes

The R plasmid contains antibiotic resistance genes such 

The antibiotic resistance genes act as marker for identifying the plasmids containing the gene of interest. For eg: plasmid containing Ampicillin resistant marker will let the host bacteria to grow in a medium containing ampicillin. 

Plasmid protects the  bacterium for hazardous condition and enable them to survive even in limited resources. They also pass some part or whole gene to another host genome or plasmid through conjugation.  

Plasmid in gene cloning

What makes the plasmid a suitable vector for gene cloning?

* Plasmids are easy to isolate, purify and can be reintroduced into a bacterium by transformation.

* They bear antibiotic resistant genes which can be used for selecting the bacterial host during screening process.

* Plasmids with a foreign DNA is said to be chimera.

* They contain origin of replication (ori) region that can allow the plasmid to replicate individually from the bacterial genome.

* plasmids with high copy number, low molecular weight and conjugation property can be produced in more numbers and thus increasing the volume of sample. 

pBR322 – First gene cloning vector

1. It contains 4,361 bp and carries two antibiotic resistance genes: Ampr (Ampicillin) and Tetr (Tetracycline).

2. Plasmid make up: Within Tet^r (BamH1, HindIII, and SalI RE recognition site), Within Amp^r (PstI site), EcoRI not within any coding DNA, and origin of replication that functions only in E.Coli.

How does this pBR322 plasmid work as cloning vector? 

How does the Alkaline phosphatase treated linear plasmid ligate together even with lose of phosphate group at 5' end? 

A phosphate group at the 5' end is important for the T4 ligase to join the plasmid and the target DNA. But due to the loss of 5' phosphate group, two nicks are formed at the joining site. Therefore the plasmid with target DNA should become unstable and linear. 

Somehow this doesn't happen, because two phosphate group present in the target DNA hold the plasmid together and once they are transformed into the host cell, the two nicks are ligated by the host DNA ligase enzyme. 

Other plasmids

Ti plasmids

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cDNA Library - A detailed note

Its a collection of  cDNA (Complementary DNA) clones generated from mRNA sequences of  a single cell population. cDNA is double stranded DNA complementary to the mRNA sequence synthesized by the enzymes reverse transcriptase and DNA polymerase. 

Steps involved in creating cDNA library: 

Extraction and purification of mRNA from the cell

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Synthesis of cDNA from the total mRNA

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cDNA cloning

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Screening of  the host containing the cDNA 

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Genomic Library- A detailed note

It is the collection of total genomic DNA from a single organism. The total genome from an organism is taken and digested into fragments. Then, they are carried into a host organism by a vector DNA and cloned into many copies. Through screening process, the cloned host organism with the gene of interest is taken and preserved for future use.

So, now we will discuss the steps in constructing a genomic library.

1) Isolation and purification of total genomic DNA from the cell (Prokaryote or eukaryote)

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2) Restriction digestion of the total genomic DNA using Restriction enzymes.

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3) Selection of Vector

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4) Insertion of the fragment into the vector using Ligase enzyme.

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5) Host Transformation.

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6) Selection. 

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Endocytosis: Import system of the cell

Endocytosis is the import system of the cell where the macromolecules and particles from the surrounding medium is taken inside the cell. Cells internalize the materials by using plasma membrane components and deliver them to an internal compartment called endosomes.  From this endosomes they can be recycled to same or different regions of the plasma membrane or can be delivered to lysosomes for degradation.

There are three types of endosomes in the cell,

Early endosomes

Endosomes which are found just beneath the plasma membrane which consists of the components obtained directly from outside of the cell like carbohydrates, fats etc. The components may go to the Golgi apparatus or may be transferred to the lysosomes via late endosomes. In early endosomes, mild digestion may start, hence many hydrolases are synthesized and delivered there as proenzymes called Zymogens which contain extra inhibitory domains at their N terminus that keep the hydrolases inactive until these domains are proteolytically removed. 

Late endosomes

Endosomes which are found close to Golgi apparatus and near the nucleus. These are actually intermediate between early endosomes and lysosomes.

Recycling endosomes

Endosomes which is found near the early endosome will be containing certain membrane proteins or receptor. It will fuse with the plasma membrane when a signaling molecule activates the signaling cascade which inturn activates the endosome to bind to the cell membrane. After finishing their function, they will get back to the cell as vesicle. Best example for this is the GLUT4 proteins which is found inside the cell bounded to the vesicle. 

Both the late and early endosomes differ in their protein compositions and transition from early to late endosomes is accompanied by the release of Rab5 and the binding of Rab7. 

The acidity inside the late endosomes is higher compared to that of the earlier one. This gradient of acidic environments has a crucial role in the function of these organelles.  Endosomes has a vascular H+ ATPase over the membrane which is responsible for making the vesicle acidic by bringing in more amount of H+ ion. The pH basically in early endosomes is 6.

Coated vesicles: They are having a distinctive cage of proteins covering their cytosolic surface. Now before the vesicles fuse with a target membrane, they discard their coat as it is required for the two cytosolic membrane surfaces to interact directly and fuse.

The coat has two functions,

1) It concentrates specific membrane proteins in a specialized patch which then gives rise to the vesicle membrane.

2) The coat molds the forming vesicle.

These proteins are assembles into a curved basketlike lattice that deforms the membrane patch and thereby shapes the vesicle. 

Three well characterized types of coated vesicles distinguished by their coat proteins:

Clathrin coated à mediate transport from the endosomal-Golgi compartments and from the plasma membrane. 

COPII coated and COPI coated àmediate transport from the ER and Golgi cistern 

Clathrin is a coat protein whose subunits consists of three large and three small polypeptide chains  that together form a three legged structure called triskelion. These triskelions assemble together into a basketlike convex framework of hexagons and pentagons to form coated pits on the cytosolic surface of membranes.

Adapter protein which is a coat component in Clathrin coated vesicles form a discrete second layer of the coated positioned between the Clathrin cage and the membrane. They bind the Clathrin coat to the membrane and trap various transmembrane proteins which include the transmembrane receptors that capture soluble cargo molecules inside the vesicle so called cargo receptors.

Dynamins: These are the cytoplasmic proteins which helps in regulation of pinch off and uncoating of coated vesicles.  The protein consists of PIP2 binding doman which tethers the protein to the membrane and a GTPase domain, which regulates the rate at which the vesicles pinch off from the membrane. 

There are three types of Endocytosis: 

• Phagocytosis
• Pinocytosis
• Receptor Mediated Endocytosis

Phagocytosis: large particles are ingested via large vesicles called phagosome, which is about > 250 nm in diameter. Phagosome end up in lysosomes and the products of the subsequent digestive processes pass into cytosol to be used as food. Phagocytosis required other than nutrition is carried out by specialized cells called professional phagocytes. In mammals two main classes of professional phagocytes are macrophages and neutrophils. Any indigestible particle inside the phagocytes forms residual bodies which will be expelled out of the cell through Exocytosis.

It’s a triggered process which required the activation of receptors that transmit signals to the cell interior and initiate the response. Eg: Antibodies triggers phagocytosis by exposing the tail Fc region to the phagocytic cells.

Localized actin polymerization initiated by Rho family GTPases and their activating Rho-GEFs shapes the pseudopods. An active Rho GTPase switches on the kinase activity of local PI kinases and initial actin polymerization occurs in response to an accumulation of PIP2. To seal off the phagosome and complete its engulfment, actin is depolymerized at its base as PIP2 is subject to a PIP3 kinase which converts it into PIP3 is required for closure of the phagosome and may also contribute to reshaping the actin network to help drive the invagination of the forming phagosome.

Don’t eat me signal: the cell surface protein binds to the inhibitory receptor of the phagocytic cell and recruits tyrosine phosphatase proteins which antagonize the intracellular signals required for the phagocytosis.

Eat me signal: Phosphatidyl serine present inside the cell membrane will get exposed to the extracellular region by the enzyme Flipase. The exposure will lead to the initiation of eat me signal and macrophage will phagocytose the whole cell.   

Pinocytosis: Fluid and solutes are ingested via small pinocytic vesicles which are about 100 nm in diameter. This process is a constitutive process where it occurs continuously regardless of the needs of the cell. The pinocytic vesicles are formed from the coated pits of the plasma membrane. The pits are mostly coated by Clathrin and it will be pinched off from the membrane by dynamins.

Not all the pinocytic vesicle is coated with Clathrin, but there are other vesicles which are not understood very well and they are caveolae. They are recognized by their ability to transport molecules across endothelial cells which form the inner lining of blood vessels. They are thought to form from membrane microdomains or lipid rafts. The major structural proteins in caveolae are caveolins which are integral membrane proteins that each insert a hydrophobic loop into the membrane from the cytosolic side but do not extend across the membrane.   The caveolins don’t not dissociate like Clathrin do because these proteins are integral proteins hence they stick with the vesicle surface even after they are pinched off. Instead they are delivered to the target compartments where they are maintained as discrete membrane domains. 

Receptor mediated endocytosis:

The macromolecules bind to complementary transmembrane receptor proteins, accumulate in coated pits and then enter the cell as receptor macromolecules complexes in clathrin coated vesicles. This process is called receptor mediated endocytosis. This happens basically because the ligands are selectively captured by receptors, receptor mediated endocytosis provides a selective concentrating mechanism that increases the efficacy of internalization of particular ligands more than a hundred fold. The LDL receptor goes to the early endosome and gives off the cholestrol molecules. Then retrives back to the plasma membrane without any coating. 

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