Endoplasmic Reticulum Er Apparatus A Closer Look Biology Essay

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Morphological sciences were developed in relation with the improvement of different preparative techniques and development of optical instruments which began in the middle of 19th century [1]. Improvements like correction of microscope lenses and finding aniline dyes as selective staining of tissue components which led to great discoveries in cell biology field that was the foundation of modern histology. The last decade of this century was called the golden age of microscopic anatomy because of using a vast variety of fixatives and dyes for better quality. After this decade the rate of new discoveries by using light microscope was declined till the middle of 20th century when the electron microscopes were discovered. Electron microscopes gave access to discovery of new structural shapes, new macromolecules and new geographical information about the organelles inside the cell which was beyond the power of light microscopes. Hence lots of different new methods for specimen preparation and staining of the cell components were discovered. The new discoveries of the cell, attracted the interests of morphologists, physiologists and biochemists to a new unified field of science called cell biology.

Endoplasmic reticulum is one of the components of the cell which works as pathways and a storage unit of protein, calcium and other substances through cytoplasm by connecting different organelles in a cell. The word "endoplasmic" means "floating in the cytoplasm of the cell" and the word "reticulum" came from a Latin word that means "little net" hence, together it means a little net that floats through the cytoplasm of a cell. The structure, function, dysfunction, isolation and visualization of endoplasmic reticulum is illustrated in this report.

History

Endoplasmic reticulum was first discovered by the french cytologist Garnier in the last years of 19th century [1,2]. He described some filamentous structures in the cytoplasm of pancreas and salivary gland cells which was stained with basic dyes. He called it ergastoplasm that varied in form and quantity. Then he included that ergastoplasm involves in synthetic function of cells and it is extended through the whole cytoplasm(rough ER). Bensly (1898) and Mathews (1899) illustrated the same thing. Later on, by development of staining method, basophilic components of the cytoplasm were named ribonucleoproteins (brown dots in cytoplasm) in 1940.

ER-1.jpg

Figure 1. Drawing of rough ER with tubules and vesicles which are the result of nonideal fixation [1].

By the help of electron microscopeIn Porter (1945) saw connections of delicate branching and strands that formed a network among the cytoplasm of the cell which was named Endoplasmic Reticulum

and he also mentioned small vesicles (50-200 nm) connected to ER or sometimes completely free in cytoplasm that were named ribosomes. Rough ER was also known as Granular ER.

Then smooth ER were discover in 1954 by Palay and Palade that was also named Agranular ER which didn't contain the ribonucleoproteins and was different in shape and function from rough ER.

The spread of cell components was analyzed with stereological and morphometric methods to electron micrographs to compare the extension of cell compartments in liver cells which was another proof of ER extension through the cytoplasm.

ER-2.jpg Figure 2 . Extension of cell compartments of liver cell [1].

The endoplasmic reticulum is a highly active organelle involved in synthesis and transport of proteins, glycoproteins and lipoproteins; synthesis of cholesterol, steroids, phospholipids and triglycerides; it is also involved in degradation of glycogen and in metabolism of xenobiotics.

As it can be seen in figure 2, ER occupies most surface area of a cell which consists of two different parts, rough ER and smooth ER, that have different functions as it's going to be discussed in next sections.

2. Rough Endoplasmic Reticulum

The rough ER or granular ER is a connective network of membrane bounded channels which sometimes is not clear under microscopes and it can be seen as circular shapes with the ribosomes attached to it or it can be seen as wide elongated tunnels (cisternae) with ribosomes attached to it [1,2]. The large amount of ribosomes linked with cisternae makes it basophilic. Rough ER works as a place for protein secretion through the cytoplasm or other parts of the cells that a protein is meant to be.

2.1. Function

It works as pathways for protein secretion [1,2]. The protein synthesis begins with the transcription of DNA into mRNA, then mRNA attaches to the smaller part of ribosomes and starts coding for amino acids. After that, it signals the tRNA to grab a specific amino acid and then tRNA attaches to the first channel in bigger part of the ribosomes and starts to bind the amino acid sequence. There are codons on mRNA which activates signals, after emergence of amino acids, to the ribosome receptor proteins on the membrane of rough ER to bind to the bigger part of ribosomes. Then the receptor proteins provide a channel through the membrane and the channel of large ribosome subunit. Hence the amino acid goes inside ER through the membrane but a protein (signal peptidase) on the inner side of ER cuts the amino acid when the amino acid sequence is complete and leaves the protein inside the ER. After that the ribosomes detach from the membrane and go to the cytoplasm again. Two proteins, ribophorin I and ribophorin II, are responsible for attachment and detachment of ribosomes.

ER-ribosome.jpg

Figure 3. The process of protein synthesis and protein insertion into the rough ER [1].

Proteins will be carried to the golgi apparatus for further secretion by membrane bound vesicles which are COP I and COP II (coat protein complexes).

2.2. Place

Rough ER exists in all eukaryotic cells but differs in shape and amount depending on cells function and the proteins that are produced. For example, there is larger space between cisternae of neuron cells which is filled with ribosomes rather than glandular cells. As it can be seen in figure 4, rough ER is attached to nuclear envelope and it spreads out through the whole cell.

Untitled.jpg

figure 4. The shape and place of rough ER and smooth ER in an animal cell [2].

2.3. Dysfunction and Diseases

Inhibition of protein glycosylation, interruption of calcium homeostasis and reduction of disulfide bonds agitate accumulation of unfolded protein in the ER and they are called ER stress [3,4,5,6]. The term unfolded protein response (UPR) is defined as cell response to ER stress. Cells normally respond to ER stress by increasing transcription of genes encoding ER-resident chaperones such as GRP78 to refold the proteins or by controlling mRNA translation to synthesize protein.

Obviously the dysfunctions of ER is caused by the diseases mentioned here but the diseases will be developed because of ER dysfunction. As the ER fulfils several functions in the cell, severe disturbance in its function can lead to the cell death. When ER is damaged by some mechanisms, it stresses the cell so it leads to an evolutionary conserved cell stress response which is aimed to equilibrate the damage to the cell. This response, itself, can trigger the cell death when dysfunction of ER takes too long or is intense. The mechanisms that cause ER not to work properly and leads to cell death have remained ambiguous.

Physiological balance of ions is required for the majority of cellular functions. A really important ion in a cell is Ca+2 which is a signal molecule that controls a lot of functions like cell growth, development, protein synthesis and regulation of methabolism of lipids and polysaccharides. The ER lumen contains a high concentration of Ca2+ which plays a role in protein folding. During Ca+2 deprivation ,ER goes under stress therefore it won't function properly. During this dysfunction, accumulation of unfolded proteins happens which is an evolutionary conservative response. This reaction is called unfolded protein response (UPR) and can lead to cell death.

Another problem that can cause ER stress in the cell is glucose deprivation (lack of glucose) which can lead to glycosilation of N-linked proteins.

Ischemia (restriction in blood supply) is a neuronal injury which is caused by ER dysfunction associated with neuronal stress. The elF-2a kinase PERK (double stranded RNA-activated protein ER kinase) is activated during brain ischemia. This protein is an ER resident protein kinase which is switched on during ER dysfunction. Then protein synthesis and protein folding is disturbed hence the accumulation of proteins is found in ischemia which results in neuronal injury. Ischemia also prevents the calcium uptake by brain microsomes. It seems that ischemia results in depletion of ER calcium stores in neurons which won't be restored back because ER Ca2+ ATPase is deactivated and protein synthesis is stopped during NO synthesis (which is resulted because of ischemia).

Activation of stress-associated ER protein 1, excess of oxygen-regulated proteins (an ER chaperone) and acidic and alkaline pH shifts are other factors that cause calcium depletion from ER stores thus they are involved in ER dysfunction and focal cerebral ischemia which is under investigation.

Alzheimer's disease is developed by ER dysfunction. Mutations in the presenilin-1 gene starts the early stage of familiar Alzheimer disease which decreases the signaling pathway of UPR (unfolded protein response). Presenilin-1 gene mutations inhibits the activation of ER stress alarm devices such as IRE1 (ER membrane protein) or PERK by inhibiting the phosphorylation of Ire1p which is responsible for the activation. Therefore, the amount of GRP78 protein levels, which is a chaperone to refold the proteins, was decreased in the brain cells of the patients. Joining Presenilin-2 genes will result in sporadic Alzheimer disease. Cells are more vulnerable in this type of Alzheimer, because they are under conditions like hypoxia or tunicamycine or calcium ionophore exposure.

A regulatory relation is seen between ERAD (endoplasmic reticulum associated degradation) and UPR (ER stress-inducing agents) which lead to pathological processes that result in cell apoptosis. Parkinson's disease gene produces Parkin, a ubiquitin-protein ligase, and mutant Parkins which lack ubiquitin-protein activity. UPR induces an up-take of Parkin and cells overexpressing Parkin, but not mutant Parkins found in Parkinson's disease patients, are particularly resistant to unfolded protein-induced cell death. Therefore the patients will suffer from the effect of neurotoxicity on their neurons in Parkinson's disease arising from ER stress.

ER stress also plays a role in diabetes. Pancreatic β-cells produce various glycoproteins and they synthesize and secret insulin. Mutations in PERK gene in association with the autosomal recessive disorder Wolcott-Rallison syndrome will result in massive β-cells loss in humans. Failure of PERK to phenocopy eIF2a inhibits the activity of eIF2a during ER stress which kills β-cells. Also, other diabetes caused by nonsecreted insulin mutant and homozygous deletion of CHOP delay disease, imply a role for this gene in β-cell depletion.

The role of ER stress in heart disease is not investigated completely but some experiments have been executed on mice to examine the relationship. ASK1 kinase activity increases aortic constriction but the mice showed decreased cardiomyocyte apoptosis under ER stress.

3. Smooth Endoplasmic Reticulum

Smooth ER was first described as agranular reticulum with smooth membrane and ribosome free local differentiation of ER apparatus which was in irregular communication with ER by vesicular transport [1,2]. It is made of dense masses of soft surfaced tubules. They were thought to be important for production of rough ER tubule but the improvements of science proved it wrong. SER has lower negative surface charge and higher concentration of cholesterol and galactose sialic acid (contributes in negative charge of the cell and binds to selectin). There are several other functions of smooth ER which are different from rough ER.

3.1. Function

Morphological observations showed different functions of smooth ER in different cell types. Smooth ER stores calcium molecules and levels the calcium concentration [1,2].

Smooth ER works in lipid metabolism in meibomian glands (lubricant secretion glands in eye lid) and in intestinal epithelium cells while fat absorption. After eating fat the intestine lumen contains a mixture of di- and triglycerides stabilized with bile salts. Triglycerides are hydrolyzed by pancreatic lipase to release fatty acids and monoglycerides. Then they go through the membrane into the apical cytoplasm of absorptive cells. Then an enzyme called Co-A ligase (fatty acid enzyme) in the membrane of SER catalyses the reaction of fatty acids into thiolesters which reacts with monoglyceride and result in triglyceride by the help of microsomal enzymes. Finally the triglycerides will be stored in the lumen of SER as droplets of osmiophilic lipids which contain triglyceride, phospholipid, cholesterol and stabilizing proteins. Later on they will be released into intracellular as chylomicrons (large lipoprotein particles) which are carried by limph to the blood stream.

Also, smooth ER works in synthesis of steroid hormones in adrenal cortex and interstitial cells of the testis and in the corpus luteum of the ovary. The isomerase, hydroxylase andhydroxysteroid dehydrogenase which are engaged in synthesis of steroid hormones can be found in smooth ER.

Smooth ER associates in glycogenolysis process of the liver because there was seen a close relationship between glycogen and smooth ER. Glucose-6-phosphatase which van be found in SER is responsible for break down of glycogen.

There are some oxidase function in the liver in relation with SER hypertrophy (enlargement of an organ because of nutrition excess) which leads to drug metabolism and drug tolerance toxicity for the liver cells.

3.2. Place

Smooth ER exists in all eukaryotic cells. But they can be mostly found in the cells that are involved in lipid metabolism. Smooth ER is attached at many sites with cisternae of rough ER but the tubules are more variable in their diameter and branches. The only difference in distinguishing smooth ER from rough ER is the ribosomes that are not attached to smooth ER and bigger softer branches of tubules in smooth ER as it can be seen in figure 5 below.

Untitled-1-1_edited-1.jpg

Figure 5. The shape and place of SER and RER in a plant cell [2].

3.3. Dysfunction and Diseases

The dysfunctions of smooth ER is in associated with rough ER because most of the diseases are caused by calcium depletion (stored in smooth and rough ER) hence resulting in protein synthesis suppression (protein synthesis in rough ER) or unfolded proteins or misfolded proteins [3,4,5,6].

So the disfunctions can not be actually separated in rough or smooth ER.

For example, the diabetes is a disease caused by accumulation of intracellular glucosamine which can be developed by ER dysfunction (mainly smooth ER) because of glycolysis process which occurs in ER.

Or accumulation of lipids can cause atherogenesis (thickness of artery wall) which is developed by ER dysfunction.

For full information about ER dysfunction, see section 2.3 above.

3.4. Sarcoplasmic Reticulum

Bowman (1840) and Rollet (1888) described that muscle fibers contain contractile fibers embedded in a semi fluid cytoplasmic matrix [1]. Then by improvements of electron microscopy Bennet and Porter (1953) described reticular structures which were made of connected tubules like smooth ER that were related to sarcomeres of myofibrils but without ribosomes attached to it. It was concluded that these tubules might carry stimuli from cell surface to specific regions in myofibrils.

Sarcoplasmic reticulum is a kind of smooth ER which can be found in muscle cells that is involved in muscle contraction and relaxation. The process begins with depolarization of muscle cell surface that goes inward through the T-tubules (transverse elements) and then the current goes across the T-tubules to the terminal cisternae of reticulum and frees the calcium which results in muscle contraction. Then the calcium is separated by sarcotubules of reticulum which results in muscle relaxation and regathering of calcium in terminal cisternae. Different part of the process are shown in figure

sarco ER.jpgFigure 6.Extention of Sarcoplasmic reticulum around myofibrils in amphibian skeletal muscle [1].

As it can be seen the dysfunction of sarcoplasmic ER will result in problems of muscle contraction. For example, myocardial ischemia results in the activation of lysosomal phospholipase C and disruption of calcium transport in sarcoplasmic reticulum mediated by oxygen free radicals.

In the next section, the methods of "how to isolate and visualize the endoplasmic reticulum" is going to be discussed.4. Isolation

Eukaryotic and prokaryotic cells contain different organelles that have different functions [7,8,10]. To examine the significant functions and significant structures of the organelles, the isolation experiments are executed to assay the organelles activity or their enzymatic activity or their structure individually. Then the information can be used for further discoveries or further uses like treatment of illnesses.

There are two main steps of organelle isolation, cell disruption and centrifugation, which are shown in figures 7 and 8.

ER-iso.jpg

Figure 7. Cell disruption step [10].

The cells that are chosen for the isolation should be first homogenized which means that the process involves breaking open the cell membrane and cell wall (if there is any) but the cells should be carefully picked depending on quantity and quality of the organelle that is going to be examined. Solid tissues or tissues from large organs of large animals can cause problems because of the connective tissues or large blood vessels. Some tissues like skeletal muscles are also hard to prepare because of the presence of actin and myosin they are hard to homogenize. But, yeast cells (i.e. Saccharomyces cerevisiae) can be used easily because of their resemblance to eukaryotic cells and easy access, although enzymatic digestion is required due to the presence of hydrocarbon polymers and glycoprotein in their cell walls.

After cell disruption, the centrifugation step is taken which depends on the density, shape and size of the organelle that is needed.

ER-iso-1.jpg

Figure 8. Different centrifugation methods [10].

When a particle is denser or heavier it will sediment faster and when two particles have the same weight, the one with a more compact shape will sediment faster.

Because there are different processes of disruption and centrifugation, one of the processes for each is going to be discussed in the following description depending on the facilities that are available.

4.1 Cell disruption

Chemical reagents like detergents are mostly used for cell disruption because of low prices and accessibility in combination with physical grinding methods for plants.

Detergents are amphipathic (hydrophilic and lipophilic) molecules that have hydrophobic parts which bind to hydrophobic parts of membrane proteins and then displace lipids which leads to cell opening. Two types of detergents are ionic (e.g.SDS, sodium dodecyl sulfate) and nonionic (e.g. Triton x100, NP 40).

Grinding is used as a pretreatment step for some sort of cells (e.g plants and yeast) that have cell walls.

Materials and methods

In plants, 20 g of leaf material (e.g. cauliflower) is weighed and poured in an ice-cold mortar with 40 ml of ice-cold manitol grinding buffer (0.3 M mannitol; 0.006 M KH2PO4; 0.014 M K2HPO4; pH 7.2). Then the whole materials are grinded for 4 minutes for breaking down the cell wall (chemical and physical damage). The whole materials in a mortar are filtrated through 4 layers of cheese cloth into another tube. The remaining is the organelles of the cells. The centrifugation step is needed for further separation of a significant organelle.

In animals, liver cells (e.g. rat liver) are homogenized in buffer A (containing 146 mM sucrose, 100 mM KCl, 1.5 mM MgCl2, 10 mM Tris-HCl, pH 7.0, 0.1 mM PMSF). Then the whole homogenate was filtrated through 3 layers of wet cheese cloth to remove cell's membrane. After that the NP 40 or Triton x100 is added to the homogenate and then the whole gradients (which are in a tube) are incubated on ice for 5 minutes while stirring continuously. Then centrifugation step is needed for separation of endoplasmic reticulum.

4.2. Centrifugation

In this step the procedure is continued with animal cells as an example of ER purification which involves both centrifugation methods, first differential and then density gradient centrifugation [7,8,10]. But neither differential nor density centrifugation result in complete separation of the organelles.

Differential separation depends on the shape and velocity of the organelles which have different diameters. And it's widely used for or fractionation of organelles at the first step because it separates large membrane organelles from smaller ones rapidly.

Density centrifugation depends on the density of particles which uses the density of organelles to float in different concentrations of sucrose buffer. Different densities and diameters of the organelles are shown in table 1 below.

Table 1.approximate diameters and densities of subcellular particles in sucrose [10].

Structure

Dimensions

(μm)

Density

(g/cm3)

Nuclei

3-12

>1.3

Mitochondria

0.5-2

1.19

Golgi app

-

1.06-1.10

Lysosomes

0.5-0.8

1.21

Peroxysomes

0.5-0.8

1.23

Plasma membrane

0.05-3

1.13

S-ER

0.05-3

1.16

Nucleic acids, Ribosomes

0.03

1.60-1.75

Soluble proteins

0.001-0.002

1.30

Materials and methods

Differential centrifugation (figure 9), the animal cell sample is first centrifuged at 700 g for 10 minutes for nuclear particles separation. Then the supernatant is removed and centrifuged again at 10.000-15.000 g for 10 minutes which results in sedimentation of mitochondrial fractions. After that the supernatant is removed and centrifuged for the third time at 100.000 g for 30 min-2 hr which results in microsomal fractions separation including ER.

ER-cent.jpg

Figure 9.Differential centrifugation steps[8].

Density centrifugation (figure 10), the supernatant is removed (from the last step of differential centrifugation) and the pellet is suspended in sucrose with different concentration layers. 2 ml of each different concentration of sucrose (in BM - buffered mannitol : 0.3 M mannitol in 50 mM

Tris-HCl, pH 7.2) is pipette into the vial in the order of 1,2,3,4,5,6 and 7% of sucrose from top to bottom (or from 1.10 to 1.22 g/cm3 of sucrose). Then the tube was centrifuged at 11.000 g for 1 hour at 4°c. The pellet contains the ER as can be seen in the figure .

Figure 10.Organelle separation by density equilibrium centrifugation [8].

After isolation, the presence of ER should be proven by using biochemical methods which is done by following the activity of an enzyme which exists exclusively in the target organelle. Hence it is done by using a marker enzyme (Glucose-6-phosphatase for ER) which oxidize glucose and the enzyme activity can be followed at 510 nm using spectrophotometer.

ER can be visualized either isolated or not isolated (in the cell) by visualization methods described in the next section.

5. Visualization

There are many methods that are used to visualize an organelle in a cell (or isolated from a cell) [8,9,10]. Considering the organelle of interest and the quality of details of an organelle or the enzymatic activity of an organelle, light or electron microscopy can be chosen.

ER-vis.jpg

Figure 11.Schematics of different visualization methods [10].

The light microscope allows us to magnify cells up to 1000 times which allows us to observe details as small as 0.2 μm which has it's own restrictions in clarity and distinguishing organelles. There 3 different systems to look at cells with light microscope, straight forward optics and phase contrast optics and interference contrast optics, the last two systems show differences of regions in a cell with differing in refractive index.

Fluorescence microscopes are basically the same as light microscopes but the light is passed through filters for letting specific wavelength to pass. Then the specific wavelength goes through the specimen and shows the place of the target organelle with the help of specific dyes which is useful to observe the extension of an organelle in a cell.

TEM or transmission electron microscopy is used for observations as small as 2 nm. The principles are the same as light microscopy but electron beams are passed through a thin layer of specimen in a vacuum conditions instead of light beams. Then a high resolution 3D image can be made by the help of scanning electron microscopy. But details of molecular structure is still beyond the power of electron microscopy.

A special method of visualization of ER is illustrated next as the antibodies to endoplasmin that is used to observe the morphology of ER by immunofluorescence on plasmacytoma (neoplasm of plasma cells usually in bone marrow) and fibroblastoid (cells of connective tissue in animals) cells. ER contains a specific glycoprotein named endoplasmin which is undetectable in other organelles and it's suitable for detecting ER by immunofluorescence microscopy.

Materials and methods

Cells were grown on Gibco medium with 10% fetal calf serum and 100 u/ml penicillin-streptomycin and 4 mM L-glutamine.

A droplet of NIH-3T3 cells (fibroblastoid cells) which were in a growth medium (107 cell/ml) was placed on a slide for 30 minutes at 37°c. Then the slide was placed in the growth medium to let the spreading occur for 2 hours at 37°c.

MOPC-315 cells (plasmacytoma cells) were suspended in PBS. A droplet was placed on a polylysine coated slide for 15 minutes to attach.

Cells were treated with 0.01% saponin in PBS for 2 minutes and then fixed with 3.5% formaldehyde in PBS for 15 minutes. After that they were permeabilized with 0.2% saponin in PBS for 15 minutes.

Cells were treated with affinity-purified antibodies (to endoplasmin) for 15 minutes at room temperature and then washed vigorously with 0.2% saponin/PBS and then treated with fluorescein-labelled goat anti-rabbit immunoglobulin (1 g purchased from sigma co.) for 15 minutes at room temperature. After intensive wash, samples were mounted in 90% glycerol with 1% phenylene diamine, and closed up with varnish. Then the were stained with fluorescein-labelled wheat-germ agglutinium (purchased from sigma co.) as above.

Samples were observed under Zeiss epifluorescence microscope with narrow band flourecein excitation filter. Then pictures were taken by Kodak Tri-X film and the pictures were scanned by confocal laser scanning fluorescence microscopy.

Next figures are the pictures that show endoplasmic reticulum in those two different cell types.

ER-vis-1.jpg

Figure 12. Immunofluorescence with anti-endoplasmin on fixed cells permeabilized with saponin [9]. A,B. 3T3 cells were fixed with formaldehyde, permeabilized with 0-2% saponin/PBS and stained with anti-endoplasmin as described in materials and methods. C. Cells prepared as as above stained with FITC-wheat-germ agglutinin. Note the perinuclear cap typical of golgi apparatus.

ER-vis-2.jpg

Figure 13. Immunofluorescence with anti-endoplasmin on plasmacytoma cells [9]. MOPC-315 cells were fixed with formaldehyde followed by permeabilizing within 0-2% saponin/PBS. Labeling was with anti-endoplasmin and FICT-anti-rabbit lg as described in materials and methods. A,B. The same field of cells examined by phase contrast and epifluorescence respectively. C. the same sample examined by confocal laser scanning fluorescence microscopy. Magnification was not measured directly for samples analyzed by this technique.

ER-vis-3.jpg

Figure 14. Immunofluorescence labeling with different reagents for ER [9]. Cells were prepared as described in materials and methods and stained with : A, D10C6 ; B, FITC-Con A ; C, anti-endoplasmin (+ FITC goat anti-rabbit lg) ; D, anti endoplasmin and anti-tubulin.

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