MDSC 1001 PBL PROBLEM 1

BY ARVIND SEECHARAN (FUTURE DOCTOR EXTRODINARE)

LEARNING OBJECTIVE 1

• DESCRIBE THE ULTRASTRUCTURE OF ANIMAL CELLS

ELECTRON MICROGRAPH OF A BASIC ANIMAL CELL

LEARNING OBJECTIVE 2

• RELATE STRUCTURE TO FUNCTION OF ORGANELLES WITHIN ANIMAL CELLS

•The largest organelle of the cell

•Often located in the central part of the cytoplasm

•It contains a nucleolus/nucleoli (which produces

ribosomal subunits) and chromatin (DNA).

•Enclosed in a double-layered nuclear membrane.

•DNA is the genetic material implicated in cell division

and in the synthesis of several molecules particularly

proteins.

•The Nucleus serves as the control centre of the cell &

contains the blueprint for all cellular structure and

activities

•The nuclear envelope is perforated by pores (3000-4000),

which facilitate communication between the nucleus and

the cytoplasm.

•Attached to the outer membrane of the nuclear envelope

are polyribosomes.

•The outer membrane of the nuclear envelope is also

continuous with the rough endoplasmic reticulum of the

cytoplasm

The Cytoplasm

The Cytoplasm•The fluid component of the cytoplasm is the Cytosol (pH 7.2) while the metabolically active contents of the cytoplasm are the Organelles.

•Organelles are metabolically active, permanent residents of the cell which survive cell division i.e. they reappear in the daughter cells following cell division.

•Organelles occur in two forms, freely within the cytosol or enclosed in membrane.

•The cytoplasm also contains substances which are not metabolically active called Inclusion Bodies.

•Inclusion bodies are the transitory residents of the cytoplasm not involved in cell metabolism and do not survive cell division.

They comprise mainly accumulated metabolites, lipid droplets, pigments and minerals and are sporadically distributed in body cells.

•The eukaryotic cell is enclosed in a limiting membrane called the Plasma membrane or the Plasmalemma.

Functions of the Cell Membrane (Plasmalemma)

• Communication through receptors on the outer surface

• Intercellular connectivity which facilitates boundary flexibility, support of cell structure and protects cellular contents.

• Provision of physical barrier between the intra and extra cellular compartments.

• Selective permeability which regulates entry/exit of materials across the membrane.

Mitochondria

• Mitochondria are membrane-bound enzyme storage organelles.

• Mitochondrial enzymes are involved in aerobic respiration, production of ATP and heat energy for maintenance of body temperature.

• It is enclosed in two sheets of membrane. An outer sieve-like unfolded membrane and an inner membrane which is thrown into long finger-like folds called cristae.

• The number of cristae corresponds to the cell’s energy needs. The space between the two membranes is the intermembranous space while the space deep to the inner membrane is referred to as the matrix.

• Mitochondria are eosinophilic, elongated rod-like organelles measuring 0.5 to 1 microns in diameter and 5 to 10 microns in length.

• The Matrix also contains chromosomes DNA, ribosomes, messenger RNA and Transfer RNA which are utilized in the synthesis of small amount of proteins for use within the matrix.

• They are wildly distributed in all cells but occur abundantly in cells with very high energy needs (Heart, muscles and kidney cells).

• Integral proteins of the outer and inner membrane provide channels for selective passage of small molecules, whiles enzymes in the matrix and on the surface of the inner membrane are involved in the production of ATP for the cell.

• However the bulk of the proteins required in the mitochondrion is synthesised in the cytosol.

• The mitochondrial matrix also contains granules which store calcium ions.

• The mitochondrion produces about 100 molecules of ATP per second

Ribosomes

• Ribosomes are small, electron-dense particles not enclosed in membrane and are located in the cytosol.

• Measure about 20-30 nanometres they are basophilic and stained by all basic dyes

• Ribosome is composed of rRNA and about 80 different proteins. It usually occur in two subunits, large and small subunits. The rRNA of the ribosome is synthesised in the nucleus while its protein is synthesised in the cytosol.

• Ribosomes are involved in protein synthesis. While cytosolic proteins (free proteins) are synthesised by polyribosomes, secretory and endoplasmic reticulum proteins are synthesised on the membrane of rough endoplasmic reticulum

• Ribosomes occur in three forms: – In isolated particles– Assembled in cluster on mRNA strand to form

Polyribosomes– Adsorbed to the membrane of endoplasmic reticulum through

their large subunit to form Rough Endoplasmic Reticulum.

• Protein synthesis by ribosomes also implicates: mRNA, tRNA and rRNA.

Lysosomes

• Contains up to 40 different acid Hydrolytic enzymes (pH – 5).

• Site of intracellular digestion.• Site of turnover of cellular components• Found in cells involved in extensive phagocytic

activities – e.g. Macrophages and Neutrophil

• Used for – intralysosomal digestion.– For Extracellular digestion/destruction e.g.

osteoclast breakdown of bone matrix.–Metabolism of several substrate all over the body.

Endoplasmic Reticulum

• This organelle is made up of anastomosing network of intercommunicating channels/cisternae/sacs

• Enclosed in a continuous membrane.• Occurs in two forms, namely Rough and Smooth which are

also interconnected.• Cisternae of smooth ER are tubular in shape. • Cisternae of Rough ER are flattened. – The roughness on the surface of rough ER is due to the

adsorption of polyribosomes on their outer surface.• Polyribosome also impacts the basophilic staining

characteristic on RER. • Its membrane is continuous with that of the nuclear envelope.

• Distribution and Functions of RER

• RER is prominent in protein synthesising cells such as; Pancreatic acinar cells, cells of the endocrine glands, plasma cells, fibroblast etc.

• Proteins synthesised in RER are stored in Lysosomes or granules; stored temporarily before exocytosis or used as integral membrane proteins.

Smooth Endoplasmic Reticulum (SER)

• This is ER not bund to polyribosomes but continuous with RER and are less abundant is cell containing RER.

Distribution and Functions of SER• SER is found in all cells where they are involved in:

– The synthesis phospholipids and cholesterol used in all cellular membranes including membranes of organelles.

• They occur in abundance in other cells where they are involved in:– Sequestration and release of Calcium ions a vital process in muscular

contraction– Biosynthesis of Lipids required for synthesis of steroid hormones– Detoxification of potentially harmful compounds such as alcohol and

barbiturates

• A complex of smooth membranous saccule usually located between the apical membrane and the nucleus of the secretory cell.

• Within its saccule are various enzymes implicated in processing proteins synthesised in the endoplasmic reticulum.

• The Golgi apparatus receives transport vesicles containing proteins from the endoplasmic reticulum and packages modified proteins into condensing vesicles for transportation to other organelle or to the cell membrane for release of modified proteins as secretory products.

• Protein modification occurring in the Golgi apparatus includes concentration, glycosylation, sulphation, phosphorylation and proteolysis.

THE CYTOSKELETON

• This is composed of a complex of microtubules, microfilaments (Actin Filaments), and Intermediate Filaments.

Microtubules

• These are tubular protein subunits involved in cellular shape, cell division, intra and extra cellular movements and transport of substances in the cytoplasm of the cell.

• Largest are 23-25nm in diameter

• Microtubules are widely distributed within the cytoplasm where they occur in various forms which include:– Cytoplasmic microtubules for intracellular transport of materials

including organelles– Centrioles which are involved in cell division– Mitotic spindles which are involved in cell division– Cilia and Flagella which are motile structures implicated in

cellular motion– Basal bodies which are located at the bases of cilia and flagella

and are involved in the configuration of these structures.

Microfilaments/Actin Filaments• Microfilament is a double-stranded helix of globular

protein subunits which is widely distributed in all body cells and is involved in:– the structural integrity and contractility of the cell– movement of organelles in the cytosol. – cell cleavage during cell division.

• Smallest is 6-7nm Diameter

• Actin filament is seen in two forms in the cell viz. polymerised F-Actin and free Globular G-Actin.

• F-Actin filaments are involved in:– Muscular contraction in collaboration with myosin filants– Cell shape integrity and locomotion (stress fibres of crawling

cells)– Movement of organelles and other cytosolic contents

(cytoplasmic steaming)– Cellular cleavage in mitotic cells

Intermediate Filaments

• Are intermediate in size to microtubules and microfilaments (10-12 nm in diameter).

• They are more stable structurally than Microtubules/filament and are composed of variable protein subunits depending on their localization and functions

Examples of intermediate filaments include:• Keratin of epithelial cell for strength and protection• Vimentin of the mesenchymal cell for structural integrity• Desmin of the muscle cell for structural integrity• Neurofilament of the nerve cell for structural integrity• Lamins of all cell nuclei also for structural integrity

Centrosome/Centrioles

• The centrosome is the region of the cytosol situated between the nucleus and the Golgi apparatus.

• It accommodates the Centrioles which are two cylindrical shaped microtubular structures oriented at right angles to one another.

• Each cylinder is made up of nine triplets of parallel microtubules.

• Centrioles are implicated in organising the microtubules of the cell as in the organization of mitotic spindles during cell division.

• They also form the basal bodies of cilia and flagella

PEROXISOME/MICROBODIES

• Are membrane-bound organelles which contain various enzymes and utilise oxygen without the production of ATP.

• Measuring about 0.5 microns in diameter they oxidise organic substrate by the removal of hydrogen ions, leading to the production of H2O2.

• This is immediately broken down by peroxisomal catalase to prevent its toxic effect on the cell.

• The oxygen atom released from this process is utilized in oxidation of other potentially toxic substances/drugs in the liver (Ethyl alcohol) and kidneys.

• Peroxisomes are also implicated in lipid metabolism (Beta oxidation of long-chain [18 carbon and over] fatty acids).

LEARNING OBJECTIVE 3

• DESCRIBE THE ULTRASTRUCTURE OF CELL MEMBRANES

THE PLAMALEMMA (CELL MEMBRANE) STRUCTURE

• The Plasmalemma is the physical external boundary of the cell.

• It is composed of Phospholipids, Protein, Carbohydrate and Cholesterol which are intricately organised into a trilaminar structure.

• The cell membranes well as the membranes surrounding the organelles range from 7.5 to 10 nanometre in thickness.

• The 3-layered structure, referred to as the Unit Membrane structure is formed from two phospholipid layers; the fatty acid nonpolar (Hydrophobic) tails of the two phospholipids are located in the middle layer while the polar (Hydrophilic) head are located on either side of the middle layer.

• The proteins of the cell membrane account for 50% (w/w) of the membrane and occur in two forms, viz. integral proteins which traverse the thickness of the membrane and peripheral proteins which are adsorbed to the outer or inner surfaces of the membrane.

• While the peripheral proteins are involved in cell recognition and interactions, integral proteins regulate passage of material and active transport of specific molecules across the membrane

• The cholesterol embedded within the phospholipid fatty acid chains their movements and also modulates the fluidity and movements of other contents of the membrane.

• The carbohydrate contents of the membrane are attached to the lipids and proteins as glycolipids and glycoproteins respectively.

• Some of the carbohydrates of the glycoprotein constitute the glycocalyx on the outer surface of the membrane.

• Others are receptors involved in adhesion, cell recognition and responses to protein hormones. Others yet are attached to the cytoskeletal components of the cytoplasm for maintenance of cell shape and integrity.

LEARNING OBJECTIVE 4

• DESCRIBE THE BASIC STRUCTURE AND PROPERTIES OF LIPIDS

CHEMISTRY OF LIPIDS

• Lipids are heterogeneous group of naturally occurring compounds, relatively insoluble in water but freely soluble in non-polar organic solvents like, benzene, chloroform, ether and alcohol.

• Formed of long-chain hydrocarbon groups but may also contain oxygen, phosphorus, nitrogen and sulfur.

Functions

• Triglycerides are the major storage form of energy

• Provide essential fatty acids; phospholipids, hormones

• Form important constituents of cell membrane and helps to maintain the membrane structure and integrity

• Absorption of vitamin A, D, E and K depends needs presence lipids in the diet

• The basic unit of lipids, acetyl CoA (the active form of acetic acid) is used for the synthesis of cholesterol and hence steroid hormones

• Its insulating effect has been utilized in the body for protecting internal organs from shock

.• Dipalmitoyl lecithin, a phospholipid act as

surfactant and is required for the normal functioning of the lung alveoli

• Helps in blood coagulation

Properties of lipids

• Oils and fats (lipids) are similar in nature. Oils and lipids are different only in their physical property. Triglycerides, which contain a higher proportion of unsaturated fatty acid or short chain fatty acid, are liquid at 20°C and are usually called as oils, e.g. vegetable oils.

• Fats on the other hand are solid at room temperature and contain saturated long chain fatty acid e.g. animal fat.

LEARNING OBJECTIVE 5

• DISCUSS TECHNIQUES USED FOR PREPARATION OF CELLS AND TISSUES FOR VIEWING UNDER A MICROSCOPE

Tissue Preparation for Light Microscopy• 1st Step – Fixation to preserve structures (This process stops cell

metabolism). Agents used for fixation include:• Formaldehyde• Alcohol (80%)• Acetone• Bouin’s fluid• Carnoy’s fluid• Rossman’s fluid• Zenker’s fluid•  • 2nd Step – Dehydration and Embedding in Paraffin. • Specimen is prepared for Embedding in paraffin to permit Sectioning

(specimen must be infiltrated with an embedding medium that allows it to be thinly sliced 5-15μm). This is done after fixation by passing the specimen through a series of alcohol solutions in ascending concentrating up to 100% to remove water (Dehydration).

• 3rd Step - Clearing • Organic solvents, which are miscible in both alcohol and paraffin, are used

to remove the alcohol prior to infiltration of the specimen with melted paraffin.

• Xylene is most commonly used solvent in this and most other laboratories. • Benzene is also used as a clearing agent•  • 4th Step – Immersion in paraffin wax.• 5th Step – Blocking and Sectioning of specimen.•  • 6th Step – Staining of sections.• Specimen is stained to permit Examination by dissolving the paraffin and

(1)rehydrated through a descending series of alcohol to water. Slides are (2)stained with Hematoxylin and counterstained with Eosin, (3)dehydrated through an ascending series of alcohol, passed through an organic solvent and covered with a coverslip to obtain a permanent section.

Tissue Preparation for Electron Microscopy

• 1. Sections of Embedded MaterialBiological material contains large quantities of water. Since the TEM works in vacuum, the water must be removed. To avoid disruption as a result of the loss of water, you preserve the tissue with different fixatives. These cross-link molecules with each other and trap them together as stable structures. The tissue is then dehydrated in alcohol or acetone.

• After that, your specimen can be embedded in plastic that polymerize into a solid hard plastic block. The block is cut into thin sections by a diamond knife in an instrument called ultramicrotome. Each section is only 50-100 nm thick.

• The thin sections of your sample is placed on a copper grid and stained with heavy metals. The slice of tissue can now be studied under the electron beam.

LEARNING OBJECTIVE 6

• DISCUSS THE NEED FOR INTRA-CELLULAR COMPARTMENTMENTALISATION AND THE BASIC PRINCIPLES OF INTRACELLULAR METABOLISM

Intracellular Compartmentalisation

• The major intracellular compartments of an animal cell are the: – Cytosol – Endoplasmic reticulum – Golgi apparatus,– Nucleus– Mitochondion– Endosome– Lysosome– Peroxisome

• The precursors of the first eucaryotic cells were simple organisms that resembled bacteria.

• These had a plasma membrane but no internal membranes, therefore the plasma membrane in such cells therefore provides all membrane-dependent functions– the pumping of ions– ATP synthesis– protein secretion– Lipid synthesis.

• Eucaryotic cells today are much larger than those cells were ( 10–30 times greater in linear dimension &1000–10,000 times greater in volume)

• Because of this increase in size, the eucaryotic cell has a much smaller ratio of surface area to volume. As a result, the plasma membrane is too small to sustain the many vital functions for which membranes are required.

• From these observations, the internal membrane compartments which eucaryotic cells contain may be viewed as an evolutionary adaptation, in order to carry out its vital functions

• Additionally, intracellular compartmentalisation has allowed for the development of specialised membrane function and organelles.

Basic Principles of Intracellular Metabolism

LEARNING OBJECTIVE 7• DISCUSS VIEWING WHOLE, LIVING CELLS vs.

DEAD “MASHED UP” CELLS.

Advantages of viewing “Whole,” Living Cells

• Able to observe how certain structures/organelles function. – E.g microvilli, golgi vesicles

• Preserves life.

Disadvantages of viewing “Whole,” Living Cells

• Difficult to observe all cells efficiently while they are alive.

• In only viewing the surface of the cell, it is impossible to develop a full understanding of its complete workings

Advantages of Viewing “Mashed Up,” Dead Cells

• Allows for better viewing methods (e.g. electron microscopy which requires the cell be dead)

• “Mashing up” the cells, allows biochemists to learn what compounds the cells and their components are made up of.– Biochemists can then apply what they know about

the behaviour of said compounds to determine the function(s) of the subject matter.

Disadvantages of viewing “Mashed Up,” Dead Cells

• The specimen being studied dies.• Researchers are unable to view the actions of

the specimen being studied.

References• http://www.ucl.ac.uk/~sjjgsca/CellOrganelles.html • http://www.biology4kids.com/files/art/cell_ribosome3.jpg • http://www.biologyjunction.com • http://micro.magnet.fsu.edu/cells/golgi • http://www.nobelprize.org/educational/physics/microscopes/tem

/preparation.html • http://www.ncbi.nlm.nih.gov/books/NBK26907/

• http://www.glogster.com/media/