Week 1 Cohort 2 - Lipids and the Cell Membrane Instructor(1) PDF

10.019 Science and Technology for Healthcare Week 1 Cohort 2: Lipids and the Plasma Membrane  Lesson Outline Four Classes of Biomacromolecules The Plasma (Cell) Membrane Mini-Activity: Crude extraction of DNA Copyrights...

10.019 Science and Technology for Healthcare Week 1 Cohort 2: Lipids and the Plasma Membrane  Lesson Outline Four Classes of Biomacromolecules The Plasma (Cell) Membrane Mini-Activity: Crude extraction of DNA Copyrights 2  Learning Objectives At the end of the lesson, you will be able to: Describe the four main classes of biomacromolecules, including the types of bonds present, and their general roles Describe the components of the plasma membrane and their functions List the different types of membrane proteins and explain their roles in transport, recognition, and communication Explain the process of extracting DNA from cells using common household ingredients Copyrights 3  Relevant Readings Alberts et al. Essential Cell Biology, 4th Edition, Garland Science, 2014. Ch 2 Pg. 50 - 79. Alberts et al. Essential Cell Biology, 4th Edition, Garland Science, 2014. Ch 11 Pg. 359 - 389. Copyrights 4  Recap: Cells and the Chemistry of Life Each cell is a highly complex chemical system The Chemistry of Life is: 1. based largely on carbon compounds (i.e., ‘organic’) 2. dependent on chemical reactions that take place in aqueous environments in a narrow temperature range 3. enormously complex in its chemistry 4. dominated and coordinated by biomacromolecules (i.e., large polymers made up of smaller linked subunits) 5. tightly regulated such that each chemical reaction occurs at the proper rate, time, and place Copyrights 5  Recap: Molecular Composition of the Cell Lipids Nucleic acids Proteins Carbohydrates Copyrights 6  Recap: Four Classes of Biomacromolecules Subunits Macromolecules i.e., carbohydrates Figure 3.1 Biology 2e. Food is a rich source of nutrients, including biological macromolecules, necessary for life. Smaller organic molecules are built up into the larger macromolecules required by the cell. Copyrights 7  Recap: Macromolecules are formed and broken down via condensation and hydrolysis reactions Subunits (monomer) are added stepwise to the end of a growing chain to obtain a polymer Copyrights 8  Video - Introduction to Biomacromolecules Copyrights https://www.youtube.com/watch?v=V5hhrDFo8Vk 9  Carbohydrates (Subunit: Simple Sugar) Play important energy storage and structural roles in the cell Carbohydrates include sugars and the polymers of sugars – Simplest are monosaccharides, or single sugars – Macromolecules are polysaccharides, composed of multiple sugar units – Characterized by a general molecular formula of Cn(H2O)n-1 Monosaccharide Disaccharide e.g., Glucose e.g., Sucrose = C6H12O6 glucose + fructose C12H22O11 Polysaccharides (hundreds or thousands of glucoslinked by covalent e subunits glycosidic bonds) Copyrights Could be branched or linear – affects packing and strength 10  Proteins (Subunit: Amino Acids) Play a diverse and important range of cellular functions, including structural support, transport, communication, etc. A protein is a biologically functional molecule that consists of one or more polypeptide chains A polypeptide is a linear polymer built from a set of 20 amino acids linked by covalent peptide bonds or = one or more polypeptide chains folded into a stable 3D conformation Dipeptide (2 aa) (with biological function) Oligopeptide (up to ~20aa) Polypeptide (20 – thousands of aas) Copyrights 11  Nucleic Acids (Subunit: Nucleotides) Role in storage, transmission and use of genetic information Nucleic acids are also called polynucleotides. A polynucleotide is made of subunits called nucleotides linked together by covalent phosphodiester bonds Khan Academy Two types of nucleic acids: DNA = deoxyribonucleic acid 4 bases: A, C, G, T Deoxyribose sugar Double stranded RNA = ribonucleic acid 4 bases: A, C, G, U Ribose sugar Single stranded Copyrights 12  Nucleic Acids: Deoxyribose and Ribose Sugars Copyrights 13  Nucleic Acids: MCQ In terms of DNA and RNA structure, what is a nucleotide? A. A nucleotide consists of a base B. A nucleotide is a sugar molecule covalently bonded to a base C. A nucleotide is a sugar molecule bonded to phosphate group and a base D. A nucleotide is a heterocyclic base bonded to phosphate group Answer: C Copyrights 14  Nucleic Acids: DNA Structure Copyrights 15  Lipids (e.g., Fats, Phospholipids, Steroids) Plays role in insulation, energy storage, cell structure, and cell signaling A class of large molecules comprising mostly of hydrocarbons (C, H) – Non-polar in nature, hence insoluble with little affinity for water (hydrophobic) Note that lipids do not form long, repeating polymers. Three biologically important lipids: Credit: BioNinja Copyrights 16  Video – A closer look at lipids Copyrights https://www.youtube.com/watch?v=5BBYBRWzsLA 17  Dehydration reaction to form covalent ester bond Triglycerides (Fats) are constructed from two types of smaller molecules: glycerol and fatty acids – Glycerol is a three-carbon alcohol with a hydroxyl group on each carbon – A fatty acid consists of a carboxyl group attached to a long carbon tail – Linked together via a dehydration reaction to form a covalent ester bond Fig. 5.10 Cambell Biology Copyrights 18  Saturated vs. Unsaturated Fats Fatty acids can vary in length (number of carbons) and in the number and locations of double bonds – Saturated fatty acids have the maximum number of hydrogen atoms possible and no double bonds – Unsaturated fatty acids have one or more double bonds Saturated fats are associated with elevated cholesterol levels and higher Copyrights risk of cardiovascular disease 19  Summary: Four Classes of Biomacromolecules Cells are highly complex chemical systems with shared fundamental characteristics Biomacromolecules are important cellular components and perform a wide range of necessary functions. Cells are mainly composed of four classes of biological macromolecules – carbohydrates, proteins, nucleic acids, and lipids Macromolecules are formed and broken down via dehydration and hydrolysis The complex cellular landscape reactions, respectively Credit: cellsignal.com Copyrights 20  Lesson Outline Four Classes of Biomacromolecules The Plasma (Cell) Membrane Mini-Activity: Crude extraction of DNA Questions on the material so far? Copyrights 21  The Plasma (Cell) Membrane The plasma membrane is the boundary of the cell Acts as a selective barrier that separates the internal components from the outside environment Comprises a phospholipid bilayer with embedded proteins – Lipid bilayer serves as a permeability barrier to polar molecules and ions – Proteins carry our other important membrane functions (e.g., sensing, transport) The plasma membrane is involved in cell communication, import/export of molecules, and cell growth and motility Fig 11.1 and 11.2, Essential Cell Bio Copyrights 22  The Lipid Bilayer is a Flexible 2D Fluid (Fluid Mosaic Model) Copyrights https://www.youtube.com/watch?v=Qqsf_UJcfBc 23  The Phospholipid Bilayer Fig 11.4 and 11.5, Essential Cell Bio Transmission electron micrograph (TEM) view 3D view 2D view Membrane phospholipids are amphipathic, i.e., they have both hydrophilic and hydrophobic parts Membrane phospholipids arrange into two closely apposed sheets, i.e., lipid bilayer, in water Copyrights 24  Membrane lipids are amphipathic Phosphatidylcholine is the Other types of membrane lipids. Each of most common phospholipid the three types shown here has a found in cell membranes hydrophilic head and hydrophobic tails Fig 11.6 and 11.7, Essential Cell Bio What happens to amphipathic molecules in water? Copyrights 25  Amphipathic molecules self-assemble in water Fig 11.11 and 11.13, Essential Cell Bio Lipid bilayer (double sheet) Hydrophilic heads orient towards the aqueous environment on both surfaces of the bilayer Hydrophobic tails orient towards each other in the interior of the bilayer and are shielded from water Spontaneously assembles into sealed compartments to avoid free edges (energetically unfavorable) Fig 5.4 Biology 2e. Copyrights 26  Selective Permeability of the Lipid Bilayer The lipid bilayer serves as a permeability barrier and “gatekeeper” to regulate the entry/exit of molecules Which of the following molecules will be able to diffuse across the lipid bilayer? Which will not? – Oxygen – Carbon dioxide – Water – Ions (e.g., H+, Na+) – Glucose Copyrights 27  Selective Permeability of the Lipid Bilayer The lipid bilayer serves as a permeability barrier and “gatekeeper” to regulate the entry/exit of molecules Selective Permeability: Small non-polar molecules (e.g., O2) diffuse across readily Smallest uncharged polar molecules (e.g., H2O) can diffuse across Larger polar molecules (e.g., glucose) hardly diffuse across Impermeable to all ions (e.g., Cl-) regardless of size So how do solutes enter/exit the cell? Copyrights 28  Structure of the Plasma Membrane Fig 4.9 Biology 2e Component Location Phospholipid Main membrane fabric Cholesterol Between phospholipids and between the two phospholipid layers Embedded within the phospholipid bilayer; may or may not penetrate Integral membrane proteins through both layers On the phospholipid bilayer’s inner or outer surface; not embedded within Peripheral membrane proteins the phospholipids Carbohydrates (components of Generally attached to proteins on the outside membrane layer glycoproteins and glycolipids) Copyrights 29  Types of Membrane Proteins Membrane transport proteins Copyrights Fig. 3.8 Anatomy & Physiology: The Unity of Form and Function 30  Solutes can cross the plasma membrane via membrane transport proteins Fig 12.3 and 12.8 Essential Cell Bio Each membrane has its own characteristic set of membrane transport proteins Solutes ( e.g., inorganic ions and small, polar organic molecules) can cross the plasma membrane through these channels Some transport proteins discriminate solutes based on size and charge (less specific) Others allow only specific molecules or ions that fit into Solutes may cross the membrane by either passive (i.e., down a binding sites to cross concentration gradient) or active (i.e., energy-dependent) transport Copyrights 31 Example: Aquaporin – Water Channel Protein Aquaporin channels allow for water transport in & out of the cell; play an important role in water reabsorption in kidneys and other cells Yellow sphere = one H2O molecule Extremely narrow central channel allows passage of water molecules in single-file Top-down view:  Larger molecules and particles enter the cell via bulk transport Wikipedia Larger molecules, such as proteins and polysaccharides generally cross the membrane in bulk, packaged in vesicles Fig 7.19 Campbell Biology Copyrights 33  Types of Membrane Proteins Copyrights Fig. 3.8 Anatomy & Physiology: The Unity of Form and Function 34  Cell-Identity Markers (i.e., Cell Surface Markers) Membrane glycoproteins, receptors, and other molecules act as markers (“ID tags”) of specific cell types Immune system can distinguish own body’s cells (‘self’) vs. foreign materials (‘non-self’); important for body’s defenses White blood cell surface markers (FYI) CUSABIO Antibodies can be used to probe for specific cell-surface antigens (markers that trigger an immune response) 35  Example: Human ABO Blood Groups “ABO” blood groups are distinguished by the carbohydrate tails of glycoproteins on the red blood cell (RBC) membrane Summary of ABO Blood Groups Different sugars on RBC surface Copyrights McGraw-Hill 2018 BioNinja 36  Types of Membrane Proteins Copyrights Fig. 3.8 Anatomy & Physiology: The Unity of Form and Function 37  Example: Sars-CoV2 entry and infection The Sars-CoV2 virus attaches to the host cell by way of its spike protein, which binds to a specific receptor protein (hACE2) on the host cell’s surface. The spike protein also contains parts that facilitate the fusion of the viral membrane with the host cell membrane to deliver the viral genome inside the cell. Understanding of the coronavirus SARS-CoV-2 spike binds to membrane fusion mechanism offers the human ACE2 (hACE2) cell-surface receptor a potential target for anti-viral development. Animation link: Cornell News 7 Apr 2020 https://www.cornell.edu/video/corona virus-membrane-fusion Copyrights 38  Summary: The Plasma Membrane The principal components of the plasma membrane are lipids (phospholipids and cholesterol), proteins, and carbohydrates (in the form of glycoproteins/glycolipids). The lipid bilayer serves as a permeability barrier to polar molecules and ions, while proteins carry out other important membrane functions (e.g., sensing, transport, etc.) The plasma membrane protects intracellular components from the extracellular environment and mediates cellular processes by regulating the materials that enter and exit the cell. The plasma membrane is also involved in cell communication, import/export of molecules, and cell growth and motility Questions on the material so far? Copyrights 39  Lesson Outline Four Classes of Biomacromolecules The Plasma (Cell) Membrane Mini-Activity: Crude extraction of DNA – Refer to Week 1 Cohort 2 In-class Worksheet Copyrights 40  Lab Activity: Crude Extraction of DNA DNA extraction is a basic procedure in laboratories and is important to the study of genetics. Extracted DNA can be used in genetic profiling (or ‘DNA fingerprinting’) to help diagnose genetic diseases, establish paternity and aid in forensics analysis. Scientists can even manipulate the DNA of organisms for biomedical purposes. backbone sugar phosphate sugar phosphate Where is the DNA and sugar how do we get it out? phosphate Copyrights 41  Very Simple Schematic of a Cell Plasma membrane Plasma membrane and 2. What is it made of? nuclear envelope are made up of phospholipid bilayers. 3. What is the first step to take if we want to extract DNA? 1. What do we Disrupt (i.e., lyse) the plasma want in here? Nuclear membrane and nuclear envelope DNA envelope 4. Which of the following should 5. How to separate the DNA from we add first? everything else we don’t want? A. Alcohol solution Filter. DNA is soluble in water and will B. Soap solution remain dissolved in the filtrate. C. Salt 6. How to get DNA to precipitate out Solution B. Soap will disrupt the lipid of solution? Adding alcohol (and salt) bilayer, releasing the DNA 42  Soap disrupts the lipid bilayer Soap is an amphipathic molecule – Comprise of hydrophobic tail and hydrophilic head Soap inserts into cell membrane; captures the lipids and proteins http://learn.genetics.utah.edu/content/labs/extraction/howto/detergent/ 43  Anti-microbial effect of Soap How does soap work against bacteria and viruses (e.g., Sars-CoV2)? Image adapted from: Jonatahan Corum and Ferris Jabr/The New York Times Copyrights 44  Video – Which is better: Soap or Hand Sanitizer? (FYI) Copyrights https://www.youtube.com/watch?v=x7KKkElpyKQ 45  Summary: What’s happening in each step? The extraction of DNA involves three key steps: 1. Cell Lysis – Cell membranes, which are composed of lipids and proteins, are disrupted or lysed using mechanical and chemical means to release the DNA and other cellular contents. 2. Separation (filtration) – The DNA needs to be separated from cell debris and other solids 3. Precipitation and Concentration – DNA is soluble in water and insoluble in alcohol. – Upon addition of cold ethanol, the DNA will form a stringy mass at the interface of the alcohol and aqueous layers. Copyrights 46  Flowchart: Crude Extraction of DNA Mash in Add extraction buffer Ziploc and mash some more Observe Work in groups of 5 to extract strawberry DNA! Filter slurry & (Protocol in worksheet) retain filtrate DNA Add COLD ethanol to precipitates filtrate slowly and gently out!!  Salt and Ethanol for precipitation of DNA backbone NaCl dissociates into Na+ and Cl- ions – Na+ neutralizes the negatively charged phosphate backbone on DNA – The neutralized DNA aggregates more easily (reduced repulsion) sugar phosphate DNA dissolves in water but has a sugar lower solubility in ethanol phosphate – With the addition of ethanol, the aggregated sugar DNA precipitates more easily out of solution phosphate 48  Worksheet Q1 Match up each step with its function: Step Function 1) Smashing of strawberry A) To precipitate DNA from solution 2) Squeeze and mix strawberry with the B) To separate components of the cell extraction buffer based on size 3) Filter strawberry slurry through the C)To physically break apart the cell wall filter bag 4) Addition of cold ethanol to filtered D)To break up proteins and dissolve extract cell membranes Answer: 1 – C, 2 – D, 3 – B, 4 – A Copyrights 49  Worksheet Q2 Where is DNA found in the cell? A. Nucleus B. Cell Membrane C. Cell Wall D. Phospholipid Micelle Answer: A Copyrights 50  Worksheet Q3 Upon adding the extraction buffer into the smashed strawberry: A. Soap in the extraction buffer precipitates out DNA. B. Soap in the extraction buffer neutralizes the negative charge on the sugar- phosphate backbone of DNA. C. Soap in the extraction buffer kills the bacteria in the strawberry slurry. D. Soap in the extraction buffer disrupts the cell membranes and binds to the lipids and proteins. Copyrights 51  Worksheet Q3 Upon adding the extraction buffer into the smashed strawberry: A. Soap in the extraction buffer precipitates out DNA. B. Soap in the extraction buffer neutralizes the negative charge on the sugar- phosphate backbone of DNA. C. Soap in the extraction buffer kills the bacteria in the strawberry slurry. D. Soap in the extraction buffer disrupts the cell membranes and binds to the lipids and proteins. Answer: D Copyrights 52  Worksheet Q4 Upon adding ethanol into the strawberry slurry, DNA precipitates because: A. DNA readily dissolves in ethanol but is insoluble in water. B. DNA readily dissolves in water but is insoluble in ethanol. C. DNA is insoluble in both water and ethanol. D. DNA is readily soluble in both water and ethanol. Copyrights 53  Worksheet Q4 Upon adding ethanol into the strawberry slurry, DNA precipitates because: A. DNA readily dissolves in ethanol but is insoluble in water. B. DNA readily dissolves in water but is insoluble in ethanol. C. DNA is insoluble in both water and ethanol. D. DNA is readily soluble in both water and ethanol. Answer: B Copyrights 54  END Copyrights 55