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University of Toronto St. George
Jennifer Harris

University of Toronto - Revision Paper - BIO230 Marcus Lam Professors: T. Harris ▯ ▯ ▯ Cell and Developmental Biology [BIO230H1F] ▯ Exam Study Guide ▯ ▯ ▯ ▯ ▯1/18 University of Toronto - Revision Paper - BIO230 Marcus Lam Contents ▯ ▯ Labs 1. Gene Regulation Part One 2. Gene Regulation Part Two 3. Rapid Isolation and Electrophoresis of RNAfrom E. Coli 4. Live Cell Imaging 5. Role of Cytoskeleton in Development (fly) ▯ Section Two: Cell and Developmental Biology 2.1 Membrane Trafficking 2.2 Cytoskeletal Networks 2.3 CellAdhesion 2.4 Tissue Morphogenesis 2.5 Tissue Patterning 2.6 Stem Cells 2.7 Principles of Cellular Signaling 2.8 Signaling via Small Molecules 2.9 Signaling via Protein Modifications 2.10 Cell Cycle 2.11 Programmed Cell Death 2.12 Cancer
 ▯ ▯2/18 University of Toronto - Revision Paper - BIO230 Marcus Lam V ocabulary ▯ ▯ Operon:Aunit made up of linked genes that is thought to regulate other genes responsible for protein synthesis.
 ▯ ▯3/18 University of Toronto - Revision Paper - BCH210 Marcus Lam ▯ ▯ ▯ ▯ ▯ ▯ Labs ▯ ▯ 1. Gene Regulation Part One 2. Gene Regulation Part Two 3. Rapid Isolation and Electrophoresis of RNAfrom E. Coli 4. Live Cell Imaging 5. Role of Cytoskeleton in Development (fly)
 ▯4/18 University of Toronto - Revision Paper - BIO230 Marcus Lam 1. Gene Regulation Part One ▯ ▯ Transform bacterial cells with a plasmid containing a reporter gene. ▯ Purpose of Gene Expression Regulation: 1. External environmental conditions 2. Developmental cues 3. In response to hormone signals ▯ Gene Regulation: Type of gene regulation in this lab: Transcriptional Regulation (most common in bacteria) 1. This occurs in first stage of gene expression, before significant amounts of mRNAare synthesized. 2. Example: Lac Operon 3. Ara Operon contains genes that encode proteins required to metabolize a sugar - L-arabinose. a. When L-arabinose is present in bacterial growth medium, the genes in the ara operon may be transcribed at high levels. b. BUT when a more easily metabolized sugar like glucose is present, there is little transcription of these genes. 4. Proteins produced are not easily detectable from lac and ara operons. 5. So how about when a marker is inserted so that when operon is switched on, an easily detectable protein was made 6. Bio-Rad constructed a plasmid called pGLO containing a reporter gene, producing an easily visualized gene product ▯ pGLO Plasmid Map 1. Arrows indicate open reading frames/genes. 2. araC gene 3. origin of replication for plasmid (ori) 4. bla gene that codes for a protein that makes bacterial cells resistant to antibiotic ampicillin 5. GFP gene that codes for green fluorescent protein, a fluorescent marker made in large amounts in bacterial cells that exhibits fluorescence under long wave UV light. 6. PBAD is the ara operon promotor, it normally regulates the transcription of genes in ara operon. But in pGLO it is located upstream of the GFP gene. 7. GFP would normally have its own eukaryotic promotor, but instead is controlled by the bacterial promotBADP. 8. Whenever environmental conditions of bacterium would have caused an increase in expression of ara operon genes, GFP will be produced instead. 9. NOTE: only gene from native ara operon present in pGLO plasmid is araC gene, and it has its own promotor. ▯ Differential Regulation of the ara Operon in the pGLO System 1. AraC is an allosteric activator protein with two binding sites. 2. When arabinose is present in the environmentAraC iinactive) binds to the sugar. 3. Binding causes a conformational change inAraC ioAraC (aative) 4. Allowing it to recognize and bind to Aral: a specific activator sequence located just upstream of theBAD promotor. ▯ ▯5/18 University of Toronto - Revision Paper - BIO230 Marcus Lam 5. It was hypothesizes thatAraC helps RNApolymerase (RNAP) bind to the BAD promotor. 6. So, the absence of arabinose: a. AraC is not activated and thus can not bind to the aral sequence b. RNAP will not bind effectively to tBADPsequence c. Very little transcription will occur -> little GFP will be made 7. Asecond mechanism to regulateaboliteActivator Protei transcription involves cyclic adenosine monophosphate (cAMP) and the Catn (CAP) a. For most efficient binding to RNAP tBAD, not only must 2 molecules ofAraC bind to the aral sequence, but CAP must bind to the cAMP-CAP binding site (CBS) located just upstream of the aral sequence. b. CAP is also found in two forms: i. When cAMP is bound to CAP, CAP is active and can bind to CBS sequence ii. When cAMP is not bound to CAP, CAP is inactive and cannot bind to CBS iii.In bacterial cells, high concentrations of glucose will reduce the synthesis of cAMP and thus CAP is less likely to be activated and bind to the CBS sequence. 1. So, even in the presence of arabinose, high concentrations of glucose will prevent the formation of active cAMP-CAP complex and the binding of RNAP to P will not be efficient, resulting in little BAD transcription of GFP. 2. But, in the presence of arabinose, low concentrations of glucose, two moleculesaofAraC will bind to aral sequence, activated cAMP-CAP will bind to CBS sequence, and binding of RNAP BADP will be efficient, resulting in high transcription of GFP. SO BACTERIAL COLONIES WILL GLOW BRIGHT GREEN when exposed to long wave UV light! ▯ Note: CBS and aral sequences are not shown on the plasmid pGLO figure, but they are present just upstream of the PBAD promotor sequence. ▯ Differential Regulation of the araBAD promotor (PBAD) with respect to carbon source availability Diagram shows the transcriptional activity fromBAD when: 1.Neither glucose no arabinose is present 2.Both arabinose and glucose are present 3.Only arabinose is present ▯ Intensity of wavy arrow extended from transcription initiation site (+1) indicated the relative level of transcript produced under each condition. 1.Circle = cyclicAMP 2.Square = arabinose ▯ ▯ ▯ ▯ ▯ ▯ ▯ Transformation of E.coli with pGLO plasmid ▯ ▯6/18 University of Toronto - Revision Paper - BIO230 Marcus Lam 1. How does pGLO plasmid get into the bacterial cell? Process of genetic transformation: insertion of foreign DNA into bacterial cell a. Plasmids: naturally occurring circles of DNAfound in bacteria and some fungi and can be transferred between cells. b. They usually code for genes that are beneficial to bacterial survival and exist separately from the bacterial chromosome. c. They are replicated by the bacterial cell’s own DNAreplication machinery, and can be replicated independently by host cell chromosomal DNA. d. So, they are often found in very high numbers within the bacterial cell, and the products of their gene expression is very high. 2. In the Lab, plasmids can be engineered (using PCR, restriction enzymes, and ligases) to contain any sequence of interest. a. Conjugation: bacteria have been found to take up DNAfrom their surroundings b. We pre-treat the bacteria with calcium ions c. Heat Shock: Subject it to brief heat shock, stimulating it to take up DNAfrom their surroundings through pores in their cell walls. d. Never 100% efficient since not all cells are competent to pick up and retain recombinant plasmids. e. We cannot differentiate between bacterial cells containing our plasmid of interest and those that do not using a regular light microscope. i. The bla gene on the pGLO plasmid codes for Beta-lactamase, an enzyme makes the bacterial cell resistant to antibiotic ampicillin. ii. This gene is called a selectable marker becasue if we include ampicillin in E. Coli growth medium, only those cells that have been successfully transformed with pGLO plasmid will be able to grow, other will die. ▯ We need to learn the basic steps in “Aseptic Technique” when working with bacteria. ▯ Schematic Diagram Showing Transformation of Bacterial Cell with pGLO plasmid and expression of GFP and Beta-lactamase ▯ ▯ ▯ ▯ ▯7/18 University of Toronto - Revision Paper - BIO230 Marcus Lam Aseptic Technique: Procedures that are used to establish and maintain a sterile environment or prevent contamination of a pure environment or pure cultures. ▯ It is practiced when working with microorganisms, so that we do not contaminate ourselves or our workspaces. 1. Wiping down workspace with bleach or ethanol solution before and after working with bacterial cultures 2. Pipetting bacterial cultures carefully to prevent formation of aerosols containing bacteria 3. Wear lab coats, safety glasses, gloves 4. Minimizing amount of time petri dishes are uncovered 5. Glass beads need to be sterile, must not touch with fingers, media and petri dish must be sterile
 ▯ ▯8/18 University of Toronto - Revision Paper - BIO230 Marcus Lam 3. Rapid Isolation and Electrophoresis of RNA from E. coli ▯ ▯ Bacterial cells contain three main classes of RNA: 1. Ribosomal RNA(rRNA) 2. Transfer RNA(tRNA) 3. Messenger RNA(mRNA) ▯ Through extracting total cellular RNAand separating it according to size by sedimentation through sucrose gradient or gel electrophoresis. Most RNAdetectable on a gradient or gel is either rRNAor tRNA. ▯ Prokaryotic cells have three species of rRNA: 1. Two in the large subunit - 23S (2900 nucleotides) and 5S (120 nucleotides) 2. One in the small subunit - 16S (1540 nucleotides). ▯ rNAoperon shoes how rRNAis transcribed into one long transcriptional unit (pre-rRNA) then processed into equal proportions of rRNAsubunits. ▯ Success or failure depends upon your ability to avoid and overcome RNase activity which will destroy your sample. RNases are everywhere, they are stable proteins that can withstand high temperatures. So, we need to wear gloves and work quickly, preparation as cold as possible to reduce RNase activity. Keep the tube closed an on ice as much as possible ▯ Hold tube near the middle, avoiding holding it near the opening or the bottom. ▯ Step Procedure Purpose A Isolation of RNAfrom Escherichia coli (E. Coli) 1 Put on pair of gloves Avoid RNases destroying my sample. 2 Transfer 1ml of E. Coli culture into 1.5ml Shaking spreads out the culture in the tube. dispensing.fuge tube. Shake gently before 3 Centrifuge 30 secs at maximum speed, blot the Pellet the cells, remove all the liquid to ensure inverted tube on paper towel correct concentration and purity 4 Use vortex to resuspend cells in 0.5mL of solution 5 Add lysozyme to a final concentration of 50µg/mlinkages that connect N-acetylmuramic acid andsidic N-acetylglucosamine in the peptidoglycan. 6 Add 10µl of 10% SDS Lyse cells, SDS is a trong ionic detergentthat solubilizes the plasma membrane.Also, SDS is a strong protein denaturer. ▯ ▯9/18 University of Toronto - Revision Paper - BIO230 Marcus Lam Step Procedure Purpose 7 Add 50µl of 5M potassium acetate, mix gently SDS plus potassium acetate is for remove protein and the other cellular constituents. Prevent damage to RNA. 8 Incubate on ice for 5 mins 9 Centrifuge 2 mins at maximum speed Pellet precipitated proteins and chromosomal DNA 10 Transfer 400µl of 5M NaCl K+ form ionic bond to produce insoluble potassium dodecyl sulphate. This will remove most of the protein unbroken cell and fragment. NaCl is used for precipitate RNA. It neutralize the negative charged RNAbackbone, decreasing the solubility of RNAin water. 11 Add 1ml of ice-cold ethanol Ethanol addition is to reduce the solubility of RNA in the aqueous solution as high salt solution discussed above has neutralized the negatively charged RNAbackbone 12 Precipitate the RNAby incubation on ice for 5 mins 13 Centrifuge 5 mins at max speed, blot the inverted To recover DNA, to remove all liquid ensuring tube on paper towel for 2 mins pure concentration 14 Dissolve pellet in 25µl of TE 15 Transfer 11µl of RNAsolution to clean tube, add Dye it for review under electrophoresis 1µl of loading dye solution and 1µl of 200X SybrSafe stain Centrifuge 3-5 seconds B Loading the Gel and Electrophoresis 1 Load 12µl from each tube into separate well in gel 2 Electrophorese in 1.0% DNAagar gel at 180V for 35-40 mins. 3 Observe fluorescent bands and make a sketch of your gel, label and identify bands. ▯ Escherichia coli (E. Coli) Because of their genetic simplicity, rapid generation time and ease to work with, they have been studied extensively on the genetic code, DNAreplication, gene expression, and protein synthesis. ▯ Chelators Bind Divalent Cations: EDTA Many nucleases require divalent cations, like Mg2+ as cofactors. EDTA(Ethylene Diamine Tetraacetate) is a CHELATOR, has a very high affinity for Mg2+. Under alkaline conditions, the acetates are negatively charged and the right distance apt to fit a divalent cation (two positive charges) between two acetates. When divalent cations are ▯ ▯10 /18 University of Toronto - Revision Paper - BIO230 Marcus Lam all bound to the chelator, RNases requiring these ions become inactive. This protects the RNAfrom digestion during its isolation. ▯ Cell Wall is Disrupted with Lysozyme Lysozyme (a glycosidase) hydrolyzes the beta-glycosidic linkages that connect N-acetylmuramic acids and N- acetylglucosamine in the peptidoglycan of bacterial cell walls. ▯ Detergents Lyse Membranes: SDS Cell membranes are composed of lipids and proteins rich in hydrophobic residues.SDS (sodium dodecyl sulfate) is a strong ionic detergent that solubilizes the plasma membrane. SDS is also a strong protein denaturant. Here, we use SDS plus potassium acetate to remove protein and other cellular constituents. ▯ Selective Precipitation of Protein and Cell Debris sing SDS with PotassiumAcetate SDS is very soluble in water, which potassium dodecyl sulfate is very insoluble. Here, we add potassium ions, which form ionic bonds to produce insoluble potassium dodecyl sulphate.All molecules previously bound to SDS now precipitate from solution with the insoluble potassium dodecyl sulphate. By centrifuging this mixture, we remove most proteins, unbroken cells and fragments of cells, as well as most of the large chromosomal DNA, which becomes trapped in the precipitate. Only very soluble molecules, such as RNA, small DNAs and small salts remain in solution. ▯ High Salt Conditions Disrupt Ionic Interactions High NaCl (5M) concentrations are used to help precipitate RNA. The Na+ neutralizes the negatively charged RNA backbone, decreasing the solubility of RNAin water. The ethanol is then added which further reduces the solubility of RNAin aqueous solution. ▯ EthanolAffects NucleicAcid Solubility By the time ethanol is added, the high salt solution has neutralized the negatively charged RNAbackbone. To precipitate the RNA, ethanol is added in a ratio of 1 part RNA, to 2 parts ethanol. Water will preferentially form hydrogen bonds with the ethanol instead of with RNA. Since RNAhas neutral charge, the non-polar parts of RNA molecule will associate with each other, decreasing solubility of RNA, causing it to precipitate. ▯ SYBR Safe Stains NucleicAcids SYBR Safe is a very sensitive stain for DNAan can also be used to visualize RNAin agarose gels. These molecules bind to double stranded DNAand single stranded RNAand emits green lights when irradiated with UV light.It is much safer alternative and has less of an impact upon the environment for disposal. The small volume of stain thats added to the DNAsample is suspended in an extremely dilute solution of DMSO, organic solvent causing irritation of skin. ▯ Electrophoresis Equipment Electrophoresis is used to separate molecules via an electric current. Molecules to be separated are placed in wells of an agarose gel medium that is submerged below the surface of a buffer. Molecules will move in the electrical field according to their charges. Positively charged move to the cathode, negatively charged to the anode. DNAand RNA carry negative charge from sugar phosphate backbone and will move towards anode. Rate of migration depends on size, shape and charge to mass ratio. DNAmolecules are same shape and very similar charge to mass ratios. Size of fragments can be used to separate DNAand RNA. The smaller the fragment, the faster it can travel through the network of pores within the gel medium, as it is less impeded by the gel matrix. Concentration of agarose in the gel can also be varied to improve resolution of particular fragments. Higher percentages of agarose can be used to increase separation of smaller DNAfragments, lower percentage can be used to separate large fragments. ▯ ▯ ▯11 /18 University of Toronto - Revision Paper - BIO230 Marcus Lam Ribosomal RNAOperon rRNAmolecules in prokaryotes and eukaryotes are produced as large primary transcripts (pre-ribosomal DNA) that require subsequent processing. They are about 30S in size and contain one copy of each 16S, 23S, and 5S RNAas well as several tRNAprecursors. So, each time the operon is transcribed the result is one copy of each rRNAneeded for the ribosome. In addition, there are multiple copies of the RNAoperon on the genome. S is the symbol for Svedberg unit, a measure of the rate at which particles move in the gravitational field in an ultracentrifuge. The sedimentation coefficient is the rate at which given solute molecules suspended in a less dense solvent, sediment in a field of centrifugal force. So the sedimentation coefficient is a rate per unit centrifugal -13ld and is a function of weight, shape, and degree of hydration in a given solvent at a specific temperatures. 1S = 10 seconds.
 ▯ ▯12 /18 University of Toronto - Revision Paper - BIO230 Marcus Lam 4. Live Cell Imaging ▯ ▯ Microscopy provides images and insights into the mechanics and biochemistry in cell processes such as cell signaling and cell-cell interaction. Optimizing illumination is key to enhanced microscope performance. The intensity and wavelength spectrum of light emitted by the illumination source is important, and light emitted from various locations on the lamp filament be collected and focused at the plan of the condenser aperture diaphragm. ▯ We will review basic cell structure using light microscopy (bright field, phase contrast, and fluorescence). In addition we will learn about fluorescent protein markers. ▯ Viewing Living Cells Including Intracellular Movement 1. Organelles are capable of complex movements by various movements of cytoskeleton 2. Plant cells: cytoplasm may show cytoplasmic streaming, resulting in broad cytoplasmic strands that transiently traverse the vacuole. 3. Mitochondria and chloroplasts may maintain position within this streaming or have short periods of abrupt movement with or against the cytoplasmic flow. 4. There is a layer of cytoplasm and organelles close to the plasma membrane that does not take part in the cytoplasmic streaming like other locations. 5. The nucleus commonly maintains its position in the cell regardless of cytoplasmic movements around it, or may move in response to stimuli such as cell wall damage. 6. Not everything that seems to move is directionally motile 7. Rapid movement may be due to capillary action or convection currents 8. Vibrating motion observed in dead cells referred to as BROWNIAN MOTION. It is random, irregular motion exhibited by minute particles of matter suspended in a fluid. This effect is ascribed to the thermal motion of the molecules of the fluid. ▯ Florescence Microscopy 1. Fluorescein Diacetate (FDA) is an uncharged and non fluorescent derivative of the fluorescent molecule fluorescein. 2. FDAis uncharged, so it passively crosses the plasma membrane 3. But once inside the cell, Esterases (enzymes) convert FDAto the fluorescent, polar molecule, fluorescein, which cannot easily diffuse back across the plasma membrane and escape from the cell. 4. Deal cells that lack esterase activity, or have damaged plasma membrane do not retain fluorescein. 5. So, FDAis commonly used as a quick method of distinguishing living cells from the dead. Living cells will fluoresce when treated with FDA, dead cells will not. 6. Fluorescein fluoresces a green or yellow color (depending on pH) when irradiated with blue light. ▯ Fluorescence Microscopy and Fluorescent Protein Markers 1. Green fluorescent protein (GFP) has revolutionized our ability to study live cells. 2. Genetically coded fluorescent proteins allowed us to tag and track an enormous range of proteins in space and real time in their natural environment without application of exogenous probes or fixation of cells and tissues that can produce artifacts. ▯ Koehler Illumination is used to ensure the best resolution and image quality for most microscopes. ▯ Lippincott-Schwartz and Patterson ▯ ▯13 /18 University of Toronto - Revision Paper - BIO230 Marcus Lam Ocular micrometer: eyepiece with a scale built into it, divided into micrometer units that are arbitrary, do not indicate actual metric micrometers. ▯ Microscope: Use visible light waves to illuminate the specimen while electron microscopy uses electrons. Resolution: the resolving power being the ability to distinguish between two very closely positioned objects. Limit of resolution is related to the shape of the cone of light entering the objective lens (NA) and the wavelength of light. The larger the NA, the lesser the resolving power; and the smaller the wavelength of light, the greater the resolving power. ▯ Light Microscopy: 1. Three things are required to view cells in a light microscope: a. Bright light focused on specimen by lenses in the condenser b. Specimen must be carefully prepared to allow light to pass through it c. Appropriate set of lenses (objective and eyepiece or ocular) must be arranged to focus an image of the specimen in the eye. 1. Magnification with 40X objective and 10X ocular is 400X ▯ Types of Image Formation ▯ Bright Field (standard light microscopy) 1. Use: view living and fixed tissue, cells, and microorganisms 2. Advantages: readily available, easy to use 3. How it works: microscope aligned to achieve the maximum lighting and resolution possible (Koehler Illumination) 4. Requirements: any light microscope, with minimum of light source, a 10x ocular, usual 4x, 10x, and 40x objective lenses (plus 100x oil immersion) ▯ Phase-contrast Microscopy ▯ Nomarsky Imaging (Differential Interference Contrast or DIC) ▯ Step Procedure Purpose 1 Set phase ring to bright field, focus with 4X, then use 10XAvoid zooming in the adjacent spaces. 2 Close the field iris diaphragm 3 Raise or lower the condenser unit to focus edge of field iris diaphragm. Condenser should be near the top. 4 Open the field iris diaphragm until light is at the edge. 5 Close condenser iris diaphragm until the best resolution anIf we close it too far, the image will show begins to darken.ined, and just until the point the image high contrast but resolution will be lost. 6 Switch to 40X, increase light slightly For more detailed view of the cell 7 Rotate ocular until lines are parallel with those of the slTo facilitate calculation of one micrometer micrometer, align the lines at the left edges of both unit. micrometers. ▯ ▯14 /18 University of Toronto - Revision Paper - BIO230 Marcus Lam Step Procedure Purpose 8 Count how many spaces on the ocular micrometer fit Deduce value of one micrometer unit. precisely in a known distance on the slide micrometer. Then calculate value of one micrometer unit. 9 Repeat procedure for 10X objective Double check the units can be converted 10 Place a drop of distilled water on slide 11 Scrape off a small cluster of hairs from tomato, transfer Create sample hairs from razor blade to water drop with needle, gently lower coverslip onto specimen 12 Focus on one cell using 10X. Switch to 40X, and switch To have a clear view of nucleus and phase ring of condenser to Ph2. cytoplasmic streaming 13 Locate the nucleus of a living cell, look for cytoplasmic streaming around nucleus, identify organelles. 14 Measure length of cell, diameter of nucleus 15 Add 5µl stock FDAto 495µl distilled water and repeat steps Make organelles florescent 16 Incubate for 10-15 mins at room temperature Identify fluorescing organelles ▯ ▯ ▯15 /18 University of Toronto - Revision Paper - BIO230 Marcus Lam 5. Role of the Cytoskeleton in Development (fly) ▯ ▯ Introduction 1. Most cells are only capable of performing one function for the organism, some cells can change from one function to another, and stay that way until they die. The FERTILIZED EGG in contrast, generates all of the different cells that make up the organism. 2. It can perform a vide variety of functions because it is already developed into a highly structured cell during OOGENESIS. 3. During oogenesis, the developing and unfertilized cell that will eventually form the egg is referred to as an OOCYTE.At the end of oogenesis, the egg is ready for fertilization. 4. entering sperm activates the egg and the haploid nuclei of the egg and sperm fuse. There fertilized egg then undergoes several rounds of cell divisions or, depending on the animal, nuclear divisions (karyokinesis without cytogeneses) as in Drosophila. 5. Eventually a layer of cells form in the periphery of the embryo. The embryo is called a BLASTULAat this stage and the layer of cells is the BLASTODERM. Embryogenesis is the name of the process by which the fertilized egg develops. 6. Embryonic genome begins to transcribe DNA, which in combination with RNAtranscripts stored in the oocyte during oogenesis, initiate the developmental pathways that structure the embryo. 7. In a process known as GASTRULATION, cells move in defined ways to set up the body plan of the embryo. 8. With continued development, populations of cells become functionally differentiated and form the tissue and organs of the animal. 9. For example, during Drosophila embryogenesis, the fertilized egg produces approximately 50,000 cells that differentiate into more than 50 cell types in only 24 hours. In humans and other vertebrates, many trillions of cells are produced that form over 200 different cell types. 10. This lab focuses on role of the cytoskeleton in the process of oogenesis (Egg formation) and embryogenesis (Development of the embryo) 11. Early in development, the oocyte generates asymmetries (polarity) that are important for proper development of the embryo. 12. We will observe the formation of cells in live fruit fly embryos. 13. Development is a very complement process. ▯ Appendix One 1. One type of fusion protein may be made when a reporter gene is attached to one end of the protein coding region of the gene of interest 2. GFP is an example of a reporter gene. 3. Transgenic Drosophila - flies that have had the gene for beta-galactosidase insertd adjacent to the protein coding region of the kinesin gene. 4. We can easily locate the resulting kinesin-beta-galactosidase fusion protein because the bacteria enzyme beta- galactosidase (a produce from part of the lac operon) metabolizes the lactose analogue X-gal and turns the colorless X-gal staining solution/substrate to blue color. So, areas of the cell that stain blue indicate the location of the fusion protein. 5. Since kinesin-beta-glactosidase fusion protein behaves like the native protein, we could estimate the distribution and dynamics of kinesin during oogenesis. 6. Fusion proteins is powerful and provides an elegant means to study the role of cytoskeleton ▯ ▯16 /18 University of Toronto - Revision Paper - BIO230 Marcus Lam Part III: Live embryo observation: Germ cell formation and Early Embryogenesis 1. first cells formed by Drosophila embryo are the germ cells (pole cells) that will give rise to eggs and sperm. The somatic tissues develop from the blastoderm that form later. Example of early segregation of germ line and soma in all animals 2. Drosophila egg surrounded by two egg shells, outer (chorion) and inner (vitelline membrane). Vitellin membrane is transparent but chorion is not. Chorion needs to be removed or cleared to observe the embryo. 3. When females deposits an egg, it is already a fertilizes and developing embryo 4. Embryos look like grains of rice with two treads attached to one side. 5. White color is the chorion and the two threads are the dorsal appendages, specialized respiratory structures of the eggshell. 6. Fruit flies usually lay fertilized eggs in rotten fruit where they may be partially or completely submerged. ▯ Labb5::RooleoffCyytoskeleton in DeevelopmeenntFloww Chhart Step Procedure Purpose 1 Add small amount of PBS to the three wells in the glass dish. Transfer 10 dead flies to one of the wells. Isolate four females and transfer to adjacent well. 2 Take a set of forceps in each hand, hold a female with one Orient the fruit fly to make set of forceps between the thorax and abdomen, grab the tip extraction of ovaries easier of the abdomen wit another set of forceps. 3 Pull the tip of the abdomen, making sure that the hole is big enough for the whole ovaries can slip out 4 Transfer the ovaries to the third well, leave the rest of the The transparent peritoneal in the second well, collect ovaries of at least 3-4 females. sheath covering the ovaries will Tease the ovarian tissue apart. inhibit C-gal uptake and therefore staining. 5 Transfer ovaries using a Pasteur pipette or forceps to the Be careful to keep the ovaries staining dish filled with enough PBS to cover the ovaries. only in the narrow part of the pipette during transfer. Or be gentle with the forceps 6 Take the dish to the fume hood, remove the PBS with TA adds fixative because it is pipette. TA add fixative, 1%glutaraldehyde to over ovaries. essential to not over fix (Wear Cover and incubate for 5 minutes. gloves in fume hood) 7 TA remove fixative with pipette and dump into glutaraldehyde waste container. Rinse 3 times with PBS. Incubate PBS for 5-10 mins. 8 TA transfer staining dish into 5ml of warmed X-gal staining Locate the kinesin-beta- solution. Incubate for 50-60min at 37˚C. galactosidase fusion protein because it metabolizes X-gal and turns blue. (Wear gloves in fume hood) 9 Since 2 times with PBS, and transfer ovaries into one well of the three well glass dish. ▯ ▯17 /18 University of Toronto - Revision Paper - BIO230 Marcus Lam Labb5::Rooe offCyyosskeleton in DevvelopmeenttFlow CChartt Step Procedure Purpose 10 Place a small drop of 70% glycerol onto a slide. Transfer ovaries into the drop with forceps. Separate the ovaries into ovarioles with dissecting needles. Place coverslip on top of preparation and remove excess glycerol. 11 Set up Koehler Illumination with compounds microscope with Observe and learn about the 10x and 40x objectives:▯ structure and physical attributes 1. Identify ovarioles and individual follicles▯ of ovarioles. 2. Identify follicles where the oocyte is about half the size of the whole follicle▯ 3. Draw the follicle and indicate the stain 12 Retrieve agar plate with eggs and add a few ml of PBT. Loosen the embryos from the Brush the embryos. agar with a brush. 13 Decant embryos and PBT into the egg collection basket and The bleach digests the chorion submerge embryos in bleach for 4 minutes. but leaves the Vitelline membrane intact. (Make sure embryos do not stick to plastic above the bleach.) 14 Remove from bleach, wash with PBT for 1 minute. Blotting Blotting removes excess PBT. on paper towel. Place the mesh with embryos onto an agar plate. 15 Place a small drop of halocarbon oil onto a slide. Transfer embryos to the oil with a brush. 16 Place footed coverslip over the egg and adjust microscope to This gives just enough space increase contrast. Footed coverslip can be built in two waysbetween the slide and the cover 1. Carefully attach small pieces of wax to four corners of tslip to avoid crushing the egg. coverslip. Wax should be first warmed by rolling between fingers into small ball. Carefully adhere small pieces of soft sax to four corners of cover glass and press against the slide▯ 2. Gently nick the four corners of the coverslip against some wax. Was should be first warmed by rolling between fingers into small ball. Crumbs of wax should remain attached at each corner.▯ Adjust size of crumbs to size of eggs. 17 Observe embryos under compounds microscope for 15-20 Learn about the gradual physical mins. transformation of embryogenesis ▯ ▯18 /18 University of Toronto - Revision Paper - BIO230 Marcus Lam Lab 5: Role ofCyyosskeleton n Devvelopmeent Foww Chaatt Step Procedure Purpose 18 Set up Koehler Illumination using 10X and 40X objectives:▯ Learn about the physical 1. Identify vitelline membrane.▯ attributes of fruit fly embryo. 2. Determine the orientation of embryo.▯ 3. Identify embryos that show a clear space between vitelline membrane and the embryo at the anterior and posterior end. Observe embryo every 5 mins over next hour to determine progress in germ cell formation, keep a record.▯ 4. Explain why germ cells form only at the posterior pole of the embryo. ▯ ▯ ▯ ▯19 /18 University of Toronto - Revision Paper - BIO230 Marcus Lam ▯ ▯ ▯ ▯ ▯ Section 2: Cell and Developmental Biology ▯ 2.1 Membrane Trafficking 2.2 Cytoskeletal Networks 2.3 CellAdhesion 2.4 Tissue Morphogenesis 2.5 Tissue Patterning 2.6 Stem Cells 2.7 Principles of Cellular Signaling 2.8 Signaling via Small Molecules 2.9 Signaling via Protein Modifications 2.10 Cell Cycle 2.11 Programmed Cell Death 2.12 Cancer
 ▯ ▯20 /18 University of Toronto - Revision Paper - BIO230 Marcus Lam 2.1 Membrane Trafficking ▯ ▯ 2.1.1 Endocytic vs. Secretary Pathways ▯ Why is membrane trafficking important? 1. Communicate with other cells and acquire resources like nutrients. 2. These processes require control and dynamic changes to the plasma membrane. ▯ Basic principles of the Biosynthetic-Secretory and Endocytic Pathways 1. Connections among the ER (makes proteins), the PM and the LYSOSOME (breaks down proteins) 2. Polarized trafficking routes: ER -> Golgi -> Lysosome - > Plasma Membrane -> Extracellular space 3. Sorting Stations: Sorted by Golgi and Early Endosome, different molecules going to different sites 4. Retrieval MechanismsAmong Routes: At PM and ER to prevent depletion of substances within the cell ▯ Two Types of Secretory Pathways 1. Constitutive: The vesicles keep budding off and is unregulated. a. The vesicles DOCK with the plasma membrane and FUSE. b. Exocytosis seals plasma membrane holes 2. Regulated: The vesicles only bud off in the presence of a regulatory signal. a. The vesicles are stored until a signal triggers their docking and fusion b. E.g. a Mast cell releasing stored histamine after induction by a soluble extracellular stimulant ▯ Regulated Vesicle Secretion Provides Extra PM 1. Cleavage Furrow: For daughter cells 2. Phagocytosis: Eat the bacteria as an immune response 3. Wound repair: Damaged hole or tear ▯ Regulated Vesicle Secretion For Extracellular Signals: Only release neurotransmitter inside synaptic vesicles by exocytosis at the nerve terminal upon arrival of action potential ▯ But the above 2 types of secretion have the same initiation biosynthetic pathway ▯. Cargo concentration is in positive correlation to level of maturity ▯ ▯ ▯ ▯21 /18 University of Toronto - Revision Paper - BIO230 Marcus Lam Endocytic pathways counterbalance secretory pathways and perform specific functions 1. Basic Steps of Endocytosis: Invagination (bud in and make a dent), then fission (break away from plasma membrane into a vesicle) 2. The endocytosed vesicle joins the early endosome compartment and is then routed to various destinations a. Recycling: Back to the PM for exocytosis b. Transcytosis: Transferring to other compartments for metabolism c. Degradation: Transferring it to lysosome for degradation ▯ Function of Endocytosis 1. Collect Resources: eg. Cholesterol (with the help of LDL receptor) 2. Internalize Pathogens by Phagocytosis: Formation of PSEUDOPOD to engulf the bacterium 3. Down Regulate Cell Surface Signaling: eg. Ubiquitin signal is sequestered into early endosome, giving a multivesicular body. Late endosome or lysosome will then attack the body and digests the signaling molecule. ▯ 2.1.2 Local Membrane Changes ▯ 1. Fusion through Exocytosis a. SNARE proteins specify which membranes fuse by pulling them together i. v-SNARE (synaptobrevin) v stands for vesicle ii. t-SNARE (syntaxin) t stands for target membrane b. Close Coiling of SNARE: In order to allow fusion of hydrophobic membranes, water must be expelled (pushed out the way) in between the membranes. c. Stalk formation (contact)—> Hemifusion (half fused)—> Fusion ▯ ▯ ▯ 2. Invagination through Endocytosis: CLATHRIN drives vesicle invagination events a. Adaptor Proteins: Bind to the cargo receptor which then selects for specific cargo protein. b. Bud Formation: From outside cell, to inside cell cytosol c. Vesicle formation involving local fusion for circular structure, different from the “fusion” above d. Uncoating of the CLATHRIN coats giving the naked transport vesicle i. Itself is a triskelion, three heavy chains and three light chains, it creates a dome like structure that bends membrane inwards ii. Aclathrin coat composed of 36 triskelions iii.COPI and COPII coats also drive vesicle invagination events. iv.DYNAMIN pinch l two lipid bilayers close together for fusion and release as a vesicle. ▯ ▯ ▯22 /18 University of Toronto - Revision Paper - BIO230 Marcus Lam ▯ 3. Budding (away From cytosol into multivesicular body) a. ESCRT Complex: Series of proteins on the cytosolic face of vesicles involved in bending the membrane away from cytosol that drives budding events. b. Purpose to change composition of cytosolic material c. Recruit cargo to those sites, fusion of cytosolic vesicles into lumen of the Late Endosome so we have membrane bound compartments. d. Similar mechanism to invagination, both can be described as budding. But difference is into or away from cytosol. ▯ Membrane invagination, fusion and budding events occur at specific sites in the system to control polarized trafficking and retrieval. 1. Clathrin: Everything thats not ER and Golgi 2. COPI: Reverse transport within Golgi and ER 3. COPII: From ER to Golgi ▯ 2.1.3 Regulation of Specificity in the Membrane Transport Machinery 1. SNAREs 2. Signaling Lipids (PIPs) 3. Small GTPases 4. Signal Sequences/Moieties on Cargo 5. Membrane Composition ▯ Signaling Lipids (PIPs): The INOSITOL sugar head group of inositol phospholipids can be phosphorylated or dephosphorylated at specific sites with a specific number of phosphates by Kinases and Phosphatases to make a variety of PHOSPHOINOSITIDE (PIP) species in membranes that binds to specific proteins. Thus, their protein partners will be recruited to these sites. ▯ ▯ ▯23 /18 University of Toronto - Revision Paper - BIO230 Marcus Lam Small GTPases 1. Small GTPases are active in the GTP-bound state 2. They ares specifically localized and they bind to activate downstream effectors 3. Guanine-Nucleotide Exchange Factors (GEFs) promote the exchange of GDP to GTP, turn ON Small GTPases 4. GTPase-Activating Proteins (GAPs) promote the hydrolysis of GTP into GDP, to turn OFF Small GTPases ▯ ▯ ▯ 5. Specific RAB SMALL GTPASES (they are proteins) localize to distinct sites in the trafficking pathway *remember the following relationships* a. Rab5A: PM, Clathrin-coated vesicles, Early Endosomes b. Rab7: Late Endosomes c. Rab11: Recycling Endosomes 6. RabGEFs activates Small GTPases, recruit Rabs to specific membranes to promotes downstream effects a. Aspecific Rab works with specific: i. PIP (Phosphoinositide: Signaling molecule) in protein recruitment ii. SNAREs protein in docking and fusion b. When lipid is inside Rab, it is inactive. When lipid is outside of Rab, it is active with GTP-bound. c. So, this structural conformation change can recruit tethering proteins, it can also recruit more Rab5- GEF recruitment in positive feedback d. We don’t know what positions the GEFs, that positions the small GTPases. ▯ ▯ Spatial Organization of individual trafficking events organizes the overall trafficking networks in addition to the plasma membrane. ▯ ▯ ▯ ▯24 /18 University of Toronto - Revision Paper - BIO230 Marcus Lam 2.2 Cytoskeletal Network ▯ ▯ Polarized cytoskeletal networks organize cells and control interactions with their environment ▯ MicrotubulesAre Inherently Polarized (from subunit, to protofilament, to network) 1. Microtubules are made of PROTOFILAMENTS, which is are heterodimers made up of two monomeric protein subunits called α- TUBULIN and β-TUBULIN a. One beta followed by one alpha and so on 2. Tubulin monomers bind and hydrolyze GTP to GDP 3. Each protofilament heterodimer is asymmetric and forms POLARIZED FILAMENTS a. Polar means one end is structurally different from the other end. Has nothing to do with charge. ▯ ▯ γ-tubulin Complexes Nucleate Microtubules 1. γ-TUBULIN (light blue) forms a ring complex as a template for tubulin heterodimers to assemble into tubes. 2. These other grey proteins, support proteins help hold them in place 4. Plus Ends: Grow away from nucleation sitesleus) of microtubules 5. γ-tubulin often associates with LARGE MICROTUBULE ORGANIZING CENTERS (MTOCs) - which is the sphere below a. Centrosomes containing 2 centrioles are surrounded by hundreds of proteins with γ-tubulin nucleation sites on the surface b. The centrioles (cylinder) are to support the structure of centrosome c. Huge 3D sphere of microtubules that extends in every direction ▯ ▯ ▯ ▯ ▯ ▯25 /18 University of Toronto - Revision Paper - BIO230 Marcus Lam Microtubule Movement by Dynamic Instability (At growing ends) 1. Single microtubule switches between growing and shrinking, which is called DYNAMIC INSTABILITY which allows them to search out the 3D structure of cell. 2. This instability facilitates the capture of cargo proteins (etc. Chromosomes during mitosis) 3. When microtubules are GDP bound, the 13 microtubules do not fit well because they tend to curve a bit, giving rise to the dynamic instability 4. Growing microtubules have a protective cap of GTP-bound tubulin 5. If GTP hydrolysis (to GDP) is faster than subunit addition, the cap is lost and it will disintegrate a. Depolymerization is 100 times faster at an exposed GDP end 6. Regaining of a GTP cap rescues growth ▯ Microtubule Networks can Form a Coordinate System on Their Own 1. Apurified centrosome was mixed with purified tubulin subunits in an artificial membrane-bound container, which is a square lipid compartment. 2. It moves to the centre of the container as microtubule plus ends push on the outer membrane and minus ends push towards the centre polarity, plus ends on the outside of the cell. 3. This may contribute to microtubule organization in cells, though many regulatory proteins are also involved ▯ How is Cargo TransportedAlong Microtubules? Motors move cargo through the microtubule networks (Walking Motion) 1. Similar to the ski resort transporting skiers to the mountain tops with cable cars. 2. The motor activity is polarized. 3. DYNEIN moves to microtubule minus ends (NEIN! its like no, so minus) 4. KINESIN moves to microtubule plus ends 5. The role for microtubules in positioning the Golgi can be seen after the addition of a microtubule inhibitor a. High concentration of microtubules near nucleus and the microtubules protruding out of the centrosomes, this shows localization of intracellular contents b. Nocodazole treatment can disrupt the microtubules c. Dynein motor is the likely key for Golgi positioning because they walk towards minus ends, center ▯ ▯ ▯ ▯ ▯26 /18 University of Toronto - Revision Paper - BIO230 Marcus Lam Balance of Signals Regulate Different Motors that Control Movement of Membrane Compartments E.g. Controlling appearance of theAfrican cichlid fish, Tilapia mosssambica through dispersion of MELANOSOMES (organelle synthesizing MELANIN which is the most common light absorbing pigment in the animal kingdom) ▯ 1. Male dominant, aggressive behavior: Turns Black a. Increase in cAMP signaling causes Kinesin and Dynein to compete for melanosomes, so they move back and forth resulting in an even spread of color. ▯ 2. Camouflage, wants to hide behavior: Turns White a. Decrease in cAMP signaling inhibits Kinesin and Dynein moves melanosomes to the minus end, the centre of the cell so most of the cell is clear. ▯ ▯ TheACTIN CYTOSKELETON also plays a critical role in organizing cell structure and controlling cell behavior (by pushing outwards or pulling inwards the cell membrane) ▯ ▯ filaments, to networks)is inherently polarized (from subunit to 1. Actin monomers are asymmetric, polarity ends arise fromATP hydrolysis toADP. 2. Actin monomers bind and hydrolyzeATP 3. Assemble head to tail forming polarized filaments in form of helix 4. The actin has a greater propensity to dissociate at theADP end and grow at theATP end. (class line-up demonstration) 5. ATP-ADP polarity along actin filaments a. After polymerization, actin-ATP is hydrolyzed into actin-ADP b. Hydrolysis reduces binding affinities to neighboring subunits increasing dissociation c. If the rate of addition of action-ATP is faster than the rate of removal of actin-ADP, a relatively stable ‘cap’of actin-ATP subunits can be formed ▯ PolarizedAssembly and Disassembly Leads to TREADMILLING 1. Subunits can undergo net assembly at plus end equal to (or greater than) the net disassembly at the minus end 2. Thus, the polymer can maintain a constant length (or grow) with a flux of subunits through the filament (treadmilling) a. Like a conveyer belt b. The firstly added subunits are displaced further down c. And they will eventually dissociate ▯ ▯ ▯ ▯ ▯ ▯27 /18 University of Toronto - Revision Paper - BIO230 Marcus Lam TheARPComplex NucleatesActin Filaments and BranchesActin Filaments to Form Polarized 2-D Networks 1. Arp2 andArp3 are drawn closer together, creating two “v” shapes template for the plus end of actin to grow from it 2. Other proteins and activating factor bind at specific sites of the existing filaments at 70 degrees to build a larger network to impact the cell. 3. COFILIN is a protein that promotes disassembly at one end. ▯ ▯ ▯ ▯ Polarized Treadmilling of LargeActin Networks Can Produce Significant Protrusive Power 1. The dissociated actin monomers flow back to be recycled and reused in later assembly 2. These networks drive polarized cell movement a. Neutrophils-Bacteria Chase: Pushes the neutrophil forward, it needs traction (or else just treadmill and maintain position) and directionality so that the leading edge can capture the bacteria. b. Branch organization 3. Treadmilling microfilaments can engage stationary anchors to create ‘PROTRUSIVE MACHINES’ a. Astationary anchor binds one part of the filament. b. The treadmilling filament extends from that point c. This extension pushes against the cell membrane and drives cell protrusion d. Large regions of actin networks are anchored to create ‘protrusive machines’ e. Disassembly at the back, reassembly at the front ▯ INTEGRINS Connect theActin Cytoskeleton to Extracellular Matrix Molecules 1. Integrins are transmembrane receptor heterodimers (non- covalently associated α and β subunits) that bind extracellular matrix protein and adaptor proteins. 2. On the cytoplasmic side, beta subunit link to adapter protein TALIN and along with VINCULIN, they attach to actin filament. ▯ ▯ ▯ ▯ ▯ ▯28 /18 University of Toronto - Revision Paper - BIO230 Marcus Lam Chemoattractant Receptors Orient theActin Networks (Tip Ends of Neurons, Neutrophil-Bacteria) 1. The chemoattractant binds to the transmembrane protein receptor, sending a chemical signal inside the cells, triggering the activation ofArc2,Arc3 formation of actin network 2. Then the cell can start pushing out in the direction of these bacterium and the back. So we have an actin-myosin contraction just like out 3. Just like food sources and ant trails, each are dynamic and can rapidly reorient to changes in the target position (disassembly and reassembly) ▯ ▯ ▯ The microtubule and actin network is very dynamic and can be reorganized. Example of mitosis with spindle fibers. 1. Completely disassemble network 2. Actin go in the middle of the cell to from CONTRACTILE RING to divide the cells 3. After the daughter cells form, they go back to their own network. ▯ ▯ ▯ Actin and microtubule networks are also integrated in cells and can crosstalk. ▯ ▯ ▯ ▯29 /18 University of Toronto - Revision Paper - BIO230 Marcus Lam 2.3 CellAdhesion ▯ Two Tissue Categories: 1. Epithelial Tissue: On the surface. a. Cells directly connected to one another with minimal extracellular matrix beneath them b. Mechanical Stresses: Transmitted from cell to cell by cytoskeletal filaments anchored to cell-matrix and cell-cell adhesion sites c. Cells form our skin and coat our organs 2. Connective Tissue: Underneath epithelial. a. Cells dispersed with extracellular matrix providing overall structure b. Mechanical Stress: Directly bears tension and compression by extracellular matrix. c. Cells include muscle, neurons and immune cells ▯ Why is Epithelial Structure Important? 1. More than 60% of the cell types in the vertebrate body are epithelial 2. Crucial for Organ Structure: It acts as a barrier i. Exterior: Prevent desiccation (extreme dryness), mechanical injury and direct pathogen invasion ii. Interior: Separate one body compartment from another 3. Epithelial have distinct apical and basal sides a. TheAPICAL surface faces the organ lumen or the animal surface b. The BASAL surface faces underlying tissue c. Epithelial polarity (difference, not charge) is critical for organ function because it allows for regulation i. E.g. Epithelial polarity controls solute diffusion between our body compartments a. Glucose is blocked from diffusing between cells by TIGHT JUNCTIONS. Instead, it must be actively transported through cells by plasma membrane channels allowing for precise regulation. Polarity is of paramount importance due to directional control of transport channels (Glucose importers at apical membrane, Glucose exporters in the basal side) ▯ Junctional Complexes 1. Occluding Junction: Tight junction seals gap between epithelial cells 2. Cell-cellAnchoring Junctions: a. Adherens junction connects actin filament bundle in one cell with that in the next cell b. Desmosome connects intermediate filaments in one cell to those in the next cell 3. Channel-forming Junctions: Gap junction allows the passage of small water-soluble molecules from cell to cell 4. Cell-matrixAnchoring Junctions: a. Actin-linked cell-matrix adhesion anchors actin filaments in cell to extracellular matrix b. Hemidesmosome anchors intermediate filaments in a cell to extracellular matrix ▯ ▯30 /18 University of Toronto - Revision Paper - BIO230 Marcus Lam Adherens Junction Structure 1. Form strong continuous adhesion belts, adhering cells to form epithelia 2. CADHERIN (Calcium-DependentAdhesion) clusters mediate the adhesion a. HOMOPHILIC (like with like) interactions between cadherin receptors in the presence of calcium ions b. Links to the actin cytoskeleton ▯ ▯ ▯ Adherens Junction Function: 1. Tissue Maintenance during development in early embryo: a. Dynamic movement of the outer epithelium of the Drosophila embryo b. Tissue structure is lost in adherens junction mutants (cells lose contact with one another) cells disintegrate 2. Tumor Suppression a. The loss of epithelial structure is a hallmark of cancer b. Cadherins are tumor suppressors, holding cells together, stopping spread of cancer cells c. NEOPLASIA(new abnormal growth) shown in three stages: ▯ Testing the Permeability BarrierAcross an Epithelia: Add a dye apically and then basally and see how far could it diffuse. If we don’t have tight junctions, the dye would flow right through. ▯ Tight Junctions Encircle theApical of Each Cell in an Epithelial Sheet: Analogy to a ziplock, we have many rows of ziplock all intertwined with each other for effective seal. ▯ Tight Junctions are Formed from Strands of Interacting Transmembrane Proteins 1. CLAUDIN: 4-pass TM protein, essential for tight junction formation 2. OCCLUDIN: 4-pass TM receptor required for barrier function, but not needed for maintaining overall tight junction structure ▯ Conclusion: Tight Junctions Function as Gates and Fences 1. Gates to prevent molecular movement across the epithelial sheet in the extracellular space between cells 2. Fences to prevent molecular movement between the apical and basolateral domains of each cell’s plasma membrane. ▯ ▯31 /18 University of Toronto - Revision Paper - BIO230 Marcus Lam Establishing InitialAsymmetry Cells use “landmarks” to break the symmetry, components will be recruited and separated to domains 1. In C. elegans, sperm entry provides a landmark for polarizing the one-cell embryo. The sperm enters at the posterior and the cytoskeletal flow towards the anterior side, giving rise to an anterior cue. The anterior/posterior cues are antagonistic and carry different cell fate determinants. One cell becomes germline, and the other cell becomes skin, neurons etc. 2. Chemoattractants polarize cells and the cells chase pray, neutrophil chasing bacterium, chemoattractant on receptor in one side of the cell. ▯ Adherins Junctions are Important Landmarks for Epithelial Polarity 1. Contacts between cell protrusions form landmarks for adherens junctions assembly between mammalian cells in culture with the help ofARF COMPLEXES. 2. AJ components are all over the place 3. two different cells create actin protrusions, as the actin protrudes out and scopes out the nvironment, they will touch each other. Where they touch is where theAJ PUNCTAare formed 4. All of the previously diffusedAJ components will go right to where the two actin protrusions have made contact. The adhesion complex feedback on itself to make continuousAJs 5. All the actin will line up to form the belt and form the belt and the cadherins will touch 6. Reorganization occurs 7. Mesenchymal-to-Epithelial Transition: These two cells previously were not epithelial because they are not lining anything. Now that they found each other, they are epithelial. ConservedApical and Basal Cues Controlling Epithelial Polarity 1. Adherins junctions form first, then tight junctions form afterwards 2. Protein sorting and development of the epithelial cells ensue 3. Scribble complex repel the apical cues and defines the basolateral side 4. Scribble is part of the adherens junction in the sense that it mediates, along with Crumbs Complex, the polarization of basolateral and apical domains. 5. The Crumbs complex attracts apical cues while Scribble complex attracts basolateral cues. Bear in mind the cues themselves also repel one another so you get a distinct basal and apical polarity (not electrical or ionic polarity, structural polarity) ▯ How is cell polarity linked to other cellular machinery? 1. The integration of polarity complexes, adhesion complexes, cytoskeletal networks, and trafficking routes is critical for the structure and function of epithelia that form our organs 2. Complexes required for epithelial structure 3. Body compartments constructed from functional epithelia
 ▯ ▯32 /18 University of Toronto - Revision Paper - BIO230 Marcus Lam 2.4 Tissue Morphogenesis ▯ ▯ Multicellular Development Requires: 1. Morphogenesis: Generation of tissue shapes that form organs and bodies. 2. Cell differentiation: Generation of the different cell types in tissues. 3. Both of them involve: Cell proliferation, specialization, interaction, and movement ▯ InAdults, Multicellular Development Continually Occurs From Stem Cells 1. Skin Epithelia (continual resupply of layers of skin every 3 weeks): Squames (that are about to flake off from surface), Keratinized squames, Granular cell layer, Prickle cell layers, Basal cell layer, Basal lamina, Connective tissue of dermis 2. Gut Epithelia (Lumen is very harsh environment, so cells constantly being lost): a. Looping in and out creating villi b. We will lose lining of gut within 4 weeks. c. Non-dividing differentiated-cells ▯ Adults CanAlso Initiate New Developmental Processes E.g. Female mammary gland epithelia 1. Gradual formation of alveoli dilated with milk in pregnancy and lactation. 2. The milk fat droplets are secreted out of the apical side into the duct so that the infant can be fed ▯ The best studied form of multicellular development is EMBRYOGENESIS. Importantly, embryogenesis, organogenesis, and stem cell development share common mechanisms of morphogenesis and cell differentiation. ▯ 2.4.1 Embryogenesis ▯ Animal Embryogenesis Begins with the Formation of a Ball of Cells called BLASTOCYST 1. Embryonic cells of the blastocyst have undergone minimal morphogenesis and cell differentiation. 2. Compaction is when cells gain cell adhesion 3. 3 main regions of embryo a. Space called BLASTOCOEL b. INNER CELL MASS gives rise to embryo c. TROPHECTODERM: gives rise to extra embryonic tissue, supporting growth like placenta ▯ ▯ ▯ ▯33 /18 University of Toronto - Revision Paper - BIO230 Marcus Lam Morphogenesis Requires: 1. Internalization of Cells 2. Elongation of the Embryo 3. Fine Repositioning of Cells ▯ 2.4.2 Mechanisms of Internalization of Cells 1. Internalization of cells during GASTRULATION creates the 3 main germ layers 2. ECTODERM: epidermis and nervous system (Ecto is outside) 3. MESODERM: muscles, connective tissue, and blood vessels (meso is middle) 4. ENDODERM: gut, lung, liver etc. (endo is inside) ▯ Ingression/Delamination 1. EPITHELIAL-TO-MESENCHYMAL(mesenchymal aka. Connective tissue) Transition: Epithelial cells separate and migrate to form mesechyma by losing adhesion and activating the basal protrusion. 2. This happens in normal cells but are also dangerous during cancer progression a. E.g. Metastases: cancerous tumors that lose adhesion invade other tissues through blood stream 3. Tightly regulated during normal development a. E.g. Quail to Chick Transplant Experiment: Extract group of cells from Quail embryo known to undergo transition and transplant them into a chick. These cells will not be rejected due to similarity, but specific protein not in chick, so we can follow where theses goes. We can determine from cross section of the developing wing that it is related to limb muscle development. ▯ Invagination/Involution 1. Intact epithelial sheets move inside the embryo: Microtubules elongate, causing cells to become columnar and the apical actin-filament bundles contract, narrowing the cells at their apices. So the top of the cells move like a series of triangles which give rise to an inward bending process a. E.g. Neural tube development to spinal cord from the ectoderm in vertebrates. Blue cells undergo constriction and form neural tube, running whole length of early embryo to form spinal cord and brain. ▯ b. E.g. mesoderm internalization in Drosophila 2. Individual nuclei marked brown, shows inward bending of tissue, move inside the embryo, in this case detach from one another and migrate around embryo. 3. The invagination/involution site is highly regulated a. E.g. ventral expression of a transcription factor called TWIST specifies the cells to undergo mesoderm internalization in Drosophila. Without Twist expression, no internalization. ▯ ▯34 /18 University of Toronto - Revision Paper - BIO230 Marcus Lam 2.4.3 Mechanisms of Embryo Elongation ▯ Convergent Extension: Specific cell rearrangement from surface cells to the midline, pushing their neighbors to anterior and posterior ends. E.g. Xenopus Embryogenesis: LAMELLIPODIA attempt to crawl on surfaces of neighboring cells, pulling them inward. The direction of cell movement is regulated. ▯ Cell Division and Cell Shape Change 1. E.g.Arabidopsis embrogenesis a. Basically makes more cells at the tip of the root due to zone of cell division. The only place new cells can go is down because the cell walls in the existing cells prevents them from going up. b. The orientation of cell elongation is regulated by the orientation of CELLULOSE MICROFIBRILS and cell shape is driven by turgor pressure. Basically inflated by water. c. B: Cell expand left and right due to pressure/resistance from top and bottom. C: opposite. 2. Adult Gut: Villus Development and Elongation a. Below is extracellular matrix, above is lumen, so dividing cells forced upwards as the bottom is crowded with non-dividing PANETHcells. 2.4.4 Mechanisms of Fine Repositioning of Cells ▯ Cell Sorting: When cells of the 3 early germ layers are dissociated and random mixed, the individual cell types re-associate to form the germ layers.. E.g. Differential Cadherin Expression and the Brain 1. Cadherins interact homophilicly, E-cadherin interact with E- cadherin, this adhesion group specific cells together 2. Different regions in the brain that expressed different cadherins: E-cadherin, R-cadherin, cadherin-6 3. The brain requires fine tuning of cell positioning because cell bodies, dendrites and axons need to go to specific places ▯ Directed Cell Migration eg. Cerebral Cortex 1. Top is going to form the brain, bottom is going to form spinal cord 2. Daughter of progenitor cells crawl up radial glial cell, and occupy a zone of tissue at the end. Then, radial glial extend further in all directions, allowing new neurons to position even further. Concentric Layers: First born neurons inside, last born neurons in the outer layer. 3. Axon does all the migration through a structure called GROWTH CONE, moving forward from actin based protrusion. Cell body stays in place so no treadmilling. 4. We can inject dyes in the eye to find axon paths, somehow neurons know how to find specific targets. ▯ ▯35 /18 University of Toronto - Revision Paper - BIO230 Marcus Lam E.g.Axon Pathfinding from the Neural Tube to the Brain 1. Axons send down to midline of floor plate at one side of the tube, then they migrate up towards the brain, forming the spinal cord. 2. The floor plate secrets attractant (NETRIN) that diffuses to the floor plate on the central midline. Growth cone receptors at the very tip of axon take that up and move towards the midline. 3. Change of Direction? Growth cone starts to express a different receptor on the surface and acts in opposite way, SEMAPHORIN and SLIT are repellants that inhibit formation of actin network, so the only way it can form is up or down. ▯ ▯ ▯ ▯ ▯ ▯36 /18 University of Toronto - Revision Paper - BIO230 Marcus Lam 2.5 Tissue Patterning ▯ ▯ Two main mechanisms of cell differentiation 1. Asymmetric Division: Sister cells born different. 2. Symmetric Division: Sister cells born the same and become different as result of after birth influences. ▯ Asymmetric Division 1. It partitions (separates) cell fate determinants to define the germ line and other tissues 2. It requires cortical polarity and proper spindle alignment 2.5.1 Extrinsic Mechanisms of Cell Differentiation ▯ Direct Lateral Inhibition 1. Creates a pattern of isolated differentiated cells: Cells begin equal, some cells randomly gain an advantage, these cells differentiate and inhibit their neighbors from differentiating 2. Lateral inhibition by NOTCH SIGNALING a. Each cell tends to inhibit its neighbor b. One cell wins after competition c. Cell with active DELTAspecializes and inhibits its neighbor from doing likewise ▯ Induction by Diffusible Signals 1. Cells begin with equal potential 2. Adiffusible signal from neighboring cells drives groups of cells into distinct developmental pathways 3. Signal is limited in duration and space so that the effects depend on the distance from the source 4. Creates a pattern of bands of differentiated cells centered around the signal source 5. Morphogens: Secreted inductive molecules that spread in a gradient. a. One type of morphogen’s concentration often dictates cell fate b. “Organizer” tissues act as morphogen sources i. Transplanted “dorsal lip” tissue of one embryo to the ventral side of another embryo. This created a duplicate embryo with two dorsal sides and a shared ventral side ii. The transplanted tissue is an organizer because it induced host tissue to generate structures it normally would not make c. Organizer function in vertebrate limb development i. Source of the morphogen is SONIC HEDGEHOG (Shh) ii. Shh spreads from the posterior, which is the polarizing region or organizer tissues of wing bud iii. The Shh gradient controls the formation of distinct digits (fingers) ▯ ▯ ▯37 /18 University of Toronto - Revision Paper - BIO230 Marcus Lam Regulatory Hierarchies 1. Regulatory hierarchies refine patterns of cell differentiation to create segmental patterns 2. Transcription factors regulate other transcription factors to generate segmental patterns of gene expression a. Bicoid:Affect the egg-polarity genes b. Kruppel and Hunchback:Affect the gap genes c. Eve and Ftz:Affect the pair-rule genes d. Engrailed: Affect the segment-polarity genes 3. Discovering the genes of the hierarchy by screening mutants a. Segmentation is fundamental to animal body plans i. e.g. Drosophila: Head, Thorax and Abdomen ii. Homeotic selector (Hox) genes specify which body parts develop from each segment iii. Hox gene clusters are highly conserved iv. Misexpression of the gene ANTENNAPEDIA(Antp) cause legs to grow from the region for antennae ▯ b. Gene order on the chromosome corresponds to expression order in the embryo i. Humans do not sprout wings because Hox genes encode transcription factors with different targets in different species c. Changes in regulatory gene expression hierarchies help distinguish species i. Same Hox genes giving the same transcription factors can give different expression of genes (as shown in protein synthesis) by binding with different genes ▯ ▯ ▯ ▯ ▯ ▯ ▯38 /18 University of Toronto - Revision Paper - BIO230 Marcus Lam 2.6 Stem Cells ▯ ▯ All structures need maintenance. Once cells and tissues are formed they continually regenerate. This can occur at the level of molecular turnover or cellular turnover. ▯ 2.6.1Architecturally Specializes Tissues Do Not Regenerate Cells ▯ Auditory Hair Cells 1. Their architecture converts sound waves into nerve impulses 2. The hair cells are embedded within the epithelium 3. The TECTORIAL MEMBRANE is made up of long strands of polysaccharides and proteins. It is considered as an extracellular molecules but not a cell itself. 4. Sound waves cause STEREOCILIAatop hair cells to tilt, tethers then pull open ion channels on neighboring stereocilia which initiates a nerve impulse 5. In mammals, these cells do not re-grow when lost. Their loss from disease, toxins or extreme noise leads to permanent hearing loss a. Remarkably, other vertebrates can replace these cells 6. Despite their stable overall structure, the hair cells and their stereocilia are very dynamic at the molecular level a. The stereo cilia are filled and supported by actin b. This actin undergoes continually polarized tread milling ▯ Human Photoreceptor Cells 1. Their architecture converts light waves into nerve impulses 2. Pulse-chase Experiment a. Cells exposed to radio labeled leucine for a short time b. They take up the labeled amino acid and incorporate it into newly synthesized proteins for a short period of time: It’s detection will gradually be lost, this shows cells do turn over at the molecular level. ▯ Bone 1. The extracellular matrix that forms bone is continually digested by osteoclasts and deposited by osteoblasts 2. The rates of the above processes causes bone weakening or strengthening ▯ 2.6.2 Tissues Exposed to Harsh Environments Renew Cells Rapidly ▯ Cell Turnover can be Stem Cell Dependent or Independent. Stem Cell Definition: 1. It is not terminally differentiated 2. It can divide without limit 3. Its daughters can remain a stem cell or differentiate ▯ ▯ ▯39 /18 University of Toronto - Revision Paper - BIO230 Marcus Lam Cell renewal can occur from division of differentiated cells E.g. liver cells and insulin-secreting cells of the pancreas ▯ The use of stem cells requires specific mechanisms. The fates of stem cell daughters can be controlled in two ways 1. DivisionalAsymmetry a. One daughter receives factors promoting ‘stemness’, and the other receives factors promoting differentiation b. Drawback: if stem cells are lost, their original numbers cannot be restored 2. EnvironmentalAsymmetry a. The cell division is symmetric and the daughters’fates are determined by the environment they are born in to b. If stem cells are lost, then their numbers can be increased by having both daughters of divisions enter the environment promoting ‘stemness’ ▯ Stem Cells Divide Slowly 1. This protects the stem cell from: a. Mutations associated with cell division b. Telomere depletion associated with cell division (telomere is a cap) 2. But large numbers of cells are needed to renew differentiated cell populations, this is solved by having transit amplifying cells expand cell numbers before final differentiation ▯ Skin stem cells and their progeny 1. The stem cells reside in the basal layer and require attachment to the basal lamina to remain as stem cells a. The basal lamina provides a niche for the stem cells 2. After detaching from the basal lamina, the cells differentiate through a linear sequence of cell types and are finally shed from the animal 3. Without renewal from stem cells, our skin would be lost in a month 4. The skin cells differentiation is linear while that in blood stem cells are more branched ▯ Blood stem cells and their progeny 1. Blood stem cells differentiate into various populations creating a branched pathway to final differentiation 2. Signals can promote specific branches depending on the need for cell types 3. Blood stem cells and their progeny reside in bone marrow 4. Identifying blood stem cells and their progeny experiment a. Homogenize mouse bone marrow to release single cells b. Expose cells to fluorescent antibodies recognizing specific cell surface molecules c. Isolate labeled cells by Fluorescence-Activated Cell Sorting (FACS) d. There are fluorescent probe in the antibodies, the cells having the fluorescence is negatively charged when being put under laser but cells that do not have the fluorescence will show positive charge e. It is to test the ability of isolated cells to restore all blood cells of an irradiated mouse f. 5 of bone marrow cells are sufficient for the complete restoration 5. Blood stem cells are maintained through interactions with stromal cells in the bone marrow a. Once the stem cells are detached from the stromal cells, they differentiate b. Stromal cells provide a niche for the blood stem cells ▯ Stem cells and their progeny must be regulated in many ways to supply the correct numbers of differentiated cells 1. Controllable parameters a. Frequency of stem-cell division ▯ ▯40 /18 University of Toronto - Revision Paper - BIO230 Marcus Lam b. Probability of stem-cell death c. Probability that stem-cell daughter will become a committed progenitor cell of the given type d. Division cycle time of committed progenitor cell e. Probability of progenitor-cell death f. Number of committed progenitor-cell divisions before terminal differentiation g. Lifetime of differentiated cells 2. Functions of stem cells and tissue renewal a. Affect how we age b. Can treat diseases and disabilities e.g. Parkinson’s disease 3. Medical uses for stem cells a. Using blood stem cells to treat leukemia by bone marrow transplant b. Problems of immune rejection i. Careful tissue matching and immunosuppressive drugs ii. If the cancer arises from a mutation in one of the progenitor populations then the patient’s own stem cells can be used after sorting c. Radiation to kill the cancerous or mutated stem cells together with other normal blood stem cells and allow the patient to regrow normal stem cells d. What if the tissue to be replaced doesn’t have a readily available supply of its own stem cells? (e.g. spinal cord injuries or neurodegenerative diseases) ▯ Can cells of a different tissue be used to make stem cells for treatment? 1. This occurs naturally during limb regeneration in newts a. Muscle cells de-differentiate, become mono-nucleated and start dividing b. Abud similar to the embryonic limb bud is formed from the cells c. Their progeny form all of the cell types needed to re-grow the limb 2. Current technology cannot do this from adult human cells at the scale or reliability needed for medical purposes, but is has been done in experiments a. This technique could avoid immune rejection by using the patient’s own cells, but cancer development is a potential problem if cell differentiation isn’t properly controlled i. Taking individual stem cell, which can divide without limit, and induce them to differentiate to different tissue. 3.1.1. What if their differentiation is not permanent or malfunction cancer ▯ Embryonic stem cells can proliferate indefinitely in culture and have full developmental potential 1. This can increase the yield of cells needed for treatments, but ethical issues, immune rejection and the potential of cancer are still concerns 2. Two potential ways to avoid immune rejection of embryonic stem cells: a. Somatic cell nuclear transfer: use a nucleus from one of the patient’s own cells and transfer it into an unfertilized egg to develop an embryo from which embryonic stem cells can be harvested i. Cells from adult tissue containing the genome to be cloned are added to a nucleus-less egg by cell fusion or nuclear injection ii. Allow time for cell division into an early embryo iii. There are 2 ways to handle the embryos 1. Cells from early embryo transferred to culture dish  therapeutic cloning 2. Embryo placed in foster mother  reproductive cloning iv. All the cells have the same genome as the patient do, so the body will regard those cells as self but not foreign and immune rejection is overcome b. Treat some of the patient’s own cells with factor known to specify embryonic stem cell character i. Treat adult cells with factors and they become embryonic cells ▯ ▯41 /18 University of Toronto - Revision Paper - BIO230 Marcus Lam ▯ ii. This will overcome ethical issue but the cancer issue is not yet solved Other tissues are between these extremes ▯ ▯ ▯ ▯42 /18 University of Toronto - Revision Paper - BIO230 Marcus Lam 2.7 Principles of Cellular Signaling ▯ ▯ Cellular Signaling 1. Cells must communicate to develop and maintain multicellular organisms 2. Unicellular bacterial-like organisms existed on earth for about 2.5 billion years before complex multicellular organisms arose 3. In part, this delay may have been due to the time needed to evolve complex signaling systems 4. However, unicellular organisms do communicate ▯ Examples of unicellular communication • Quorum sensing in bacteria: o Many bacteria release and respond to chemical signals o This signaling coordinates motility, antibiotic production, spore formation and sexual conjugation in bacterial populations • Mating in budding yeast o Signaling between yeast cells prepares the to mate Their morphology are changed when they detect signals • o Aggregation of ameboid cells o Signaling between Dictyostelium cells draws them together to form a fruiting body ▯ How do cells communicate to develop and maintain complex multicellular organisms? • Mammals, flies and worms use similar communication pathways • Many core pathway components were first discovered in mutants affecting cell communication in Drosophila, C.elegans and yeast • By genetic screening, it allows determination of genes that are important for development of embryo ▯ The basics of sending and receiving signals • Cells can send out hundreds of different types of signaling molecules o E.g. proteins, small peptides, amino acids, nucleotides, other small molecules, and dissolved gases o They are exocytosed, emitted by diffusion, or displayed on the cell surface • Cells receive signals in 2 main ways o It depends on the signaling molecules can pass across the membrane or not, which in turns on the hydrophobicity and hydrophilicity of the signaling molecules o Cell-surface receptors Intracellular receptors • Signaling occurs over short distances o Contact-dependent signaling: signals are retained on the cell surface o Paracrine signaling: signals are released from the cell but act locally  • Internalization by neighboring cells • Signal instability or destruction by extracellular enzymes • Binding to extracellular matrix molecules e.g. fibrous molecules • Signaling occurs over long distances (has long trajectory)
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