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Kenneth Yip

Lectures 1-3: Organizing the Cell and its Outside Interactions We're asking: How do cells and tissues organize themselves spatially? 1. Membrane Trafficking Required for: – cell-cell communication – Resource acquisition NB: Requires dynamic changes to the plasma membrane Basic Principles of the Biosynthetic-secratory and endocytic pathways: 1. The presence of polarized trafficking routes (travel from a place to another in the cell) 2. Sorting Station (similar to bus stations) - Sites of arrival events from many places with many materials - Multiple different departure events - Early endosomes: Multiple outputs (in from the plasma membrane, out to the lysosome, Golgi and ER) – How does a cell know where to go – i.eCisternae of the golgi (trans golgi network), secratory vesicles, and early endosomes (but not lysosomes) 3. Retrieval mechanisms are needed so there is a general balance between all of these routes - Material is sent from ER to Golgi but some need to be sent back (if not will deplete cells of materials) Ex. From ER to Cis Golgi network and endosomes comming into the cell from external env. Secratory pathways (out of cell) = Two types I. Consitutive Secratory Pathway: Continuous flow of material outside of the cell (no regulation at all). Will dock and fuse with plasma membrane II. Regulated Secratory Pathway: Starts in the same way, but changes. A vesicle buds off but waits for a signal (hormone or neurotransmitter or maybe even a ligand) until its told to bud with the plasma membrane and exit the cell. Alll vesicles are stored until a signal triggers docking and fusing of all (i.e. Allergies and histamine release). Provides extra plasma membreane when needed (i.e in the cases of Cleavafe furrow, phagocytosis, and wound repair). – Regulated secretion in terms of extracellular signals 1. Delivery of synaptic vesicle to component of plasma membrane 2. Endocytosis of synaptic vesicle compoments to form new synaptic vesicle directly 3. Endocytosis of synaptic vesicle components and delivery to endosome 4. Budding of synaptic vesicle from endosome 5. Loading of neurotransmitter into synaptic vesicle 6. Secretion of neurotransmitter by exocytosis in response to an action potential In both Pathways, the concentration of the secretory vesicle is due to the fact that after it buds, parts of the membrane with the clathrin coats break off and go back to the trans Golgi network thus concentrating the content of the vesicle Endocytic pathways counterbalance secretory pathways and perform specific functions Steps to endocytosis: Invagination and Fission ItStarts at early endosome - Endosome has choices to make on where contents go – Can either be destroyed by lysosome, can be recycled back into the basolateral domain of plasma membrane or leave through another part of the plasma membrane through transcytosis) Cells collect resources by endocytosis (ex. Cholesterol) - Bringing things into the cell - Needs cholesterol from fluids outside of cell - This cholesterol is brought in by the Low Desnsity Lipoprotein receptors (binds to cholesterol - cell has receptors that are TM proteins and recognize and grab LDL and drags it into an endocytic pit and is brought into the cell through endocytosis. - Now, coated vesicle, then targeted to go to sorting station. - Receptor is recycled (sent back to the membrane to be used again) - LDL is targeted to the lysosome, once targeted there, the lysosome (low pH and digestive effects) then release LDL as free cholesterol into the cell, used to build membranes – Want receptor to go to the membrane and not lysosome So LDL comes in and goes to the lysosome Cells down regulate cell surface signalling by endocytosis - Phagocytosis - Can control signaling with endocytosis - If it wants so shut signaling complex down it can just completely remove the signaling complex - When that happens, there are signal on outside of the cell, the membrane, receptors and and adaptor molecules and such on the inside of the protein. This way you can pull the whole complex into the cell - After endocytosis through specific sorting can be converted into a multivesicular body by pulling the whole membrane into the lumen of a larger vesicle (vesicles inside of vesicles) – This membrane composes the entire signaling complex, and can be sent to lysosome and fuses with it and shuts down the signaling complex Know how budding and invagination occur Also be careful of the wording BUDDING AWAY FROM CYTOSOL = ENDOCYTOSIS BUDDINGAWAY FROM THE LUMEN = EXOCYTOSIS Formation of Lysosome: Early endosome + ubiquitin --> Pinches off to form a multivesicular body --> Lysosomal lipases from late endosome/lysosome binds and releases contents making multivesicular body a lysosome – 1 v-SNARES (vesicles) and 3 t-SNARE (target membrane), proteins that pull two membranes together by coming closer and squeezing water out. Clathrin Drives vesicle invagination: – Cargo receptor in membrane attaches to aptor proteins, this complex binds to cargo and pulls into the structure, clathrin then binds atop all of that and helps bud the vesicle. – Clathrin triskelion is composd of 3 heavy chains and 3 light chains; form a cage-like structure – COPI and COPII drive invagination – Made of: selected membrane proteins, Sec23/24, Sar13/31 and Sar1-GTP – Sec23/24 binds to the selected membrane proteins, next to sec23/24, Sar1-GTP is bound the membrane. Sec13/31 binds to Sar1-GTP which then composes the outer coat. Dynamin drives fission after vesicle invagination (helps exclude water so membrane can bind with itself. ESCRT Complex Using clathrin to pull out of the cytosol by use of protein complexes and PI(3)P and PI(3,5)2 Viruses typically can use this Proteins Coating per section of network COPII only used in Cis Golgi Network (so from ER TO CGN) COPI for return from CGN to ER or to directly transport stuff out of cytosol from TGN Clathrin is for the TGN and and signals that vesicle go to an early endosome Be sure to understand the above content Phosphatidylinostiol (PI, with phophorylative modification it becomes PIP) can have modification at different positions of the inostiol -OH head group PIPs bind very specific proteins which then ensure specificity (ex. Different PIPs are used at different stages to control trafficking networks What posititions Phosphatases and Kianases that position the PIP Must know what GEFs do (they promoate exchange of GDP to GTP) and GAPs (GTP->GDP) Specific Rab small GTPases localize to distinct sites in the trafficking pathway – Rab11 = recycling endosome – Rab5A = Plasma membrane, clathrin-coated vesicles, early endosomes – Rab 7 = late endosomes - Rab5, its GEF exchanges GDP for GTP, gives it a lipid anchor (in inactive state tucked into the protein - due to conformational change) localizes it to target membrane - One of these downstream regulated molecules itself can be Rab-GEF which results in positive feedback loop – Can interact with PIPs and attract other proteins to the chain etc. – Also, specific Rab with a specific PIP will is a control mechanism Rab-GTP on vesicle (with v-SNARE also on vesicle) binds a Rab effector which is called tethering, and then it allows for the fusion of the vesicle and release of contents. This all works together in a combinatorial effect (specific rabs to SNARES) Organization of cell is based on the organization of the trafficking networks that run throughout the cell Lecture 2 Notes: Cortical Polarity, the Cytoskeleton and Cell Migration – The cytoskeleton is polarized (from the subunits to the protofilaments to the network even) – Each protofilament is made of heterodimers of the monomeric proteins a-Tubulin and B- Tubulin (tubulin monomers bind and hydrolyze GTP). – The heterodimers are asymmetric (thus, assembly head-to-tail results in polarized tubules) – y-Tubulin binds tubulin heterodimers assembling these protofilaments into tubes – The latter also nucleates microtubules at the (-) end – The (+) end grows away from the centriole (for example) – The Microfilaments are made up of a y-tubulin ring (makes a positively charged ring) which allows the a-B-tubulin heterodimers to associate via the (-) end allowing the (+) end to grow outwards – Microtubules are made up of a 13 dimer circular structure – Dynamic instability allows growth and shrinkage of the microtubule – When they grow they have a proactive growth ratio (the rate of growth is greater than the rate of loss) – If GTP hydrolysis (i.e. Rate of loss – bc hydrolysis occurs after the dimers bind)> subunit addition, then the y-tubulin ring (cap) is lost and depolymerization occurs (100x faster with exposed GDP end) – so... loss of cap = rapid shrinkage, regain of cap = rapid growth – We know microtubules are polar because they arrange themselves at the negative end and grow outwards (tested in artificial cell environment) – Leads us to believe that this property is a key element in cellular organization, but, in vivo there are many proteins associated with this – Basically the microtubule coordinate system is used to transport things like a ski lift (remember dynine and kinesin? Yeah they're back :) ) – Dynine (think of it as moving backwards): moves positive to negative end. – Kinesin (think of as moving forward): moves towards the positive end (kinesin is basically the important one we should know for vesicular transport) – Microtubules are also key in positioning the Golgi – nocodazole expoeriment: the chemical nocodizole can depolymerize microtubules – the experiment showed that when treated with the latter, the golgi quickly lost its positioning inside the cell. The motor protein responsible for this is dynine (because the golgi is towards the inside of the cell, next to the ER) – Agood example of motor proteins use is in theAfrican cichlid fish, which changes colour – In this model the two motor proteins compete for the melanosomes (gives fish pigment) and when camouflages, theres a decrease in cAMP which allows of dynine to outcompete kinesin (kinesin inhibited) and pulls melanosomes to center making the fish an albino – Cytoskeleton in cell division – Actin is mostly found around the perifery, they are dimers, and a have asymmetric monomers which give it polarity, they hydrolizeATP and are sort of similar to enzymes – After the dimers bind it hydrolyzes ATP (soATP hydrolysis entails that the dimer will be lost after this event) – So when changes ATP>ADP the subunits tend to fall off (ATP bound near plus end,ADP bound near minus end) – ie net assembly at plus end and net disassembly at negative end – ARP COMPLEX NUCLEATES ACTIN FILAMENTS – ARP complex made of two proteins:Arp 2 andArp 3 which form a dimer and act like a cap (similar to y-tubulin) – IT NUCLEATES AT THE MINUS END (soARP complex is positive in polarity) mimicking a positive-ended microotubule, – they can also form at 70 from each other – There can be treadmilling of networks of microfilaments (even separtate ones what attach at 70 ) – The networks drive polarized movement – Allow for traction, i.e. Treadmilling in spot (think of the belt wheel on a tank). Filaments need a stationary anchor (ie. It doesnt move while treadmilling but stays associated with the actin filament while it changes dimeric subunits) – Actin makes up the contractile ring that makes cells split during division Lecture 3: Structures of cells – Epithelial cells are a barrier from the rest of the worls – Apical = top ; Basal = Bottom (order from top to bottom = apical to basal) – Tight Junctions: Occlude/seal the cells not allowing the diffusion of large molecules – Adherins Junctions: cell-cell anchoring, connects acting filaments to keep cells together (like velcro) – Actin filaments make up the cadherins (associate via homophillic interactions) – Links cells together at the N-term of the actin filament. There is a hinge region which is needed to be flexible at first, but rigid when associated together to allow for tight binding, to do so Ca is added b.w the two cells to allow the flexible hinge to become rigid – Adherin junctions very important in the developing embryo, allows positioning of the cells – Important for tumor supression too – Desmosomes: Connects intermediate filaments with those of a neighbouring cell (anothe cell- cell anchoring) – The three above junctions make up the Junctional complex that seals the cell (adhesion belt) – Gap Junctions: allows passage of small H2O-soluble molecules to pass from cell to cell – Hemidesmosomes: Connect the cell to the extracellular matrix which makes up the basolateral side of the cells – they anchor the cell to the ECM – How glucose gets into the cell? Via Na/Glucose symporter in the apical domain of the intestinal villi, bring glucose in against its gradient by using the energy generated from the transport of Na into the cell – Glucose then moves to the basal domain where there are glucose carrier proteins which move it down it [gradient] to the basal lamina into the blood – How do the membrane proteins get there? Via vesicular transports to different aread of the cells, proteins produced and embedded int the membrane and transported to various areas in the cell which then become a part of the membrane when the vesicle fuses – If dye added under tight junction, you can see it go right up to the apical domain but it wont permeate through – Tight juncrions formed from strands of interacting TM proteins – These TM proteins would be like claudin and occludin which sort of sew the cells together (tight junctions can go b.w the leaflets of the cell) – Claudin: 4-pass TM proteins that is essential to the proper formation of tight junctions – Occludin: 4-pass TM receptor which is essential to barrier function of cell, but not needed for maintaining structure of junction – Claudin+occludin are both homophillic (will only bind with themselves) – Tight junctions = Gates to prevent molecular movement across epithelial sheets in the extracellular space between the cell. ; fences Cell Polarity – Cells use landmarks – In the nematode the negative end is denoted by where the sperm enters the egg (posterior side) – So anterior and posterior cues allow for cell polarity and movement of proteins and cells to either ends of the zygote – Contacts bw the cell protrusions from landmarks forAJ assembly bw mammalian cells – diffuseAJ components (everything all over the place) with the addition of the actin protrusions (They make the hole where the two cells are touching each other) which make theAJ Puncta (where theAJ will be – where the two cells are touching) which then modifies the actin structure and makes continuous Ajs (this pocess is a mesenchymal-to-epithelial transition) – Kind of like two blind- mute guys trying to find eachother in a room) – Structure of Ajs in order: Cadherin –Arm – alpha-cat – F-Actin (F-actin is slightly below the apical domain, with a small end of it in peering into the nasolateral domain). – Crumbs Complex, Par3-Par6-aPKC complex and Scribble complexes determine the polarity of the apical domains of the cells, sending inhibitory signals to the baolateral proteins Lecture 4-6: Multicellular Development (Tissue Morphogenesis, Tissue Patterning, and Stem Cells): We're asking: How do multicellular organisms develop? Lectures 4&5: Multicellular developmeng Morphogenesis needs: Internalization of Cells; Elongation of the Embryo; and Fine Repositiong of Cells – Requires Morphogenesis and cell differentiation – Cell proliferation = making more cells, cell specialization = different cell in groups of others – Cell interaction = cell change due to interactions with environment or other cells cell movement= cell movement – In adults multicellular development occurs from stem cells (eg. Skin, or in the lumen of the gut) – The skin (of a mouse) has different layers, Demis and epidermis. – The basal cells are the ones that divide – Cells in the mamaries will priduce milk – Embryogenesis is the best studied form of development (similar mechanisms of morphogensis and cell differentiation) – Polar body = where egg is formed, will be degraded – Blastocyst still has the zone pellucida – trophectoderms will form other tissues like placenta – Inner cell mass = chald – By 2.5 days there should tight junctions connecting all the cells – Gastrulation: Formation of the inner gut Mechanisms of Cell Internalization – 1 Ingression/Delamination – Ingression = Moving inwards – Delamination = Splitting or separating into layes – idividual cells separate from the early outer epithelium – Epithelial-to-mesenchymal transition = Were epithelial cells but are now cells that make up the inner body (cells from outside migrate inside) – The latter is dangerous in terms of metastasis – Moving divinding somites in the quail embryo to a region in the chick embryo will result in the quails cell formation in the chick body (think Shh) – highly regulated – 2 Invagination/Involution – Invagination: the formation of an inner layer – Microtubules elongate causing cels to become columnar; actin and myosin arrange around the area and begin contracting causing involution and making a hole, or invaginating the area (considered both invagination or involution) – Involution = the ingrowth and curling inwards of a group of cell, as in the formation of gastrula from a blastula – Eg. Mesoderm internalization in the drosolphila initiated by transcription factor Twist which moves a sheet of cells into the inner cell mass Elongation of the Embryo 1. Convergent Extention – Thinness or thickness of cell – Cells (lamelopodela – cells that can crawl) move inwards to stack onto eachother through the movement inwards forcing the other cells to line up (horizontally) onto eachother, thus elongating at the end. (cell movement is regulated) 2. Cell Division and Cell Shape – Cells divide at tip resulting in elongation (ie. In arabidopsis) – Orientation of elongation is regulated by the orientation of cellulose microfibrils and driven by tugor pressure – Cellulose microfibril depends on the direction of the shoots – When extending a hormone causes the extention of the cellulomicrofibrils (i.e can grow taller or fatter) – ex. from the crypt of the villus with the two stem cells which continuously divde to replenish cells (moves the cells upwards) Fine Repositioning of Cells 1. Cell Sorting – Take frog embryo, shake it up, cells will reassociate in the right order – this is mainly due to the reactivity of cadherin (or lack thereof) to other types of cadherin – Since they are only homophilic in association , it allows for the cells of the same type/class which are essential to development to form together 2. Directed Cell Migrtion – When progenerator cells divide and move up a glial cell to position themselves along the axon. – First born cells will be on the lower reaches of the axon, and later formed ones will be near the top. – Allows for the type of cirle, tunnel we see when looking at neurons – can also direct using hormones – They use red and green dyes to see how far the cells in the eyes travel long the optic nerve – Roof plate = dorsal end of the organism, floor plate = ventral – Neuron spinal cord (going up ventral plate cause our spinal cord is on the inner side of our backs) makes a right angle turn at the floor plate and goes to the brain – Agrowth cone expressing netrin (the commisual neuron) approaching the floor plate (where the attractant netrin is secreted) – so netrin pulls the neuron towards the floor plate and the wall of the neural tube. – Growthcone expressing ROUNDABOUT which repels slit and downregulates netrin (doesnt let it hit the walls) – Neuron the secretes Slit (a repellant) and semaphorim (another repellant that causes the 90 o angular shift. – Slit and semaphorin repel the neuron as to not come into direct contact with the wall of the neural tube and the floor plate. Lecture 5 – Mechanism of Cell Differentiation Two main mechanisms to cell differentiation: 1. Asymmetric Division: Proteins move to opposite ends of cell, two cells are born different (stem cell and differentiated cell) 2. Symmetric Division: Sister cells are born the same but external influences cause them to change/differentiate over time – Asymmetric Cell Division – Differentiation in C. elegans - Lineage tree – origins of different cells – a lot are asymmetric – all from a single clone or egg – lots of asymmetric cell divisions from a single cell to make up the cells that make up the gut – Asymmetric cell division partitions the cell fate determinants to define the germ line and other tissues – Requires polarity at the periferi of the cell and proper spindle alignment Extrinsic Mechanisms of Cell Differentiation 1. Direct Lateral Inhibition: A pattern of differentiated cells – All the cells begin equal but some randomly gain an advantage and then they differentiate and inhibit their neighbouring cells from doing so – Example: Notch Signalling – When delta ligand binds notch, notch sends and inhibitory signal to the production of its own ligand delta (Delta from one cell stops delta from being produced in another cell) – This is competition between the two cells – When one cell is going to be differentiated its because its Notch receptor no longer functions and thus the neighbouring cells delta ligand has no effect on the cell – this allows the cell (that has the non-functioning notch receptor) to differentiate/specialize - cell at bottom has delta which activated the notch receptor on the top cell. - When notch is activated it stops this cell from specializing and it inhibits the expression of delta - top cell already had some delta and that will have the same effect on the bottom - it will activate notch on the bottom cell which stop cell specialization and suppression of the expression of delta - Has lots of delta, will activate a lot of notch on the top cell - very powerful notch, and will stop cell from producing delta (top has no delta, or little delta) - inactive notch means it cannot stop cell specialization (cell differentiates) 2. Induction by Diffusable Signals: Signal is like a wave, it only has energy for a certain time and distance before it disappears – Cells start with equal potential but a signal is sent which can only travel a certain distance – This created a pattern of bands around the cells which are the center of the signal – Morphogens: Secreted inductive moleculs that speads in a gradient; [Morphogen] dictated the cell fate; high, medium, or low morphogen levels can induce different cell differentiation programs – Organizer Tissue is a type of morphogen: When transplant of dorsal tissue from one embryo to the ventral side of another will result in a duplicate embryo with two dorsal sides and a shared ventral side (shakerspeares poetic “beast with two backs” LOL) – The transplanted tissue will generate tissue that it would not normally make – Need to move ORGANIZER TISSUE, not the cells only – The reverse would be moving ventral tissue to the dorsal side?Aduplicate with two ventral sides – Chick Wing Experiment: Took polarizing tissue from where the wing buds during embryogenesis and transplanted it slightly above it – Due to the secretion of a morphogen known as Shh (sonic the hedgehog) it forced the chick to develop with 4 digits compared to 2 (in regular chick wings there are 2 digits – big and small), 2small and 2large; the small one have lower morphogen concentrarion, which is why they're small. – So. the Shh gradient controls the formation of digits 3. Regulatory Hierarchies – One cell becomes 2 different cells, 2 different cells become 3, 3 become 5 and so on – Main idea: Cells can divide and produce different morphogens which cause each cell to differentiate differently when the time is right creating many cells types from a single daughter – These hierarchies refine the patterns of cell differentiation to create a segmental pattern (think drosophila development) – Transcription factors influence other transcription factors which result in development of segmental patterns – Egg Polarity Genes: Set the regulatory mechanisms in order – Gap Genes:Allows the formation of gaps between the abdominal segments of the fly – mutation of Kruppel will make the embryo develop without any gaps in it and only forms the Thoracic parts of the body: Two Genes that regulate – Kruppel and Hunchback – Segement-Polarity Genes:Allows for the formation of spaces between the vertebrae of the fly (Wnt). Two genes that regulate – Even-Skipped (Eve) and Ftz – Segmentation – essential to body plans – Genes are literally in linear fashion from head to tail – Hox (Homeotic Selector) Genes specify which body parts to decelop from each segment – i.e the antennapedia gene (Antp) – order in the chromosome is the expression order in embryo – Like an organizational system which relies on gene duplication. So if one copy is mutated it is possible for the subsiquent ones to acquire mutations form it as well – Hox genes are highly conserved – We start the same but the TF with different targets in differentated species (thats why we dont have wings and flies do) – In two species two genes are related, but different, and also differently expressed – do so using silensor protein (sit on the DNAand don't allow the TF's to read the DNAseq.) Related species - Regulatory modules: seq. of DNA with enhancers or silencers - green protein goes to red seq. to produce gene 3 proteins – It is the changes in the regularoty hierarchies which change the gene expression and differentiate different species (why drosophila has halteres and not wings on its second set) – So the mechanisms require 1. Intrinsic Change –Asymmetric Cell Division and 2. Extrinsic Signaling – Lateral inhibition; induction by diffusable signals and regulaotry hierarchies Lecture 6 – Cell Death and Renewal Some Tissues contain the same cell for the entire life of the organism, but the molecular components do turnover – Organ of Corti, the inner ear of mammals - movment of fluid makes what we call sound - supporting cells are epithelial cells - tectoral membrane is a huge mass of extracellular matrix - Inner hair cells, detect sound and send it to the brain - stereocilia - specialized cilia – This architecture allows us to convert sound waves into nerve impulses Single cell - many stereocilia sticking out - when its shifted, it causes stereocilia to tilt - has two channels that are linked together by a tether - stereocilia will tilt, the tether pries open the channel opens and allows ions in, initiating a cell impulse – Sound waves cause stereocilia on hair cells to tilt, allowing teathers connecting two stereocilia (a bundle) to tilt causing the opening of channel on the second one which then initates the nerve impulse – this movement is due to the basiliar membrane shifting causing the stereocilia to tilt (as it is attached to tectorial membrane that doesnt move) – so the vibrations cause it move – these cells do not regrow – Even though the hair cells and stereocilia are very stable, they are dynamic on a molecular scale – Stereocilia filled with actin (which gives it shape), which is polarized and exhibits lots of treadmilling – Another example of this is the human photoreceptor - rods have rhodopsin (detects low levels of light) - Cones, opsin (for colour and fine detail) - goes to the back of the eye, to the cones and rods, then forward to the interneurons, then to th front where the ganglion cells are and back to the brain – Opsin or rhodopsin make up the disc of the photorecetive membrane (which is part of the cone/rod) – In a pulse-chase experiment of the cones/rods, its detection will gradually be lost. – Cellular turnover example: Osteoclast (bone degredation and Ca uptake); osteoblast: makes new bone from Ca in blood; Osteocyte: Maintenance of bone cell Other Tissues Renew Their Cells Rapidly – Definition of “Stem Cell”: 1. It is not TERMINALLY differentiated; 2. Can divide without limit; 3. Daughters can remain as stem cells are differentiate – Cell renewal can also occur from division of differentated cells (like liver cells and pancreatic cells) – The use of the stem cells require specific mechanisms: 1. DivisionalAsymmetry – One daughter recieves factors the promote stemness and other recieves factors which influence differentiation – If stem cells are lost their origial numbers cannot be restored 2. EnvireonmentalAsymmetry – Cell division is symmetruc and the daughters cell fates are determined by the environment they are born into (like tofu) – If stem cells are lost, their number can regernate due to the fact that both daughter enter the environment promoting stemness Slow Division of Stem Cells Protects the cells from: 1. MutationsAssociated with Cell Division 2. Telomere DepletionAssociated with Cell Division Large number of stem cells needed to renew differentiated cell populations TransitAmplifying Cells: Build up cell numbers before cell differentiation (makes daughter stem cells for a number of divisions and then they differentiate via environmental signals) Skin Stem Cells: Basal Lamina provides a niche for stem cells Blood Stem Cells: Differentiate into various populations creating a branched pathway to final differentiation; certain signals can promote specific branches (i.e. Neutrophils, T cells, RBC etc) of needed cell types. The stem cells reside in the Marrow. – There are blood cell precursors in bone marrow – If all blood stem cells were killed by X-irradiation, would new bone marrow restore them?A: YES – Did the experiment on a rat, RBCs turned over and generated a steady supply of new RBS Identifying Blood Stem Cells and their Progeny: 1. Homogenize mouse bone marrow to release single cells; 2. Expose cells to fluorescent antibodies recognizing cell surface molecules; 3. Isolate labeled cells by FACS (fluorescence- activated cell sorting). FACS: ultrasonic nozzle vibrator, cell suspension, sheath fiels. Drops of the sheath fluids and cells will be detected and analyzed by a computer and small groups of cells will be negatively charged due to detection of single fluorescent cells, then they are separated into two test tubes, while the remaining solution will collected in a flask. The two TT are then tested to see if they can regenerate RBCs – Blood Stem cells are maintained by interactions with stromal cells, while the Kit receptor in the stem cell binds to the Kit ligand on the stromal cell, it can divide, then the resulting daughter cell will have to differentiate or die Stem Cells Must be Regulated to Supply the Correct Number of Differentiated Cells 1. Frequency of Stem cell division 2. Probability of stem cell death 3. Probablity that the stem cell daughter will become a committed progenerator cell (not a terminally differentiated cell, but one that gives rise to a specific cell type) – must eventually differentiate or die but can divide for sometime before it dies. 4. Division cycle time of committed progenerator cells 5. Probablity of progenerator cell death 6. Number of committed progenerator cell divisions before terminal differentiation 7. Lifetime of differentiated cells Medical Uses of Stem Cells – Leukemia Treatment – Drawbacks: possinbility of rejection; must match specific tissue type and immunosupressive drugs – If cancer arises from a mutation in one of the progenerator populations then the patients own stem cells can be used after sorting – What about the spinal cord/neurodegenerative diseases? Cells from other tissues can be used as stem cells to help regenerate those parts of the body (i.e. Frog leg amputation, or shaving off part of your rib to use as a graft – both will grow back quickly) – Doing this could bypass the immune rejection (these are known as iPs cells) – Other examples are embryonic stem cells that can proliferate indefinately in culture (need to remove the inner cell mass from the blastocyst). – The latter is not able to be done because some religious zealots think that 32 cells= a human (10^14 cells) – Ways to bypass idiots: Take an egg, remove the genetic material, add cells from adult containing the genome to be cloned, allow for some cell division, when blastocyst forms implant it into a faster mother (this is called reproductive cloning) or grow cells in culture (theraputic cloning) 1. 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 ES cells can be harvested 2. Treat some of the patient’s own cells with factor known to specify ES cell character (e.g. a combination of Oct3/4, Sox2, Myc and Klf4 can convert differentiated cells into cells with ES cell characteristics) Lectures 7-9: Cell Signalling Lecture 7: Principles of Cellular Signalling – unicellular bacteria on earth for 2.5 billion years before multicellular came along, thought to be time delay used to evolutionarily develop these mechanisms – but unicellular organisms do communicate! – Ex. Quorum Sensing in Bacteria: The chemical signal coordinates motility, antibiotic production, spore formation and sexual conjugation – Ex. Mating in Budding Yeast: Signalling between yeast cells to make prepare for mating – Ex.Aggregation ofAmeboid Cells: Signalling between Dictyostelium cells draw them togeth
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