Entire BME 50B Lecture Notes

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Biomedical Engineering
Y U Liu

BME 50B 1 Vesicle signaling: protein localization and secretary pathways • • amino acids know where to go in a cell based on their signaling sequence o • the process of secretion/transporting transmembrane proteins to the cell membrane o ribosomes are chilling in the cytosol, then latches onto an mRNA o if the synthesized protein (aka polypetide chain) has a signaling sequence, the ribosome + mRNA + polypetide will go to its place o we will focus on the signaling sequence bringing it to the ER BME 50B 2 o  SRP finds signal sequence and latches  SRP finds the receptor on the ER membrane and latches  this process feeds the signal sequence through the translocation channel in the ER membrane  the SPR leaves to find its next victim  the polypetide chain can grow into 2 different types of protiens o  secretion proteins are fed by the ribosome into the ER lumen  once the ribosome feeds the whole protein through the membrane, it leaves to find its next victim  a signal peptidase dislocates the secretion protein from its signal sequence, which is forever stuck in the ER membrane BME 50B 3 o  transmembrane proteins differ from secretion because they get stuck into the membrane of the ER by a start/stop hydrophic region of amino acids  act as receptors etc.   BME 50B 4 o  these guys can pass many more times then this with start/stop signals which are hydrophobic and want to be inside the membrane BME 50B 5 o • the vesicle budding/fusion process o COP and Clathrin are 2 coats use to bud: one between the ER and Golgi, the other between the Golgi and Cell Membrane BME 50B 6 o  first, the formation of transport vesicles (from ER to golgi) use COP coat  adaptin finds the cargo receptors on the ER membrane's outsides  these adaptin have the COP attached to it  once enough COP surrounds a vesicle, dynamin cuts the stretched membrane from the ER  the adaptin + COP detaches from the vesicle to allow it to go find the Golgi (transport vesicles)  clatherin acts the same way, but removes the buds from the Golgi and to be transported to the membrane (secretory vesicles) o BME 50B 7 o • why it goes from ER to Golgi o ER  disulfide bonds  gylcosylation  quality control o Golgi  modification of sugars  aka can add mass  sorting • secretory pathways o constitutive  happens all the time, no regulation o regulated  will bud off the golgi like normal, but will be stopped before reaching the membrane  will only go if the signal reaches it  if no signal goes off, the secretory vesicles can store up and get rather big BME 50B 8 o • endocytosis o opposite of secretion o • figuring this stuff out o temperature sensitive mutants BME 50B 9  all these pathways are needed for a cell to function, so if they are created with a mutation, then there will be no cell to test on  so, the cell proceeds like normal at 25 C, but once they are heated to 35 C, protein bonds in specific places break  we can then see what breaks in the cell and can blame it on the mutant o Green florescent Protein (GFP)  a protein found in jelly fish that, if placed on the end of a protein in the cell, do not affect its function  glow under a UV light so that we can watch were stuff goes • How can you block secretion? o kill Rab and SNARE  but that will overall kill the cell o Killing COP, however, will allow everything else to work find BME 50B 10 Electric Potential signaling • membrane transporters o things that can/cannot pass through lipid bilayers o  o membrane transporters allow things to pass through a lipid bilayer which could not otherwise  o protein orientation  transporters need a hydrophobic side outside (to fit inside the lipid bilayer)  therefore, the proteins orient themselves inside the cell membrane in a alpha helix BME 50B 11   polypetide backbone of a protein is polar and hydrophilic  it gets hidden in hydrophobic amino acid side chains  in this way, multiple alpha helixs' can makeup a pore or channel  • understanding diffusion o concentration gradient: diffusion  things in high concentration have a 'force' exerted on them to move to low concentration  happens because of Browian motion (thermal energy in liquids make things randomly bounce everywhere)  diffusion constant (D)  a large D means that the substance will diffuse faster, and therefore have a bigger 'force'   BME 50B 12   Fick's law   a large concentration difference and a large difficusion constant will cause a big flux to occur (large 'force')  ion gradient: a comparison between ions inside and outside the cells of mammals   when something has a large gradient, then a 'force' will try to restore it back to being 50/50. bigger gradient, bigger 'force' • leak channel o leak channels will only let out and in their designated ion (for our example, we use a potassium leak channel) o 'forces'  concentration gradient - diffusion 'force' BME 50B 13   'pushes' potassium out  this happens because for every about 30 times a potassium hits the channel from the inside, maybe 1 hits the channel from the outside   electric field - drift force   potassium ion causes excess positive charge to accumulate on the outside of the cell membrane  this causes a type of capacitor to occur, which creates an E force pushing against the diffusion 'force'  o potential equilibrium BME 50B 14   happens when Jdiff = Jdrift ~ -60, -70 mV   change of potential = [(Diffusion constant)/(mobility)]*ln[(concentration of the outside)/(concentration of the inside)]  plugging in some numbers:  • pump o specific pumps, takes only a certain 2 molecules (for our example, we will use potassium and sodium) BME 50B 15 o energy burned (ATP) with a pump to go against the concentration gradient  o  • voltage gated channels o the channel proteins shift shape depending on voltage BME 50B 16 o o there is also a voltage gated K+ channel, which opens at +50mV, and is what causes the drop in membrane potential o the 3 stages  in closed state, the channel will open once it reaches the threshhold  the channel will then become inactivated at the peak of the action potential  it is important that it becomes inactivated instead of closed otherwise it would just fire again and be stuck at the peak  the protien then goes back to closed after the membrane potential drops below -60 again/at a certain time  repeats • Action potential o caused mainly by these 3 channels and 1 pump o BME 50B 17 o • How they figured this stuff out o 1) they took a squid axon (which is large), squidgied all the insides out of it, refilled the insides and outsides, and tested the potential using electrods   findings   less outside concentration ='s smaller gradient ='s smaller force driving Na into the cells  hence, the slope is less steep  also, the action potential peak is much lower  delta V = 62*log(Ci/Co), where Ci is lower (initial concentration) so delta V is lower BME 50B 18 o 2) Patch clamping: allows us to study the electrical measurements of individual ion channels o • Action potential significance o allows signal to go from the cell body, down the axon, to the axon's terminal branches, then interact with other cells to tell them what to do o • How? o one action potential kicks off the next and they travel down the axon o  BME 50B 19  the action potential only propagates in one direction because of the inactivated state of the Na+ channels  otherwise it would oscillate o Myelin Sheath  makes this process faster  the faster this happen the quicker your reflexes etc...so its good   How?  acts as a capacitor  low capacitance, things respond fast  a fat Myelin sheath = 's a lower capacitance  a low capacitance creates a faster respond time  it does this by making the action potential only happen in the Node of Ranvien instead of across the whole axon   also, the Myelin sheath acts as a capacitor = 's a lower capacitance  a fat Myelin Sheath means its capacitance is low  at low capacitance, things respond faster • Once the action potential gets to the end of the axon o it moves down the nerve terminals until it hits something (like a muscle) o BME 50B 20  o at the end of the nerve terminal: specifically a nerve/muscle junction  1) the action potential will open up a  2) Ca++ channel, allowing Ca to rush in (10k times more calcium on the outside of the cell compared to the inside)  3) Ca++ activates the enzyme Kinase  4) Kinase sticks a phosphorous group to synapsin to create a phosphoralated synapsin  synapsin's job is to hold acetylcholine (ACh) vesicles back from fusing with the cell membrane  5) ACh vesicles are released to fuse with the cell membrane because of the phosphoralation of synapsis and ACh is realeased into the synaptic cleft  6) ACh binds and opens ACh gated (excitatory) Na+ channels on the muscle cells, which allows Na+ to rush into the cell  7) an action potential in the muscle cell then is fired off  8) the action potential opens a voltage gated Ca++ channel  9) the Ca++ that just rushed into the muscle cell causes muscle contraction  10) outside of the cell, ACh is degraded by acetylcholine esterase (AChE), allowing the muscle to relax BME 50B 21   all these steps seem silly to get one reaction, but this happens everywhere in the cell pretty much BME 50B 22   advantage of using chemical signaling at a synapse rather than electrical connection  a cell is able to integrate the inputs of a bunch of other cells this way (inhibitory, excitatory, ect.) and can figure out what to do from there  if it was electrically connected, then only that one input matters and it can't know what is happening in other regions of the body • 2 types of synapse ion channels: excitatory and inhibitory o they regulate how far or close a cell is to the threshold to fire o BME 50B 23 • the first firing of a neuron cell: caused by dendrites o tons of inputs go onto one dendrite, and tons of dendrites go onto one cell neuron o dendrites don't have action potentials (no voltage gated Na+ channels)  therefore, in order to talk to the neuron, a dendrite is either + or - depending on its own inputs o the axon hillock integrates all these inputs from the dendrites  enough excitation (excitatory signals) from the dendrites causes the axon hillock to fire an action potential o • reflexes o a sensory neuron kicks off the action potential in motor neurons o this does not require the brain o shown is a knee when you smack it o • Long Term Potentiation: Memory and Learning o we know that an input into a neuron results in an output BME 50B 24  o It was found, though, that after repeating the same input for a long time, the output increased with the same strength of input   the rise is called long term potentiation o this happens because of an increased signal transmission at that particular synapse  causes more neurotransmitters to appear on the cell membrane  o types of inputs to strengthen memory/learning  before: the result after one or two inputs of that same thing  after: the result after that same input has been stimulated many times  Cooperativity  a weak input won't produce a great output after continued stimulation by itself, but when coupled by more weak inputs will create a strong input, which causes long term potentiation   Input specificity  what we don't want strengthened can and should stay inactive so that our other inputs can become more active and long term potentiate BME 50B 25   Associativity  sometimes a strong signal can strengthen a weak signal  • BME 50B 26 Engery Generation • ATP o a storage place for energy  it stores the energy in a chemical bond o ATP provides energy when a phosphoanhydride bond is broken  ATP --> ADP o o each cell has about 1 billion ATP which get used every 1~2 minutes • mitochondria o energy factory of a cell  the placement and number of mitochondria a cell has depends on the type  heart, liver, and nerve cells require alot of energy, and therefore have alot of mitochondria o structure   outer membrane has a lot of porin which allow a lot of small molecules to flow through freely  ATP for example can freely pass through the outer membrane BME 50B 27  the inner membrane  has ATP synthase  high surface area allows more ATP synthase to be present, which makes the yield of ATP higher  establishing the gradient in the intermembrane space  the the electron transport chain is responsible through oxidative phosphorylation   this oxidation/reduction reaction drives this transport:   oxidation: loss of e-, reduction: gain of e-  the H+ gradient is more electrical then chemical  • overview on how a cell obtains energy (ATP) from food BME 50B 28 o o • 1st step: Glycolysis o the breakdown of glucose into two molecules of pyruvate o the process takes 2 and produces 4 ATP (net 2), and produces 4 NADH o produces 2 pyruvate BME 50B 29 o • 2nd step: pyruvate oxidizes to acetyl CoA • 3rd step: citric acid cycle o each cycles produces 3 NADH, one GTP, one FADH2, and 2 CO2 o • 4th step: ATP synthase uses the H+ gradient to convert ADP to ATP BME 50B 30 o Chemiosmosis - the harnessing of energy formed by chemical gradients across the membrane to form chemical bonds o o  Protons pass through channel within stationery portion of carrier (light green) causing the rotation of the stalk (red)  Rotation of stalk causes conformational changes in the head proteins (darker green), which causes bond formation between ADP and Pi o Complex can also work in reverse, using ATP to pump protons across in the reverse direction. o Electrochemical energy --> Mechanical energy --> Chemical (bond) energy • 5th step: ATP escapes the mitochondra to go help the rest of the cell o Voltage gradient drives the pumping of ATP out of mitochondrial matrix and ADP into mitochondrial matrix BME 50B 31 o o once out of the matrix, ATP is free to go through the outer membrane of the mitochondria • experiment to confirm the chemiosmotic hyothesis • o • overview BME 50B 32 o o BME 50B 33 Cell Growth • cell cycle overview o Interphase (when the cell isnt dividing)  G1 phase  S phase (synthesis)  DNA replication  G2 o M phase  mitosis, the division of the cell o • M Phase: 6 steps of cell division o Prophase: the cell prepares  inside the nucleus  DNA inside the nucleus has already been replicated  a protein comes and loops the DNA super close together to make a chromosome    the chromosomes are identical strands held together by a kinetochore  outside the nucleus - mitotic spindle  centrosomes being migrating to the polls of the cell  cell microtubules begin organizing in this way, forming the mitotic spindle  BME 50B 34  the mitotic spindle pulls apart chromosomes  o Prometaphase: the cell pulls  the nuclear envelope breaks  the mitotic spindle is much more organized  kinetochores of chromosomes attatch to the mitotic spindle  this phase takes the longest (about 3 out of the 6 hrs it takes for the division of this particular cell)  o Metaphase: the chromosomes organize  happens when the prometaphase yields a perfect line-up of the chromosomes at that equator of the mitotic spindle  there are many 'checks' that happen before the next phase so that one cell doesn't get over/under its share of chromosomes  BME 50B 35 o Anaphase: the chromosomes tear apart  happens relatively quick  the chromosomes get pulled apart by motor proteins moving along the mitotic spindle  the spindle stays put  o Telophase: the cells start seperating  o Cytokinesis: the cells are ready to be on their own  BME 50B 36 • Interphase: chill time between dividing o the cell grows  everything must be duplicated EXACTLY (not 3x, not 1.5x, exactly 2x)  how the cell does this is not quite understood • regulating cell mitosis o the faster the cell cycle happens, the more growth, so something must regulate its growth o  in places that need rapid growth (a wound, lining of intenstines), the cell cycle needs to happen faster  in other places, the rapid growth is not needed o a protein called M-Cdk (mitosis - Cyclin dependent kinase) is the 'switch'  switches on during mitosis, off during interphase   Cdk needs M-cyclin to do its job (Cdk is always there, its the cyclin that are changing concentration)  the cell can only make cyclin so fast  once it hits the certain concentration, Cdk can pair up with it to do its job   at the end of mitosis, the cyclins are destroyed by ubiquityation   once M-Cdk is activvated by cyclin  it can runs around and phosphoraltes (activates) the proteins that  group the DNA into chromosomes BME 50B 37  breaks the nuclear envelope  induces microtubules to form mitotic spindles  kinase review  kinase uses an ATP to add a phosphoric group to a protein  protein phosphatase removes the phosphoric group    whether kinase turns the protein to be phosphorylated on or off depends on that specific protien   Cdk is the first (off w/out P, on with it) o it happens in the cytoplasm  • shifting between the other phases o also controlled by cyclin BME 50B 38  o they can stop shifting through phases (growing) by withdrawing to G0 phase during G1 phase  o checkpoints  • S phase: DNA replication o prior to DNA replication  P53 checks for DNA damage BME 50B 39   if there is damage, the S phase is pushed back  if the damage is beyond repair, the cell self destructs o if everything if fine, S phase can start: o  • How long is each phase? BME 50B 40 o although the internal clocks keep cells in the same phase with the cells around them during their early life, they end up out of phase o measure how much DNA is in each cell with flow cytometer  cell DNA is stained with fluorescents  cells drop one by one between a light source and a microscope that measures the DNA light    each cells had a different amount of DNA inside them depending on their phase  double for G2, and the beginning of M  half for the end of end of M and G1  normal for S (because the DNA is being replicated, but is not yet double) o • synchronizing a population o the drug colcemide blocks microtubule spindle forming  doesn't let cells go past the M phase  BME 50B 41 o when colcemide wears off, the cells will be in phase again, for a time o once a population is synchronized, we can measure the amount of cells in the S phase  tag thymine (DNA component) with H3 mutant  meausre when that tag ... INCOMPLETE LECTURE 8 LAST SLIDE  • the decision to grow o a cell spits out growth factors and is received by the receptors of other cells o some cells also spit out inhibitory growth factors o during the G1 phase, each cell integrates all the information it is recieving  the G1 phase ends when the integration reaches the conclusion (checkpoint): divide  o types of growth factors  EGF - epidermal growth factor  NGF - nerve growth factor  PDGF - platelet-derived growth factor o types of growth inhibitory factors  TGF - B o in the lab  culture cells in liquid medium  they don't multiple, just move around a bit  add serum to the culture  they start the cycle and multiple  serum is from a blood clot  when blood coagulates, platelets stick together BME 50B 42  the platelets then burst open and release PDGF, which tells the skin cells to grow • in depth signaling o Tyrosine Kinase Receptor  o Ras signaling - tells the cell to go from G1 to S phase (DNA replication)  ligand attaches to receptor  receptors dimerize  receptors cross phosphoralate  adapter protein docking  Ras-activating protein attaches to adapter  signaling protein causes Ras protein to swap its GDP (inactive) for a GTP (active)   Ras deactivates itself by self hydrolyzing the GTP to GDP  the turned on Ras causes a signaling cascade  MAP kinase kinase kinase to use an ATP to phosphoralate MAP kinase kinase which uses an ATP to phosphoralate MAP kinase which phosphoralates other protiens BME 50B 43   the phosphoralated transcription regulated protein (Rb) goes from its active to inactive state  inactive Rb causes an active transcription regulator starts transcription  the transcription regulator starts transcription  • Classes of cell-surface receptors BME 50B 44 o • types of cell-cell signaling o endocrine  secretes into the blood stream to hit receptors  long range o paracrine  a cell will say something to a cell next to it which will react and carry the message  short range o neuronal  tells target cell to activate or deactivate  very specific, short range o contact-dependent (aka jextacrine)  instead of the growth factor being secreted, the ligand is expressed on a protein that is transmembrane  only signals to cells that are touching it, short range BME 50B 45 o • tumors and cancer: o something is wrong with the growth factor  autocrine signaling  the cell tells itself to grow   overexpressed receptor  the receptors are so dense that they don't need the GF to dimerize  auto phosphorylation   constitutive signaling BME 50B 46  truncated EGF receptor doesn't need to dimerize (always active)  o tumor transformation  monoclonal  the tumor originates from one cell that doesn't know how to stop growing  usually cells stop proliferating when they touch  contact inhibition  anchorage dependent (want to stick to the bottom of the dish)  transformed cells don't care  lose their contact inhibition  lose their anchorage dependence  change shape  don't need growth factor  o chemical carcinogens  recent studies suggest carcinogens are mutagenic   somatic mutation  means they aren't necessarily passed through heredity, but rather circumstance  soma - everywhere in the body except the gonads  (germline mutations are in the eggs or the sperm that get passed to the kid) o early studies: Rous Sarcoma Virus (RSV)  most cancers aren't viral, but this on
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