Bio 203 Exam #4 PDF

lOMoARcPSD|50420626 BIO 203 Exam #4 Intro to Cell Biology (Miami University) Scan to open on Studocu Studocu is not sponsored or endorsed by any college or university Downloaded by Lily Rose ([email protected]) ...

lOMoARcPSD|50420626 BIO 203 Exam #4 Intro to Cell Biology (Miami University) Scan to open on Studocu Studocu is not sponsored or endorsed by any college or university Downloaded by Lily Rose ([email protected])  lOMoARcPSD|50420626 Intermediate filaments (intermediate in size) - Found only in animal cells - Used for cell/tissue support - Stable components made up of many polypeptides joined together - Does not require nucleotides or motor proteins - Assembly 1. Fibrous alpha helicase PP is made having different ends making it polar 2. Two monomers form coiled-coil dimer (polar; different ends) 3. Two dimers stagger to form tetramer (non-polar; same ends) 4. 8 tetramers form diameter (not polar) 5. 8 tetramers determines length by addition to growing filament - Different proteins form IF in different tissues depending on type of cell - Epithelial- keratin filaments Cells are connected through junctions where the IF allow for cells to stretch If IF are interrupted then skin starts to pucker due to no IF holding them together → blister of skins - Connective tissues- vimentin proteins - Nerve cells- neurofilaments - Nuclear- lamin proteins are found in all animal nucelis Lamins come together to form IF that lie underneath nuclear envelope and anchors chromatin If lamin proteins are not present or disorganized then an aging disorder(progeria) can exist where there is not an attachment site for chromatin Microtubules (largest and found in all euk cells) Can be found in certain areas depending on their function Assembly - Assembly occurs at MTOC where there is the centrosome and basal body Centrosome is where it makes MT that will be in cytosol and its closest to nucleus in a non-dividing cell Basal body is where cilia and flagella will be made near the membrane - Composed of tubulin, globular proteins( alpha, beta and gamma) where alpha and beta will form a dimer unit and gamma is used to make a template - Gamma tubulin is a nucleating protein that will orient alpha and beta dimer correctly. Gamma protein will form a circle to form a template and give correct diameter - Gamma will bind to the alpha of the dimer unit that contains GTP near/in the centrosome. GTP allows the dimer unit to bind. When dimer units bind to the gamma template and is added to the + end, it forms a protofilament where alpha beta dimers alternate. We need 13 of them to form correct diameter of MT - Will have polarity (- end is where alpha end is/in centrosome and + end is where beta is) Functions Downloaded by Lily Rose ([email protected])  lOMoARcPSD|50420626 - Mitotic spindle - Cilia and flagella Movement involving motor proteins so cell can move Assemble in a basal body where the gamma tubulin will form a 9+2 arrangement of 9 doublets of gamma tubulin with an additional 2 in the middle Dynein motor proteins and ATP will be used Dynein motor proteins will be moving towards - region, dynein tries to carry the other protofilament but through linkage proteins that hold doublets together results instead of sliding they will bend - Transport Involve motor proteins that have a head and a tail Kinesin will move cargo towards the + end/away from nucleus and dynein moves toward - end/towards nucleus Tail region is where cargo will bind Can move proteins, organelles, microtubules, etc… 1. Head region of motor protein will have ADP bound before attaching to MT 2. When one of the heads binds to MT and ADP is exchanged to ATP 3. Exchange causes zipper of neck region and will swing second head forward 4. ATP on the first head will be hydrolyzed while second head exchanges ADP→ATP Dynamic instability (unique to only MT) Regulated in the cell so we can grow and shrink microtubules as needed Growth and shrinking will occur on the beta end GTP will control dynamic instability by being part of dimer unit When dimer binds it needs GTP to bind to grow protofilament making GTP hydrolyzed to GDP when dimer binds GTP cap/ GTP is high will promote growth where GTP dimers are added to the end and the GDP dimers are contained in the middle; GTP=higher affinity If GTP is low/ GTP cap is lost growth decreases and GTP cap gets lost resulting in all GTP to be hydrolyzed and thus dimers start to separate instantaneously → dynamic instability due to no affinity to keep dimer together MAP proteins bind to MT or dimers to regulate assembly and disassembly; prevents dynamic instability Actin filaments/ Microfilaments Assembly - Composed of actin proteins that are tertiary globular structures - Contains nucleating protein ARP ⅔ that allows the first couple of actin to join together to help get orientation correct - One end will have plus/barbed end (actin-ATP) and minus/pointed end(actin-ADP) thus making polar Downloaded by Lily Rose ([email protected])  lOMoARcPSD|50420626 - Plus and minus end are controlled by hydrolysis of ATP - Treadmill affect Instead of complete disassembly actin fall off one by one ATP-actin is added to + end and treadmilling effect occurs at - end due to lower affinity of actin-ADP More actin will be added to plus end and lost at - end because of critical concentration where the actin-ATP end has lower concentration and higher affinity This treadmilling affect maintains actin length Can have proteins that can promote assembly or disassembly Profilin- binds to actin-ADP to increase exchange to form actin-ATP Cofilin- bind to actin-ADP as it comes off and inhibit exchange to actin-ATP; promote disassembly - Functions Cytokinesis Cell migration - Not all cells can migrate 1. Extension of front of cell by polymerization/assembly of actin filaments (requires ARP ⅔ and profilin) 2. Adhesion of front end to protein in ECM 3. Contraction where myosin motor protein will shorten by pushing actin filaments closer together 4. Retraction of back end which will release from proteins - Found in all eukaryotic cells Endomembrane functions ER - Lipid and protein production - Detox of drugs - Steroid hormone production Golgi - Modification of proteins - Sorting - Production of non glycerol containing phospholipids Lysosome - Degrade macromolecules and organelles - Digestion of material coming into cell Modifications in the ER Disulfide bonds - Can only form in lumen and will function in ECM Glycosylation(carbohydrate sequences added) - Addition of sugar unit to protein - Important for membrane proteins and for transport to lysosome Downloaded by Lily Rose ([email protected])  lOMoARcPSD|50420626 - Transport to lysosome N-linked glycosylation is added to the nitrogen of asparagine side chain that has mannose residues Folding - ERAD Quality control in ER to check for folding If any proteins are not folded correctly then they are sent to cytosol and destroyed - UPR Unfolded protein response When there are too many abnormally folded or unfolded proteins within a given cell it will lead to protein production to shut down and can lead to cell death Vesicle formation for soluble ER proteins Will need proteins called coat proteins (either clathrin or COP proteins) COAT proteins need the receptor in the membrane to bind to the soluble proteins(adaptin or cop bind to receptor in this situation) Membrane proteins, no receptor is needed and COP or adaptin will bind to protein directly 1. Coat proteins bind to membrane - Coat protein needs receptor to carry cargo - Receptor will bind to cargo then coat proteins will bind to receptor directly or indirectly - If clathrin then needs adaptin to bind to receptor - If COP then it can directly bind to receptor 2. Coat protein causes membrane to pucker 3. Dynamin pinches off vesicle from membrane using ATP 4. Coat proteins are removed making receptor accessible 5. Motor proteins (usually kinesin) transport vesicles to destination (target membrane) 6. Rab protein on vesicle binds tethering protein on membrane that has specific binding to know that vesicle is at right location and pulls vesicle close to membrane (docking) 7. V-snare on vesicle and t snare on membrane intertwine and vesicle able to be pulled closer causing water to be expelled and fusion to occur 8. Soluble proteins will be released into organelle or secreted and membrane proteins will become part of new membrane Vesicular transport After ER they can travel to golgi then to lysosomes or plasma membrane ER to golgi - ER to golgi will require COPII coat proteins - COP II proteins used to transport through golgi as well Golgi to ER Downloaded by Lily Rose ([email protected])  lOMoARcPSD|50420626 - Some proteins may leave ER accidentally or proteins need modifications that can only be done in ER - Uses COP I proteins and dynein motor proteins - ER resident proteins all have KDEL (AA sequence) that COPI will recognize and bind to receptor and form vesicle and transport it back to ER Golgi to lysosome/endosome 1. N linked glycosylation with mannose added in ER 2. COPII vesicles transports protein to golgi 3. Mannose phosphorylation in golgi turning into M6P 4. Mannose 6-P bound by receptor that is bound by adaptin/clathrin 5. Vesicle transported to endosome and endosome fuses with lysosome Golgi to Plasma membrane - Transported through regulated and constitutive secretion - Don’t know coat proteins used - Constitutive secrete (ECM proteins) Unregulated manner Moderate/small amount of cargo that will be released on a regular basis No signal for it to occur - Regulated (hormones, enzymes) More cargo secreted at one time due to waiting for a signal Need a signal from outside of the cell that we want to release the molecule Receptor mediated endocytosis 1. Cargo must bind to LDL receptor 2. Clathrin forms vesicle and buds inward 3. Clathrin is removed 4. Vesicle fuses with endosome 5. Cargo delivered to lysosome 6. Receptor is recycled Cell signaling types Endocrine - Release hormones in blood - Long distance Paracrine - Local - One Signal secreted into general area and activate many cells Synaptic - Seen in NS - Cell to cell Contact dependent - Membrane bound signal that will not leave cell and receptor has to come in contact with other cell Downloaded by Lily Rose ([email protected])  lOMoARcPSD|50420626 Water vs Lipid soluble signaling molecules Lipid - Receptor is inside the cell - Molecule has to cross membrane to bind to receptor Water - Receptor is in the membrane - When bonds it changes shape of receptor in membrane - Effector: membrane bound molecule that is activated by receptor - Secondary messenger- cytosolic protein that is activated by effector and carries out activation or inactivation of other proteins Effects of signaling pathways Activation/inactivation through phosphorylation - Phosphate is added changing the shape that requires ATP - Fast acting - Use of G proteins Large: trimeric (alpha, beta, gamma subunits) that do not need GAP or GEF Small: these require GAP and GEF to hydrolyze GTP or exchange GDP to GTP to turn gene off and on since can not do it by itself GAP(turn gene off) GEF(turn gene on) Making a new protein - Activate TR factor - Slow acting due to TR→TL etc… Outcomes of altering protein synthesis TR or protein synthesis Cell survival Cells move Cell death Metabolic changes Cell division Differentiation Cytosolic signaling pathway Contain cytosolic receptors (cortisol, estrogen, steroid hormones) Lipid soluble molecule that is a slow process 1. Hormone crosses lipid membrane and binds to cytosolic receptor in cytosol 2. Causes a change in shape and complex will then move inside the nucleus 3. It will then bind to gene and activate TR of that gene Membrane bound receptors G protein coupled receptors - Trimeric receptor spans membrane 7 times; alpha helicase Downloaded by Lily Rose ([email protected])  lOMoARcPSD|50420626 Activation of PKA 1. Signal molecule binds receptor (GPCR) 2. G protein (trimeric) interacts with receptor (GPCR) G-Alpha is the only one that has GTP/GDP associated with it Beta and gamma stay together When G-alpha GDP is bound they are together and inactive When G-alpha GTP is bound they are separate and receptor is active G-alpha can have many types 3. Signaling molecule will activate alpha subunit to turn into alpha-GTP and gamma-beta will separate 4. If GS- alpha then will activate adenylyl cyclase (effector) 5. When effector is activated it will take ATP →caMP (2nd messenger) and move through cytosol and activate protein called protein kinase A 6. PKA is activated and can activate or inactivate any proteins inside of the cell through phosphorylation GPCR pertaining to increasing blood glucose - Signaling molecule will either be adrenaline or glucagon 1. PKA is activated by cAMP and will P phosphorylase kinase making it active. Phosphorylase kinase will activate glycogen phosphorylase. This will breakdown glucose 2. PKA will also activate TR factors for gluconeogenesis (CREB). When CREB is activated it binds to enhancer of genes for gluconeogenesis to increase glucose 3. PKA will also P glycogen synthase(enzyme needed for glycogen formation) and inactivate it so no glycogen is made from glucose Inactivation of G-proteins - Removal of signaling molecule - Hydrolyzes GTP (this makes alpha inactive) - Arrestin: inhibits from GPCR from being activated - Receptor endocytosis/destruction; receptor brought inside cell - Cyclic AMP phosphodiesterase- inhibits cAMP Enzyme coupled receptors - RTK Structure - Single membrane spanning proteins - 2 of these proteins come together to form a dimer when signaling molecules bind (1 or 2 signaling molecules). Cross P occurs where they phosphorylate each other Activation of AKT 1. Signaling molecules binds RTK 2. RTK forms dimer and cross phosphorylates 3. Pi3K binds phosphorylated RTK and is activated (effector) - Pi3K is intracellular protein that contains SH2 or PTB sequence Downloaded by Lily Rose ([email protected])  lOMoARcPSD|50420626 4. Pi3K phosphorylates PIP2 to form PIP3 - P lipids in membrane 5. PIP3 is docking site for AKT and PK1 where they will bind and become activated 6. Docked PK1 and cytosolic PK2 bind AKT 7. AKT activated (2nd messenger) - RTK-AKT decrease blood glucose Signaling molecule is insulin that binds to RTK 1. Once AKT is activated, it will P proteins holding vesicles with glucose transporter at trans golgi. This causes the release and insertion of glucose into cell 2. AKT will P PP1 that removes activated phosphate (dephosphorylates) from phosphorylase kinase and glycogen phosphorylase. This prevents glycogen breakdown 3. AKT will P GSK-3 (usually inhibits glycogen synthase) and will inhibit it so glycogen synthase is active Glycogen synthase will be dephosphorylated and active Result: No PKA=no P CREB=no gluconeogenesis - Regulation of RTK RTK can be removed from membrane by ubiquitin or being sent into the cell PTen removes activating phosphate from PIP3 Downloaded by Lily Rose ([email protected])