Cell Bio- Exam 2

Slide Set 5: Vesicular Traffic, Secretion, and Endocytosis

Vesicular Transport

• Proteins are synthesized in the ER, then are moved from ER to golgi, once mature proteins are formed, they need to leave the ER (Secretory)
• After golgi, they have multiple different pathways   * Constitutive secretion- constant secretion of proteins from cell, golgi to out of cell   * regulated secretion- secretory vesicle takes protein out of cell from golgi   * Endocytic- early endosome takes proteins from membrane to late endosome and then sometimes to lysosome   *
• Microscopy study with GFP   * studied $trafficking via GFP virus particles$   * use temperature, $if temp inc, protein mvmt blocked$   * you can track proteins via fluorescent microscopy   * results: $there is trafficking within the cell, you can get a rough est of the time that it takes$   * tracking total fluorescence signal over time
• Oligosaccaride modification   * mannose trimming occurs when oligosaccaride moves from ER to golgi   * treated with endoglycosidase D which cleaves sugar from protein
• Vesicle Budding and Fusion   * transport vesicle leaves donor compartment   * transport vesicle fuses with target compartment
• Coated Vesicle Budding   * SNARE protein helps transport vesicles recognize target membranes   * membrane cargo protein and soluble cargo protein bind together   * coat proteins surround vesicle
• Uncoated vesicle fusion   * V SNARE proteins will interact with T SNARE proteins on membrane   * $Rabs protein- can help recognize which target mem they should fuse too$, assists with docking

What is the mechanism by which vesicles are formed?

• Three types of coated vesicles   * Clathrin coated - helps with transport from trans golgi network to late endosome and helps transports obj entering the cell via endocytosis     * have heavy and light chains, as well as binding site for assembly particles     * soccer ball structure     * Functions:       * help form $mechanical force to form vesicle$         * coat subunits bind to surface of donor membrane         * clathrin and other proteins help form bud/vesicle and help with the mechanical force of budding off       * capture membrane receptors         * $clathrin and adaptin (bound together) bind to cargo receptor bound to cargo molecules in membrane, and then start budding process,$         * adaptin helps transmem receptor bind to coating proteins           * certain aa are carried that signals adaptin to bind, these are then phosphorylated     * $Dynamin$       * $required for pinching off of clathrin vesicles from donor membrane$       * polymerizes around the neck and then hydrolyzes GTP, conformational change initiated in dynamin that stretches vesicle neck until the vesicle pinches off   * $COP 1- in charge of moving protein from trans golgi back to ER$     * coatomer coated     * intra golgi traffic, golgi to ER     * $ARF$ plays a role in coat formation   * $COP 2- helps with protein leaving ER to cis golg$i     * coatomer coated       * $Sar 1 uses COP 2 components$
• GTPases   * Active- when protein binds to GTP   * $GAP- hydrolyzes GTP to GDP$     * Sar 1 initially binds to GTP, then binds to Sec 12 to hydrolyze GTP, then recruits COP2 components to have GTP bound to mem     * $Sar 1- controls coat assembly on COP2 vesicles$   * inactive- off, GDP bound   * $GEF- releases GDP so GTP can be made$     * $ARF$- also a GTPase, $plays role in coat formation in COP1 and Clathrin coated vesicles, intitially binds to GDP$       *

What are the molecular signals on vesicles that cause them to bind only to the appropriate target membrane?

• $SNARES and RAB GTPases$ play a role in vesicle traffic and fusion   * generate tight interactions, help vesicles fuse to the donor membrane
• RAB GTPase   * donor mem: RAB receptor, vesicle: RAB   * mediate diff transport vesicles fused to diff transport membranes   * many diff RABs in eukaryotic cells

How do transport vesicles and their target organelles fuse?

• $SNARE and RAB help vesicle recognize donor membrane$
• $RAB will not help fuse$, will help recognize membrane
• Vesicle Fusion Machinery   * Vesicle Docking: V SNARE and T SNARE associate, RAB binds to RAB receptor   * Assembly of $SNARE complex:$     * SNAP 25- snare complex, includes $V SNARE and Syntaxin$     * generates strong force to help fusion to the membrane     * $twisted very tightly together$   * Membrane Fusion     * proteins work to untwist SNAP 25     * fusion of membranes occurs   * Disassembly of SNARE complexes     * SNARE complexes disassociate and are free for another round of vesicle fusion, RAB also disassociates from the RAB effector

Steps in Secretory Pathway cont

• Vesicular Transport from ER to Golgi
• protein always goes from cis to trans face of golgi
• cis cisterna→ medial cisterna → trans cisterna
• $ER retention signal$- four aa, KDEL; if added at c term of protein it will return to ER from cis golgi bc it will bind to place on cis golgi and be recognized
• $Cisternal progression through golgi glycosylation$ and other mods in golgi   * removal of 3 mannose residues in cis golgi $(-3 Man$)   * protein moves to medial golgi by cisternal maturation   * 3 GlcNAc residues added , 2 more mannose removed, single fucose is added $(+ 3 GlcNAc, -2Man, + Fucose)$   * processing completed in trans golgi by addition of 3 galactose residues and linkage of N-acetylneuraminic acid residue to each galactose $(+3 Gal, + 3 NANA)$   *   * $Role of glycosylation$     * $post translational modification$       * helps protein become hydrophilic→ aids in folding       * aid in transport (rarely- targeting to lysosome)       * resistance to proteases (stability)       * protein protein interactions
• Vesicular sorting at trans- golgi network
• Vesicular Trafficking to Final Destination (golgi to ___)   * Endosome   * Plasma Mem     * constitutive secretion- unregulated membrane fusion     * regulated secretion- regulated membrane fusion   * Lysosome     * some proteins go here     * very acidic environment     * v class pumps used with ATP to pump proton inside     * lysosomes form a functional hub for cellular trrafficking pathways       * ER→ Golgi→ lysosome       * Pinocytosis→ lysosome       * Phagocytosis→ lysosome       * autophagy→ lysosome     * $How does cell know which proteins are sent to the lysosome?$       * $M6P residues!$       * receptor on trans golgi network that will bind to M6P and will incorporate into vesicle and then will go to late endosome       * if pH low in late endosome, M6P transferred to lysosome     * Lysosomal Storage diseases       * can be due to absence of 1 or more lysosomal hydrolases or the mistargeting of lysosomal hydrolases       * characterized by tissue destruction or accumulation of undigested macromolecules       * $I cell- protein stuck in trans golgi, severe tissue destruction, GlcNac Deficiency$
• Endocytosis   * goes through plasma mem, through early endosome then late endosome, then lysosome   * $pinocytosis$-     * very tiny things; proteins, lipids. Goes through $early, late, then lysosome$     * continuous process, rate depends on cell type     * pinocytotic vesicle forms from clathrin coated pits in plasma mem     * $receptor mediated endocytosis- ligand binds to cell surface receptor, clathrin helps to form vesicle, clathrin coats vesicle$   * $phagocytosis$-     * large things like bacteria; $phagosome then to lysosome$     * feeding for lower single celled euks     * multi celled orgs- used as a defense against invading microbes     * requires surface receptors, triggered event   * $autophagy$-     * from ER, if we do not need certain organelles anymore, $autophagosome forms then transported to lysosome$   * LDL Uptake     * LDL- byproduct of fat transport, have ApoB protein     * $ApoB and LDL receptor bind$     * vesicle begins to form with help of clathrin coat     * transported to early endosome→ late endosome→ lysosome     * Disorders- LDL receptor missing, receptors do not associate with clathrin coat   * Fate of cell surface receptors after endocytosis     * recycling of receptor to same domain       * receptor transported back to surface of membrane and pH will change→ receptor ready to bind to another LDL particle     * degradation of receptor after endocytosis       * in lysosome     * $transcytosis$       * $any protein that is missent to basolateral side will be resent to apical membrane side$       * $the vesicular transport of macromolecules from one side of a cell to the other$

Slide Set 6: Microfilaments

The Cytoskeleton

• Functions of cytoskeleton   * cell shape, mvmt, and contraction   * organelle mvmt and organization   * cell division   * intracellular org and vesicle mvmt   * interacting with signaling pathways
• basically like the bones of the cell
• Components   * Microfilaments     * actin filaments, thinner   * Microtubules     * tubulin dimers, thicker   * Intermediate filaments     * various, diff proteins combined together
• Cell signaling   * signals tell cytoskeleton abt organization and mvmt of organelles as well as changes in cell shape, mvmt, and contraction

Actin Microfilaments

• Functions   * org of intracellular organelles and transport of vesicles (myosin)   * intracellular mobility (bacteria)   * cellular stability   * cellular motility   * muscle contraction
• Lamellipodium   * supported by growth of actin filaments, generates a protrusion structure to adhere to surface and move cell forward
• Polymerization and Dynamics   * 1 actin filament= 2 strands   * one + end (0.12 M), one - end (0.6)   * g actin is monomer, microfilament polymer of actin   * ATP binding cleft in actin structure   * alpha, gamma, and beta actin: all associated with diff structures   * $G actin polymerization$     * $g actin binds to f actin, elongating existing filament$     * $can be added to + and - end, and leave from both sides$     * $g actin dec to critical con→ polymer shrink$     * $g actin inc above critical conc→ polymer inc in length$
• Actin Binding Proteins   * Polymerization- Profilin and Thymosin B4     * $Profilin- promotes polymerization$     * $Thymosin b4- blocks polymerization$ of ATP   * $Length- Cofilin, Gelsolin$   * $Nucleation and branching- Arp2/3$   * Crosslinking- Filamin   * Motor Proteins- myosin   * stability/cap end of filaments- capz and tropomodulin     * $CapZ- caps at + end$     * $Tropomodulin- caps at - end$   * $org of filaments/muscle contraction, binds to side of filaments- nebulin$
• Actin based Motility   * $Formin - leads to assembly for long actin filaments$     * will form $dimer structure$     * actin binds to structure and elongation commences   * $ARP2/3 complex can be used for polymerization to power motility, mediates branching$   * $listeria monocytogenes uses actin polymerization to move through cells$ and from cell to cell→ hijack actin machinery and polymerize it to move around     * $ActA protein activates Arp2/3 to nucleate$ new filament assembly from preexisting filaments     * $filaments grow at + end$ until capped by Cap Z     * $actin recycled through cofilin,$ which enhances depolymerization at the - end of the filaments     * this process $propels bacterium forward$   * Toxins that perturb pool of actin monomers     * $Cytochalasin D- depolymerizes actin by blocking further addition of subunits$     * $Latrunculin- inhibits g actin from adding to filament end$     * $Jasplakinolide-$ stabilizes and binds actin dimers, $lowers critical conc ba$r     * $Phalloidin- prevents actin filaments from depolymerizing by locking F subunits together$   * $Actin also interacts with itself$     * types of lateral attachment of microfilaments to membranes       * ankyrin- binds to Band 3 and then spectrin, forms network       * band 4.1
• Actin Motor Proteins   * $myosin$     * can bind to actin and help generate contraction in muscle cells     * $composed of heavy chains and light chains,$ diff myosin has diff amts of each     * myosin heads can bind to ATP and actin     * $Myosin 1$       * $small, single head$       * $step size 10-14nm$       * $works with membrane association and endocytosis$     * $Myosin 2$       * $dimer (2 heavy chain)$       * $8nm step$       * $bipolar filaments$       * works with contractions     * $Myosin 5$       * $bigger, 2 heavy chains and more light chains$       * $36nm step$       * $responsible for organelle transport$     * $Myosin mvmt process$       * $ATP binds to head grp, head group not associated with actin yet$       * $ATP hydrolyzed$, head grp rotated into position to bind, $head grp binds to actin$       * power stroke occurs, $Pi released and myosin straightened, moving actin filament left$       * $ADP released, the ATP bound and head grp released from actin$     * Step size vs neck length       * is myosin step size/velocity proportional to neck length?       * $YES, velocity inc with inc neck length$     * contractile ring       * myosin 2 takes a large part in forming when cells are splitting, myosin 1 is on outside of cells   * Sarcomere (not protein, just structure of skeletal muscle)     * vertical component is Z band, in between is A band, myosin in between actin filaments     * actin end facing inside is - end     * sarcoplasmic reticulum- specialized region of the ER, regulates and stores Ca (Ca helps muscle cells to contract)   * Cap Z- binds to + end of actin   * Tropomodulin- binds to - end of actin   * Nebulin- binds to side of actin filaments   * Titin- binds to myosin and Z disk proteins
• $Rho GTPases$   * $membrane bound Rho proteins can bind effector proteins that cause changes in the actin cytoskeleton$   * dominant active rho- always keep making actin   * $Cdc42$- filopodia formation     * $works at the front of cell, activates Rac$     * guys see a Rac and are activated   * $RacGTP$- lamellipodia formation     * $leads to activation of Arp2/3 and Rho$   * $RhoGTP-$ Stress fiber formation     * leads to $myosin 2 activation$

Slide Set 7: Microtubules and Intermediate filaments

MIcrofilaments vs Microtubules vs Intermediate filaments

• microfilaments   * actin binds ATP   * form rigid gels, networks, and bundles   * tracks for myosin   * contractile machinery and network at cell cortex
• $microtubules$   * $tubulin binds GTP,$ rigid and not easily bent   * $trasks for kinesins and dyesins$   * organization for long range organelles
• Intermediate filaments   * great tensile strength, less dynamic, unpolarized   * no motors   * cell and tissue integrity

Microtubules

• play a role in….   * organization of organelles and transport of vesicles   * mvmt of cilia and flagella   * nerve cell, RBC, and flagellar structure   * alignment and separation of chrom during mitosis
• $Tubulin$   * $monomer of microtubules, alpha and beta make up monomer$
• Two populations of microtubules   * Unstable short lived- assembles and disassembles rapidly   * stable and long lived- remain polymerized for a long time (sperm flagella, RBC, nerve cells)
• Polymerization and Structure of Microtubules   * Structure     * tubulin has alpha and beta parts     * $bind to 2 GTP$     * $alpha T GTP is never hydrolyzed$     * $beta T GTP can be hydrolyzed$     * $one end is beta T exposed→ + end$     * $one end is alpha T exposed → - end$     * microtubules made up of 13 protofilaments → singlet     * can have doublets (cilia/flagella) and triplets (basal bodies and centrioles) as well   * Polymerization     * $microtubules assembled from MTOC$     * MTOC-any structure used by cells to nucleate and organized microtubules       * centrosome falls into this category       * $neg end of microtubules at MTOC$     * gamma tubulin ring nucleates microtubule assembly
• Dynamics of Microtubules   * Length over time: Assembly stage→ Catastrophe stage→ Disassembly stage→ Rescue Stage   * $Polymerization of tubulin into microtubules$     * $protofilament first formed$     * $alpha T first binds to protofilament, then beta T$     * $sheet$ assembly     * then form $tube formation$     * $GTP cap at top,$ GDP microtubule is the rest       * GTP cap bc $alpha and beta T carry GTP, addition of another Alpha and beta T will cause hydrolysis$         * \           * end more smooth (assembly), - end more rough (disassembly)   * Disassembly and reassembly of microtubules     * cool to 4 deg, microtubule will disassemble     * warm to 37 deg the microtubule will repolarize   * Drugs that disrupt microtubule dynamics     * $colchincine- binds btwn alpha and beta T dimer so it cannot be used for polymerization,$ causes depolymerization     * $taxol- bind to side of tubules$ - stabilize the microtubule structure
• Binding Proteins   * $MAPs$     * $can stabilize microtubules,$ similar to taxol     * $side binding$     * MAP2- longer     * Tau- shorter     * +TIPS       * can regulate + end of microtubules   * Motors     * $Kinesins$       * ferry cargo around the cell       * $ferry towards + end$       * have light chain, bind to ATP for energy resource       * bind to microtubule with head groups, bind to vesicle via kinesin receptor       * hydrolyze ATP to drive mvmt       * Kinesin 1 and 2- organelle, mRNA, and chromosome transport       * $Kinesin 5- bipolar structure, 2 head grps, can bind to 2 diff microtubules, microtubule sliding$       * $Kinesin 13- can regulate microtubule end disassembly$       * Process         * first head group, $(leading head),$ no ATP, $bound to microtubule$         * $leading head then binds to ATP$         * $conformational change induced, following head swings forwards$         * $following head becomes leading head$         * $new leading head releases ADP which it was originally bound to, and new following head hydrolyzes ATP to ADP$ and then process restarts     * $Dyneins$       * $ferry cargo around the cell towards - end$       * Power stroke of dynein- ATP hydrolysis causes change in orientation of head→ mvmt of MT       * $dynactin- bind cargo, make dynein more processive$       * LIS1 protein- interact with ATPase domain of dynein to elongate power stroke

Intermediate filaments

• heterogeneous
• great tensile strength
• no known motors use them as tracks
• more stable than filaments or tubules
• no intrinsic polarity
• made up of protofilaments that can form diff structures   * $have N term and c term and head and tail end$
• $keratin, lamin, vimentin$

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