Cell Bio Final Study Guide PDF

​ What are the 3 properties of the cytoplasm? -​ 1.Viscoelastic ( like the school doors. Spring like, closes gently/can't slam close) -​ 2. Thixotropic -​ 3. Crowdedness ​ What do each of the cytoplasm properties refer to? -​ These features of cytoplasm are largely due to com...

​ What are the 3 properties of the cytoplasm? -​ 1.Viscoelastic ( like the school doors. Spring like, closes gently/can't slam close) -​ 2. Thixotropic -​ 3. Crowdedness ​ What do each of the cytoplasm properties refer to? -​ These features of cytoplasm are largely due to components of the cytoskeleton. ​ How did we determine the cytoplasm is thixotropic? -​ Feed macrophage iron molecule -​ Put a magnet at the bottom and move the iron around -​ Moving it slowly (low shear stress), Its stiff -​ Moving it quickly (high shear stress), Iron bead exhibits only a little resistance -​ Actin filaments, microtubules are apart of the cytoskeleton and moving the iron bead quickly breaks them into liquid ​ What is the significance of the 50 nm barrier in the experimental analysis of diffusion/crowdedness? -​ Dextran particles with a diameter of less than 50 nm exhibit free diffusion (you’ll see them all throughout the cell) -​ Dextran particles with a diameter of more than 50 nm diffuse really slowly or don't diffuse at all (in cytoskeleton rich areas, the size limit was even smaller ~20 nm) -​ !!Vesicles are uniformly below 50 nm and communication has to occur through vesicles. They have to be able to move through the tracks. If the matrix was TOO crowded that they can't fit through, then you wouldn’t be able to communicate within the cell through vesicles. ​ What are the 3 major fibrous polymers of the cytoskeleton? Define and describe 1)​ Actin -​ Smallest (7 nm diameter) -​ Polymerized globular (G)-actin and associated binding proteins 2)​ Intermediate filaments -​ 10nm diameter -​ Polymerized IF subunits (many different types) and associated binding proteins 3)​ Microtubules -​ Biggest (24nm diameter) filament  -​ Composed of polymerized alpha-beta tubulin dimers and associated binding proteins ~ Both actin and microtubules are polarized polymers meaning they have direction (plus end or minus end) ~ IF’s have no direction ​ What are the types of actin filament arrays? 1)​ uniformly polarized bundles within microvilli 2)​ mixed polarity actin bundles are termed stress fibers. 3)​ loosely ordered cortical actin 4)​ sheet-like arrays of rapidly turning over filaments that extend the lamellipodia of a crawling cell. ​ What is a centrosome? -​ Centrosomes are structures found inside of cells. They are made from two centrioles. Centrioles are microtubule rings. The main purpose of a centrosome is to organize microtubules and provide structure for the cell, as well as work to pull chromatids apart during cell division ​ How are the fibrous polymers arranged in epithelial cells? -​ Intermediate filaments course out from surface of nucleus to the plasma membrane -​ Microtubules extend out from a perinuclear MT organizing center: the centrosome -​ Actin filaments are uniformly bundles and provide the structural integrity for the finger-like projections -​ Intermediate filaments are linked in the nucleus and provide nice contact points to the plasma membrane ​ What are the cellular functions mediated by the cytoskeleton -​ 1. Generation/maintenance of asymmetric cell shapes; cell polarity. -​ 2. Spatial organization of cytoplasm. -​ 3. Regulation of membrane structure, topography, lateral mobility. -​ 4. Establishment/maintenance of cell-cell and cell-substrate contacts. -​ 5. Generate tension, transmit and sense both intra and intercellularly. -​ 6. “Down stream” effectors of signal transduction cascades. -​ 7. Subcellular localization/ regulation of cytosolic enzymes. -​ 8. Mechanoregulation of membrane physiology (e.g. channel gating;) -​ 9. Cellular movements; cellular contractility; protrusive activity; cytokinesis; -​ 10. Organelle movements—including chromosome movements. -​ 11. Exocytic and endocytic membrane traffic. -​ 12. mRNA localization/transport; regulation of translation. -​ 13. host cell-microbial pathogen interactions. ​ Describe some of the places where actin is found and its role in the cell -​ Actin is primarily found in the cytoplasm of eukaryotic cells, where it forms a network of filaments known as the actin cytoskeleton (muscle and liver cells) ​ What is listeria monocytogenes? -​ Listeria monocytogenes (Lm) has been responsible for several outbreaks of foodborne illness domestically. -​ Cantaloupe, Unpasteurized cheeses, Raw or uncooked Foods etc. -​ Each year approximately 2500 people become seriously ill due to Lm infections. -​ Nearly 500 of these die from their infection. -​ Listeriosis only accounts for about 0.02% of illnesses due to foodborne disease, but it causes 27.6% of all deaths due to foodborne infection. -​ Pregnant women and newborns are most susceptible ​ What is the principle of the Portnoy and Tilney experiment? -​ The study explores how Listeria monocytogenes spreads in a macrophage cell line. The first step of the study is phagocytosis of the Listeria. After phagocytosis, the bacteria escape the phagosomal membrane, likely through hemolysin secreted by Listeria, which  breaks down the vacuole. Within two hours, the bacteria are surrounded by actin filaments. At first they are short, but later they form a long “comet” tail at one side of the bacterium. The actin filaments push the bacteria towards the surface of the cell and reach out to extend into a neighboring macrophage. The Listeria inside the new cell are surrounded by the original phagosome membrane and a new membrane from the neighboring cell. Both membranes are dissolved by the bacteria, which allows the cycle to repeat. This process lets Listeria spread between cells without the host’s immune system detecting it. When the cells are treated with cytochalasin D, Listeria are unable to form “comet” tails and remain embedded deep within the cytoplasm. ​ What is the structure of polymerized actin? -​ a double helical structure composed of two strands of globular actin monomers (G-actin) twisted around each other, forming a flexible filament with a defined polarity, meaning one end is different from the other ​ How does actin push viruses out of the cell? -​ by forming "actin tails" or "comets" around the virus, which are essentially structures of rapidly polymerizing actin filaments that propel the virus towards the cell membrane, effectively pushing it out of the cell; this process is often facilitated by viral proteins that can hijack the cell's actin polymerization machinery to create these actin tails, allowing the virus to spread to neighboring cells. ​ What are the 2 versions of actin? -​ Unpolymerized ‘Globular’ (G) -​ Filamentous (F) ​ What are the structures of the microfilament? -​ two-stranded helix - each actin monomer has 4 contacts -​ – polarized filament - all actin monomers oriented in same direction -​ – actin in filament hydrolyzes ATP to ADP -​ G-actin Mr: 42kDa: actin monomer binds 1 molecule of ATP or ADP -​ (hydrolysis occurs after addition to polymer) -​ The subunit is oriented with plus/barbed end surface at the bottom. ​ What gets hydrolyzed when actin binds to the monomer -​ When actin binds to a monomer, the molecule ATP bound to the actin monomer gets hydrolyzed to ADP ​ Describe the polarity of actin -​ Plus end grows faster and at a 5-10 fold lower critical concentration than the minus/pointed end. -​ Polarity dictates the direction a myosin can pull on or walk along an actin filament. Most known myosins are plus-end directed motors. The one exception: Myosin VI. ​ What is a critical concentration? -​ The minimum concentration of units needed before a biological polymer will form -​ SO the plus end only needs 5-10 fold lower than minus end ​ Which end of actin grows faster? -​ plus end ​ What are the phases of actin polymerization? -​ nucleation - requires trimer formation (3-hit) -​ – elongation - addition to both ends - mostly (+)end -​ – steady state - gain=loss ​ What is the CC for actin? Tubulin? -​ Subunit concentration above which polymerization will occur. The concentration of subunit (actin monomer; tubulin dimer) that is in equilibrium with polymer once polymerization has reached steady state equilibrium -​ For pure Actin: 0.2 μM Tubulin: ~ 10 μM; -​ ​ What are the key features of the kinetics of actin and microtubule assembly? -​ Subunit concentration must be > C c Assembly must occur in three distinct kinetic stages: 1)​ Nucleation: rate limiting step; extremely slow for pure tubulin assembly; very slow for actin assembly 2)​ Elongation; rapid linear formation of polymer mediated by end-on addition of subunits to both end of growing polymer. 3)​ Steady state equilibrium: Although there is no net change in polymer concentration; polymer is in constant -​ exchange with the C c pool of subunits at both ends of the polymer. [Polymer] [subunit] Cc -​ Rate of polymer elongation is faster at the plus end (also called barbed end for actin filament) than at the minus (pointed) end of the polymer. -​ The C c for subunit addition is also lower at the plus end than at the minus end, although this difference is much greater and functionally more important for actin. -​ The plus end is also the fast disassembly end for both MTs and actin filaments. -​ During elongation phase rate of polymer elongation is linear and is linearly dependent on the total subunit concentration ​ How do we measure actin/tunulin polymer concentrations? -​ Sedimentation: Filamentous (F)-actin and Microtubules pellet at 50-100kg; monomers alone do not -​ Change in viscosity: viscosity increases as [polymer] and length increases. -​ Turbidity: Spectrophotometric measurement of light scattering (change in turbidity); used for Microtubules -​ Change in fluorescence: pyrene-labeled actin fluorescence increases ~ 20-fold when G-actin incorporates into polymer. -​ Microscopy: EM or fluorescence you can see polymers ​ Go through and rewatch/work through the exercises we did in class Look at end billionaire questions on lecture 22  ​ What causes the lag in hydrolysis? -​ At the plus end, there is an NTP(nucleotide) cap containing subunits under conditions of rapid assembly -​ Hydrolysis occurs at a rate slower than subunit addition when high free subunits are available for addition, at least at the growing plus end. ( For both G-actin and tubulin dimer, nucleotide (NTP) hydrolysis occurs upon incorporation into polymer.) -​ As a growing filament ages, subunits in the oldest part of the filament will contain the most ADP subunits -​ However, if the available pool of subunits is at or below the C c, then the plus end will contain subunits with NDP rather than NTP; (key for MT assembly dynamics); for actin filaments, regions of the filament containing ADP bound subunits are structurally different from subunits with bound ATP or ADP.Pi. ​ What do the actin drugs do? -​ Cytochalasins: Bind barbed/plus ends, inhibits polymerization -​ Latrunculin: Binds monomers and inhibits polymerization -​ Phalloidin: Bind and stabilize filaments, phalloidin can be labeled with a fluorescent dye, making it very useful for staining actin filaments in cells ​ How do cells maintain a pool of up to 40% unpolymerized actin -​ A cell would do it naturally by a protein interacting with actin to thin out its function ​ How do G-actin binding proteins regulate depolymerization? -​ by controlling the pool of available actin monomers, either by sequestering them (preventing polymerization) or by promoting their release at the pointed end of actin filaments, which facilitates faster depolymerization; the most well-known example of this is the "Actin Depolymerizing Factor" (ADF)/cofilin family, which binds to G-actin and enhances its dissociation from the pointed end of filaments, effectively accelerating depolymerization ​ What is thymosin β4? -​ G-actin-binding proteins - maintain unpolymerized pool of G-actin - polymerized in response to signaling  -​ thymosin β4 – binds G- actin 1:1 - buffers ~70% actin in cells -​ Basically it will inactivate 70% of actin in cells. Won't allow addition to the filament ​ What is profilin and how does it work? -​ binds G-actin 1:1 - ~20% actin in cells - blocks (+) end of monomer - blocks Spontaneous polymerization -​ Nucleotide exchange factor - promotes actin polymerization -​ allows addition to (+)end of filament - Blocks polymerization to (-) end and polymerization on (+) end dissociates profilin -​ Regulation of Actin polymerization- depolymerization in cells ​ What are formins? What inhibits its activity? -​ nucleate growth of long actin filaments in stress fibers and circumferential rings – associate with (+)end -​ Formin activity is auto-inhibited by N-terminal interaction with C-terminal domains -​ Interaction of Rho-binding domain with Rho-GTP activates -​ Rho activated by external signal receptor (GEF) to GTP form Actin filament nucleation proteins -​ Formin FH1 domain binds profilin-ATP-actin -recruits pool of polymerizable actin monomers -​ Formin FH2 dimer donut rocks to allow addition of actin monomers to ends of two strands ​ What is Rho? What regulates its activity? -Formans have a signal to activate them. They're called Rho which is a binding domain. - Formins nucleate growth of long actin filaments in stress fibers and circumferential rings - associate with (+)end - Formin activity is auto-inhibited by N-terminal interaction with C-terminal domains - Interaction of Rho-binding domain with Rho-GTP activates - Rho activated by external signal receptor (GEF) to GTP form -Formin FH1 domain binds profilin-ATP-actin recruits pool of polymerizable actin monomers -Formin FH2 dimer donut rocks to allow addition of actin monomers to ends of two strands ​ What are actin filament nucleation proteins? -​ proteins that help actin filaments form by stabilizing actin polymerization nuclei ​ What is cofilin? How does it work? (When the cell wants to disassemble actin really quickly, Cofilin will find sites with ADP. And those are the sites where it breaks the filament into small pieces.) -​ binds to 2 ADP-actin subunits near filament (-) end -​ twists filament and breaks off pieces – more (-) ends disassemble faster -​ releases from ADP- G-actin ​ What are capping proteins? What purpose do they serve? -​ proteins that regulate actin assembly dynamics by capping the barbed ends of actin filaments -​ Capping on plus end makes them not grow as quickly and falling off of the minus end -​ Capping on minus end promotes plus end to grow and minus end to not fall off -​ The more plus ends you have, the more growth, branching mechanism ​ What are crosslinkers? -​ Not going to influence the graph of how actin filaments would grow quickly -​ When filaments are being formed, you might not want to assemble and reassemble, you can link them together as a bundle or a network. -​ chemically joining two or more molecules by a covalent bond. ​ What is fimbrin? Where is it at? -​ Bind filaments and put them in the same orientation. It will cross breed them to create a bond between actin filaments -​ Fimbrin - 2 CH- domains(Calponin homology) -​ crosslinks actin filaments into tight bundle of parallel (same polarity) filaments -​ This is in one direction, minus to plus, so there needs to be another protein to give the opposite : α-actini ​ What is α-actinin?  -​ Anti-parallel dimer of α-actinin (each with one CH-domain) crosslinks actin filaments into loose bundles with mixed polarity (anti-parallel- contractile systems -​ Opposite binding ​ Spectrin? -​ Protein -​ Anti-parallel tetramer (2xα-β of spectrin (each β with one CH-domain) crosslinks actin filaments into loose networks -​ HUGEEE domain that expands it: triple-helical domains in spacer region - flexible, but inextensible -​ Found in cell cortex -​ Gives red blood cells their distinctive shape ​ What makes RBCs flexible? How do the proteins interact to give it that flexibility? -​ Distinctive biconcave shape -​ important for fluid dynamics of flow through blood stream -​ Flexibility required for RBC to squeeze through capillaries with smaller diameters -​ Spectrin molecules - crosslink short actin filaments -provide a structural network underlying the RBC membrane How? Membrane cytoskeleton establishes cell shape -​ Spectrin filaments binds through CH-domains to short actin filaments -​ Spectrin anchored to Glycophorin and through binding of central domains to Ankyrin-Band III complexes -​ flexibility of spectrin molecules provides strength and flexibility to RBC membrane ​ How is dystrophin associated with muscular dystrophy? -​ Dystrophin - single CH domain with long triple helical tail -​ Associated with PM of muscle cells -​ Mutation causes several forms of MD How? -​ Dystrophin - anchored to glycoprotein complex-ECM on muscle cell membrane -​ anchors non-sarcomeric actin filaments to membrane -​ crucial for membrane mechanical stability -​ Anchoring complex contains signaling proteins - NOS, GRB2 ​ Do the breakout rooms ​ How many myosins are there? -​ 15 have been identified but theres about 20 right now ​ Where does motor activity come from? -​ Motor has a head domain that uses ATP to create force on actin filaments -​ The motor activity of myosin comes from the energy derived from the hydrolysis of ATP ​ What is the head domain? -​ interact with actin filaments to produce force through ATP dependent crossbridge cycle of steps along actin filament -​ most step toward the (+)end of actin filament -​ Its the actin binding site and ATPase binding site ​ Neck region? -​ IQ motif - binds light chains, variations of length -​ dictate step size ​ Tail domains? -​ relate to specific functions in cells -​ There's 3 classes that increase in size. (one arm, 2 arm with small ridges, 2 arm with thick ridges) Functions: Bind to vesical and walk towards plus end -​ Class one: membrane association, endocytosis -​ Class two: contraction  -​ Class three: Organelle ​ What is myosin II bipolar filament? -​ A myosin II bipolar filament, found within the sarcomere of a muscle cell, is a thick filament composed of multiple myosin II protein molecules arranged with their tails pointing towards the center of the filament and their globular heads projecting outwards at either end -​ Has two heads and a really long tail that coils together. Has a bare zone where everything happens on the two sides. Polarity: they want to crawl towards the plus end. As you crawl the sarcomere shortens. Left (-) , Right (+) -​ Only head and tail is sufficient for actin filament movement ​ Work through the JamBoard from the lecture and understand the results -​ ​ How to we study the force generated from actin/myosin? -​ in vitro motility assays where single actin filaments are propelled by myosin molecules on a surface, allowing direct measurement of the force produced by the interaction between the two proteins ​ Which direction is actin propelled on myosin? -​ towards the barbed end (plus end) of the actin filament ​ What is the myosin crossbridge cycle? -​ ATP binding opens cleft and dissociates head from actin filament -​ Hydrolysis of ATP =ADP+Pi and cocks head toward actin (+)end -​ head and rebinds to actin filament upstream of original position -​ Pi release cause pivot of head on neck (powerstroke) -​ release of ADP (rigor position) -​ Energy: 1 ATP/powerstroke 1)​ Cross bridge formation: the activated myosin head binds to actin forming a cross bridge - inorganic phosphate is released and the bond between myosin and actin becomes stronger 2)​ The Power Stroke: ADP is released and the activated myosin head pivots sliding the thin myofilament towards the center of the sarcomere 3)​ Cross Bridge Detachment: when another ATP binds to the myosin head the link between the myosin head and actin weakens, and the myosin head detaches 4)​ Reactivation of Myosin Head: ATP is hydrolized to ADP and inorganic phosphate. the energy released during hydrolysis reactivates the myosin head returning it to the cocked position -GOOGLE : ATP hydrolysis for myosin II ​ How much ATP is needed for the powerstroke? -​ one ATP molecule is needed for each power stroke during the myosin cross-bridge cycle; -​ meaning that each time a myosin head binds to actin and generates force (the power stroke), it utilizes one ATP molecule to detach and reset for the next cycle ​ What is the difference between skeletal/cardiac muscle and smooth muscle? -​ highly aligned sarcomeres Skeletal - syncytium of fused cells. (many cells that are fused together like a plasma membrane) -​ Many nuclei, common cytoplasm Cardiac -​ Cellular (connected by gap junctions) Smooth muscle: -​ non-striated -​ connected by attachment plaques -​ They don't have defined sarcomeres, instead they have dense plaques that mimic sarcomere edges ​ What is myofiber? Where is it found? -​ muscle ‘cell’ or muscle fibers -​ fusion of many cells into multinucleated cell with large interior cytoplasmic space filled with myofibers -​ elongated cells that make up muscle tissue and are responsible for generating muscle force -​ Myofibers are found in skeletal muscle -​ Myofibers are multinucleated cells that are formed when multiple myotubes fuse together. They contain myofibrils, which are large cellular assemblies that give muscle fibers their contractile properties -​ use chemical energy from metabolism to shorten and return to their original length, which allows muscles to move ​ Myofibril? -​ end-to-end linkage of sarcomeres  -​ long, cylindrical organelle found within a muscle cell, specifically in skeletal muscles, that is composed of repeating units called sarcomeres and is responsible for muscle contraction by containing the contractile proteins actin and myosin, which slide past each other to generate force; ​ Sarcomere? -​ Contractile unit structures -​ the smallest contractile unit within a muscle, and it is found within skeletal muscle tissue, where it is responsible for muscle contraction by the sliding of actin and myosin filaments along its structure; this arrangement gives the muscle its striated appearance under a microscope. ​ What are the thin filaments? -​ Actin filaments attached to each Z-disk by + end -​ ​ Where is Cap Z? -​ Capper for the plus end -​ in the Z band of the muscle sarcomere ​ Where is tropomodulin? What is it? -​ Capper for minus end -​ Stabilizes filament ​ Where is the long nebulin at? -​ Long nebulin molecule lies along actin filament -​ length of nebulin = length of thin filament ​ What are the thick filaments? -​ "thin filaments" refer to the protein actin, while "thick filaments" refer to the protein myosin Thick: -​ myosin bipolar filaments in center of sarcomere -​ Thick and thin filaments overlap in each half sarcomere Thin: Located in the I bands, or light bands, of the sarcomere. Thin filaments extend from the Z discs toward the center of the sarcomere, where they overlap with thick filaments. Thick filaments: Located in the A bands, or dark bands, of the sarcomere. Thick filaments are centrally positioned and partially overlap with thin filaments. ​ What is the I band? -​ space between end of thick filament and Z-disk ❖​ In a rigor state, myosin heads are still attached to the actin. Its dead so there's no ATP to displace it ​ Understand the sliding filament theory of contraction -​ When we go through the cross bridge cycle, that's how the myosin is able to hydrolyze the ATP to form the binding sites to bind the actin -​ So as each side crawls to the plus end, it forms a contracted state from relaxed -​ It can't stay contracted, the muscle needs to relax -​ So there is a protein that help relax it, “Titan” ​ What is titin? What are its properties? -​ It is the largest polypeptide known -​ Elastic property of Titin allows it maintain its relaxed state -​ passive tension of relaxed muscle is maintained when thin filaments removed -​ experimentally by gelsolin -​ structural protection - prevents overstretch of sarcomere -​ Titin is required to maintain structural integrity: when removed, it takes away bipolar filaments. Acts like a spring to keep filaments in place -​ ​ What is tropomyosin? What does it do? Where is it at? -​ Thin filament regulation -​ Tropomyosin is one of those filaments that like to bind actin -​ Lies on both sides of the actin filament -​ 2 positions -​ ‘off’ state - blocks myosin head interaction with actin filament (without calcium) -​ ‘on’ state - allows myosin head interaction with actin filament (with calcium) -​ ​ What are the 3 proteins in the troponin complex? What is the troponin complex? -​ Troponin complex sits on top of the tropomyosin complex and that is what's pushing the tropomyosin out of the way 1)​ TN-T binds complex to Tropomyosin 2)​ TN-I required to position TM in an ‘off’ state. Inhibits tropomyosin from moving off 3)​ TN-C related to calmodulin and binds Ca 2+ with KD ~ 10 -6 M ​ Where does Ca2+ bind? What is the purpose? -​ On TN-C of troponin complex -​ Moves TM out of the way ​ What are all the roles that Ca2+ plays in the muscle -​ Increase of calcium makes it go to the contracted state -​ Its a t 10^-7 M at relaxed state, squeezing you're at 10^-4 -​ Sarcoplasmic reticulum holds all the calcium -​ GO BACK TO THIS ​ Understand the myosin II actin mediated sliding filaments -​ 1. Contraction of cleavage furrow during cytokinesis -​ 2. Stress fiber contraction in cultured cells and in vivo, vascular endothelial cells -​ 3. Contraction of adherens junction‐associated actin ring for regulation of tight junction permeability in epithelial and endothelial cells -​ Muscle: The basic unit of muscle contraction is the sarcomere, where actin and myosin II filaments slide past each other when ATP is hydrolyzed. -​ Non-muscle cells: Actin and myosin II filaments are also present in non-muscle cells, where they form contractile assemblies that resemble miniaturized muscle fibers. -​ Cytokinesis: Actin, myosin II, and other proteins assemble in a contractile ring that constricts the cell into two daughter cells ​ How do we measure myosin crossbridge force? -​ Purified myosin put on a bead at low density -​ Put actin filament on latex bead connected on both ends then levitated the beads with lasers holding them up -​ by tuning force of trap - measure force of myosin and step size -​ breaks bead out of trap - 1-5 pN – ​ Hand over hand mechanism? -​ a movement pattern where two opposing parts, often resembling hands, alternate leading and trailing positions to achieve continuous motion, like how humans walk  -​ Myosin V has a step size of 36 nm. -​ Single labeled heads show 72 nm step size -​ Must be hand over hand ​ What does myosin V do? -​ transports cargo within cells by moving along actin filaments -​ Tails of several myosins (incl. I, V, VI, XI) interact with membranes/vesicles - responsible for membrane/vesicle movement in cells ​ What's the structure of microtubules? -​ They're a tube composed of protofilaments -​ Organized as a flat sheet. Once there's enough it turns into a tube -​ They create tubuoles using tubulin dimers -​ Microtubule - open 24 nm diameter tube -​ Microtubule wall - linear protofilaments of - dimers - all aligned with same polarity ​ What is the purpose of microtubules? -​ ​ Know the polarity of MT -​ The centrosome/MTOC defines MT polarity; generally plus ends pointing out. An important exception; mixed polarity in dendrites ​ What is MTOC? What are the purposes? -​ "Microtubule Organizing Center," and it is a cellular structure responsible for organizing microtubule networks within a cell, primarily by initiating microtubule assembly and anchoring their minus ends, which allows for the proper positioning and function of microtubules in various cellular processes like cell division and intracellular transport ​ How do we experimentally seethe MT -​ Add a depolymerizing agent (a drug) , then view polymerization -​ Drugs have the capability to regulate whether or not they shoot off the green hairs or collapse back down. That's how you define where the MTOC is. ​ What drugs affect MTs? -​ Taxol, Colchicine/Colcemid, Vinblastine/vincristine, Nocodazole Colchicine (vinca plants) – blocks (+)end dimer addition – depolymerizes MTs in cells, blocks mitosis Nocadozole - – blocks (+)end of MTs – blocks mitosis of fungi (athletes foot medicine) Taxol - (pacific yew trees) -​ blocks loss of tubulin from MT -​ polymerizes all tubulin in cell irreversibly -​ blocks mitosis ​ How do MT polymerize? -​ tubulin dimers (-tubulin-GTP) associate and dissociate at each end -​ tubulin dimer in wall of MT (-subunit) hydrolyzes GTP to GDP+P i -​ Unpolymerized tubulin - added to flagellar axoneme (‘seed’) which contains uniformly polarized MTs polymerizes greater at end with the -tubulin subunit exposed = (+) end ​ What is the Cc of MT? -​ MTs (like MFs) - critical concentration for polymerization is 10 M -​ below Cc MTs do not form -​ above Cc all excess tubulin assembles into MTs ​ Understand the breakout session from lecture ​ Which end of MT polymerizes faster? -​ (+) end is more active for both polymerization and depolymerization -​ (+) end individual protofilaments may project beyond end of growing MT -​ Protofilaments may splay when depolymerizing ​ What causes the lag in assembly? -​ Lag in hydrolysis of bound nucleotide after subunit addition to polymer -​ At the plus end, there is an GTP(nucleotide) cap containing subunits under conditions of rapid assembly -​ Hydrolysis occurs at a rate slower than subunit addition when high free subunits are available for addition, at least at the growing plus end. ( For both G‐actin and tubulin dimer, nucleotide (NTP) hydrolysis occurs upon incorporation into polymer.) -​ However, if the available pool of subunits is at or below the Cc, then the plus end will contain subunits with GDP rather than GTP; (key for MT assembly dynamics) ​ What is GTP? -​ a tubulin protein molecule where the beta subunit is bound to a molecule of GTP ​ What is dynamic instability? -​ individual MTs oscillate between phase of elongation and catastrophic shortening -​ intrinsic property of cytoplasmic MTs in cells ​ What are the conditions that promote polymerization? -​ Promotes assembly: Salt, Low calcium, GTP, cold temperature ​ What are MAPs? What do they do? -​ Microtubules associated proteins -​ proteins that bind to and interact with microtubules within a cell ​ What are the classes of MAPs? -​ 1. Decreasers of critical concentration (e.g. neuronal MAPs) -​ 2. Reducers of dynamic instability (e.g. neuronal MAPs) -​ 3. Promoters of dynamic instability (e.g. certain kinesins that bind to plus end and destabilize) -​ 4. Plus end binders (+TIPs) e.g. EB1) -​ 5. MT severing proteins (e.g. katanin) -​ 6. MT dimer sequestering proteins (e.g. stathmin) -​ 7. MT nucleating complexes of the centrosome (gamma tubulincomplex) Crosslinkers -​ 1. Other MTs (e.g. certain neuronal MAPs -​ 2. Intermediate filaments (e.g. plectin) -​ 3. actin filaments: (e.g. plectin, myosins V and X) MT associated motors -​ 1. dyneins -​ 2. kinesins ​ What are MAP2 associated MTs? -​ a protein that stabilizes microtubules (MTs) by cross-linking them with other MTs and intermediate filaments -​ MAP2-associated MTs in dendrites are more widely spaced than Tau-associated MTs in axons ​ What's the difference between stabilizing proteins and destabilizing proteins? -​ Stabilizing proteins help maintain a protein's structure, while destabilizing proteins reduce a protein's stability ​ What are +TIP proteins? -​ Plus End Binder: EB1 enriched at MT (+)end only when MT is elongating ​ What is kinesin-13? What does it do? -​ Destabilizing protein -​ induce depolymerization uniquely from both ends of the microtubule -​ Kinesin-13 binds curved end of protofilament ​ What's stathmin? -​ Destabilizing protein -​ Stathmin curves end by binding two dimers -​ regulates MT assembly by dimer seques ​ What does katanin do? -​ MT severing in vitro by the (triple A) ATPase katanin. Katanin activity is upregulated upon entry into mitosis and may play a key role in remodeling the MT cytoskeleton. -​ Purified rhodamine labeled MTs 30 sec.(A) and 3 min. (B) after addition of ATP and katanin. Katanin destabilizes end in mitotic spindle -​ Basically–It will cut microtubules to activate it. When katanin and atp are added, it chops it up into little pieces. Which cytoskeleton and microtubules have to be chopped up for mitosis ​ How does MT affect mitosis? What role does it play? -​ forming the "mitotic spindle," a structure that actively separates and distributes replicated chromosomes to the poles of the dividing cell, ensuring each daughter cell receives a complete set of genetic material; essentially acting as the "ropes" that pull the chromosomes apart during cell division ​ What are the differences between anaphase A and B? -​ Anaphase A refers to the movement of chromosomes towards the spindle poles during cell division, primarily driven by the shortening of kinetochore microtubules, while  anaphase B involves the separation of the spindle poles themselves, caused by the elongation of interpolar microtubules, effectively pulling the separated chromosomes further apart; essentially, anaphase A moves chromosomes to the poles, while anaphase B pushes the poles apart -​ A) Chromosome moves to pole as kinetochore MTs shorten -​ B) Anaphase B requires ATP -​ Kinesin-5 (+)end-directed motors slide interpolar MTs and push poles apart - IP MTs may elongate by assembly at (+) ends -​ Dynein (-)end -directed motors associated with cell membrane may pull on astral MTs ​ What are the MT motors involved with each? -​ The primary microtubule motors are kinesin and dynein; kinesin typically moves towards the plus end of a microtubule, while dynein moves towards the minus end, allowing for bidirectional transport of cellular cargo within the cell. ​ What cleaves the cell in cytokinesis -​ a "cleavage furrow" is the indentation that effectively cleaves the cell in two, formed by the contraction of a ring-like structure called the "contractile ring" which is composed of actin filaments and myosin proteins located just beneath the cell membrane; essentially, the contractile ring pulls the cell membrane inward to create the cleavage furrow and divide the cell into two daughter cells. ​ What is cancer? -​ family of diseases characterized by uncontrolled cell proliferation (growth and division) -​ Cancer can result from transformation of a single cell in any tissue of the body -​ Dividing cancer cells can generate a confined tumor -​ Cancer cells may metastasize by leaving their original location to colonize other areas of the body ​ How is mitosis a target for cancer therapies -​ It prevents cell division -​ Drugs that inhibit MT polymerization - e.g colchicine, nocodazole, vinblastine, vincristine -prevent spindle formation -​ Drugs that inhibit MT depolymerization - e.g., taxol - prevent anaphase