Unit 2 Exam Flashcards

Question

How do microtubules have polarity? How does it grow?

Answer 1

Polarity - orientation of their subunits gives microtubules polarity - the microtubule plus end grows and shrinks much more rapidly than its minus end Growth - Rapid microtubule growth occurs by the addition of tubulin dimers at the ends (first lag phase, second elongation phase then lastly plateau phase) - addition of GTP-tubulin to plus end of a protofilament causes the end to grow in a linear conformation that assembles into the cylindrical wall of the microtubule

Answer 2

Dynamic Instability - Individual microtubules alternate between cycles of growth and shrinkage Catastrophe - change from growth to shrinkage Rescue - change from shrinkage to growth

Answer 3

Addition of GTP-Tubulin - This addition to the plus end of a protofilament causes the end to grow in a linear conformation that assembles into the cylindrical wall of the microtubule Hydrolysis of GTP - occurs after assembly - changes the conformation of the subunits, forces the protofilament into a curved shape that is less able to pack into the microtubule wall.

Answer 4

- Nucleation depends on the γ-tubulin ring complex. - Microtubules are generally nucleated from the microtubule-organizing center (MTOC) where γ-tubulin is most enriched - Unless the cell is dividing, γ-tubulin is in the centromere

Answer 5

- Its a type of microtubule-organizing center (MTOC) - composed of two centrioles and surrounded by a dense mass of protein termed the pericentriolar material - γ-tubulin is in the pericentriolar material

Answer 6

- Microtubule associated proteins (MAPs) bind and stabilize microtubules - Map2 and Tau set the spacing of the microtubule bundles Note: - Tau Mutations cause Neurodegenerative Diseases (e.g., Alzheimer's disease)

Answer 7

Kinesin - motors that move towards the plus ends of microtubule - long-range movement (progressive same size steps, one head is attached to microtubule) - are apart of a large protein superfamily (common element = motor domain of the heavy chain) - tetramer protein - must be inhibited for minus-end transport Structure - Two heavy chains and two light chains - Heavy chain has many domains - Head splits ATP and converts the energy into motion - Tail is the cargo-binding

Answer 8

- Induces depolymerization from both ends of the microtubule. - are incapable of movement. - regulate microtubule dynamics to control spindle assembly

Answer 9

- unusual as it moves from microtubule plus-ends towards the minus-ends in motility assays - tail of kinesin-14 can bind microtubules and allows it to organize microtubule bundles

Answer 10

- Dynein steps are big but irregular and it moves toward the minus ends - Dynein is ~4 times bigger and more complex than Kinesin - Head is force generating motor(AAA = ATPase domain) - stalk contains the microtubule binding site at its tip (so tail binds cargo) - ATP changes the conformational structure to dissociate microtubule binding

Answer 11

Kinesin - Small - Towards (+) end - Regular steps Dynein - Big - Towards (-) end - Irregular steps Both: - move vesicles in the secretory pathway - ATP driven

Answer 12

- hairlike cell appendages that have a bundle of microtubules & associated proteins at their core (aka the axoneme) - flagella are found on sperm and many protozoa and have an undulating motion. - cilia beat with a whiplike motion - axonemal dynein bends the axoneme which move the cilium and flagellum

Answer 13

Known: - helical polymers of the protein actin actin subunits, (G-actin) are polypeptides carrying a molecule of ATP or ADP - Actin is an ATPase - Actin subunits assemble head-to-tail to form a tight, right-handed helix, forming a structure about 8 nm wide called filamentous or F-actin Polarity: - At minus end the ATP-binding pocket is exposed - At the plus end the ATP-binding pocket is buried in the filament - Growth happens mostly from the + end

Answer 14

Rate-limiting Step - two actin molecules bind relatively weakly to each other, but addition of a third actin monomer makes the complex more stable. - the filament then undergoes a net addition of subunits at the plus end while simultaneously losing subunits from the minus end. (always going to + end) - actin treadmilling pushes membranes forward

Answer 15

- actin-binding proteins regulate polymerization and length of filaments - migrating cells make protrusive structures termed filopodia and lamellipodia (crosslinking results in these structures) Note: - filopodia are made of actin bundles - lamellipodium has complex actin networks

Answer 16

Capping proteins - prevent G-actin addition and loss binds to the plus ends Cofilin (scissors) - cuts actin filaments into small pieces A-Actinin - crosslinking protein that makes F-actin bundles - it is a dimer & rigid - crosslinkers must have ≧ 2 actin binding sites Filamin - a flexible actin crosslinker protein crosslinkers must have ≧ 2 actin binding sites - a dimer - flexibility allows different angles

Answer 17

- The Arp2/3 complex promotes actin branching - Arp2/3 is composed of 7 subunits that are structurally similar to G-actin - initiates a new branched filament by binding to the side of a filament and recruiting actin monomers.

Answer 18

- actin cytoskeleton is linked to the membrane by ERM proteins - ERM proteins: Erzin, Radixin, Moesin Note: - binding sites for actin and membrane proteins are hidden but revealed when Erzin is phosphorylated

Answer 19

- signal the formation of different actin structures - Ex: Rho Rac and Cdc42 - GTPase activating proteins (GAP) Guanine nucleotide exchange factor proteins (GEF) proteins regulate the activity of the GTPases

Answer 20

- All myosins share similar motor domains (dark green), but their C-terminal tails (light green) are very diverse. Myosins are conventional or unconventional - Each myosin head binds and hydrolyzes ATP, using the energy of ATP hydrolysis to walk toward the plus end of an actin filament

Answer 21

Myosin-V - two-headed molecule that steps processively along actin filaments, transporting and dispersing cellular cargos in a wide variety of cell types. Myosin I - has only one head and binds membranes - mutants are left/right inverted Myosin II - forms bipolar filaments - composed of two heavy chains and four light chains. - dimerization occurs when the two α helices of the heavy chains wrap around each other to form a coiled-coil. MLCK - regulatory subunits of myosin II - phosphorylates the myosin light chains and unfolds myosin II into an active state

Answer 22

Stress fibers are contractile actin bundles found in non-muscle cells. They are composed of actin filaments and non-muscle myosin II.

Answer 23

1. Without ATP myosin is attached to the actin filament in Rigor. 2. ATP binding to the myosin head domain causes the release of actin. 3. ATP hydrolysis causes a large conformational shift in the ‘lever arm’. 4. Associates with actin again. 5. Releases the phosphate and does the power stroke, the force-generating step! 6. ADP is released myosin head remains tightly bound.

Answer 24

- tail–tail interactions form large, bipolar thick filaments that have several hundred myosin heads, oriented in opposite directions at the two ends - skeletal muscle cells (also called muscle fibers) are big multinucleated cells form by the fusion of many muscle cell precursors

Answer 25

a muscle fiber contracts when myosin filaments pull actin filaments closer together and thus shorten sarcomeres within a fiber Notes: - The A-band (black) does not change - The I-band (white) disappears

Answer 26

Sarcomere - Each is formed from a miniature, a precisely ordered array of parallel and partly overlapping thin and thick filaments Myofibril - is a cylindrical structure 1–2 μm in diameter that is often as long as the muscle cell itself. - consists of a long, repeated chain of tiny contractile units of sarcomeres

Answer 27

- sarcomere shortening is caused by the myosin filaments sliding past the actin thin filaments, with no change in the length of either type of filament - myosin and actin filaments are packed together with almost crystalline regularity

Answer 28

Thin filaments - are composed of actin and associated proteins, and they are attached at their plus ends to a Z disc (build by Capz) at each end of the sarcomere - α-actinin holds actin thin filaments together in a regularly spaced bundle Thick filaments - are anchored at the M line

Answer 29

Titin - molecular spring, with a long series of immunoglobulin-like domains that can unfold as stress is applied to the protein. - keeps the thick filaments poised in the middle of the sarcomere Nebulin - maintains the length and the stability of the thin filament - consists almost entirely of a repeating 35-amino-acid actin-binding motif.

Answer 30

A Sudden Rise in Cytosolic Ca2+ Concentration Initiates Muscle Contraction Steps: 1. signal from the nerve triggers an action potential in the muscle cell plasma membrane, and this electrical excitation spreads swiftly into a series of transverse tubules, or T tubules—that extend inward from the plasma membrane around each myofibril. 2. When the incoming action potential activates a Ca2+ channel in the T-tubule membrane, it triggers the opening of a Ca2+-release channel in the closely associated sarcoplasmic reticulum membrane 3. Ca2+ flooding into the cytosol then initiates the contraction of myofibrils through the Troponin complex and Tropomyosin 4. In a resting muscle, the troponin complex pulls the tropomyosin into a position along the actin filament that interferes with the binding of myosin heads. - When the level of Ca2+ is raised, troponin C—which binds to Ca2+—causes troponin I to release its hold on actin 5. The increase in Ca2+ concentration is transient because the Ca2+ is rapidly pumped back into the sarcoplasmic reticulum by an ATP-dependent Ca2+-pump

Answer 31

- establish an apical-basal polarity, which results from the differential distribution of phospholipids, protein complexes, and cytoskeletal components - within the epithelium, cells are attached to each other directly by cell–cell junctions, where cytoskeletal filaments are anchored, transmitting stresses across the interiors of the cells

Answer 32

1. Tight junctions - molecules cannot leak freely across the cell sheet (sealed by these junctions) - visualized by freeze-fracture electron microscopy (branching network of sealing strands encircling the apical end of each cell) 2. Adherins junctions - associate with the actin cytoskeleton - Cadherin/Catenin complex is the main component of the adherent junctions - cell–cell junction in which cadherin receptors bridge the neighboring plasma membranes via their homophilic interactions 3. Gap junctions - clusters of channels that join two cells together and consist of building blocks of two connexons or hemichannels, one contributed by each of the communicating cells - have a pore size of about 1.4 nm, which allows the exchange of inorganic ions and small molecules, but not of macromolecules

Answer 33

Catenins - link classical cadherins to the actin cytoskeleton - Cytoplasmic protein - Heterophilic cadherins are called atypical (ex: Fat and Dachsous) Cadherins - Aka calcium adherins - are homophilic (bind to each other) - transmembrane protein - cadherin superfamily members all have extracellular portions containing multiple copies of the extracellular cadherin domain - mediate highly selective recognition, enabling cells of a similar type to stick together and to stay segregated from other types of cells. Notes: - Cadherin-Cadherin binding is Ca2+ dependant - E-cadherin = epithelial - N-Cadherin = neurona

Answer 34

- Each cadherin domain forms a more-or-less rigid unit, joined to the next cadherin domain by a hinge. - Ca2+ ions bind to each hinge and prevent it from flexing. - When Ca2+ is removed, the hinges flex, and the structure becomes floppy

Answer 35

- Assembly of strong cell–cell adhesions requires changes in the actin cytoskeleton - Cadherins generate local signals to inhibit the GTPase Rho and activate the GTPase Rac

Answer 36

Strands formed by.. - claudins (are transmembrane proteins, related proteins are occludins) - they form selective channels allowing specific ions to cross the tight-junctional barrier, from one extracellular space to another Insects - Tight junctions in insects = septate junctions - organized differently, but have the same function (also made of claudins)

Answer 37

- Each connexon or hemichannel is formed of a complex of six connexin proteins. - Vertebrate gap junctions are formed by connexins, while invertebrate gap junctions are formed by innexins. - Both connexin and innexin form similar sized pores

Answer 38

- Non-cellular component present within all tissues and organs - large network of proteins and other molecules that surround, support, and give structure to cells and tissues - Proteins from the extracellular matrix are secreted (ex: Collagen, proteoglycans, laminin) - provides essential physical scaffolding for the cell

Answer 39

1. Proteoglycans - have huge oligosaccharide chains at least one of the sugar side chains of a proteoglycan must be a GAG (Glycosaminoglycan) Note: - O-linked glycosylation at the Golgi - Addition of GAG happens outside the cell 2. Fibrous proteins - polypeptide chains organized approximately in parallel along a single axis, producing long fibers or large sheets - mechanically strong and resistant to solubilization in water - Ex: Collagen 3. Glycoproteins - contain relatively short oligosaccharide chains - large scaffold proteins containing multiple copies of specific protein-interaction domains - have multiple domains and crosslink other matrix molecules and cell receptors

Answer 40

GAG - Aka type of proteoglycans - are unbranched polysaccharide chains made of repeating disaccharide units (in ECM) - absorb large quantities of water and swell Example: Hyaluronan (also called hyaluronic acid or hyaluronate) - the simplest of the GAGs - made out directly from the cell surface by an enzyme complex embedded in the plasma membrane. - Role = space filler and compression protection

Answer 41

Collagen - the molecule is along, stiff, triple stranded helical structure, in which three collagen polypeptide chains, called α chains. - many molecules assemble at the ECM into thick long Collagen fibers - collagen fibrils form structures that resist stretching forces - Not all collagens form giant fibrils, some are fibril-associated collagens Note: - Defects in the structure or processing of the protein collagen affect the elasticity of the connective tissue

Answer 42

Fibronectin - a dimer joined by disulfide bonds Laminin - large glycoprotein composed of three chains and links the ECM to cell surface receptors

Answer 43

Role - They are transmembrane heterodimers that link ECM to actin cytoskeleton - cluster to form strong and dynamic connections to the ECM called Focal adhesion sites - Epithelia without integrins detach from the basal lamina How they link - The extracellular portions of integrin bind ECM proteins like fibronectin or collagen - The intracellular integrin tails bind to a complex of adaptor proteins that link the actin cytoskeleton (eg, Talin and Vinculin) Structure - Two integrin conformations (inactive=binding sites hidden and active=binding sites shown) - Integrins are dimers - The cytoplasmic region binds talin Activation - can be activated from the outside or from the inside fibronectin peptide talin - Ie: Binding to a ECM substrate or binding to talin in the inside

Answer 44

Focal adhesions - are large macromolecular assemblies that form mechanical links between intracellular actin bundles and the ECM - All the mechanical signaling makes focal adhesions very dynamic Talin and Vinculin (main components) - Talin is an elastic scaffold protein and a tension sensor at cell–matrix junctions - Talin brings a protein called focal adhesion kinase (FAK) to the focal adhesion - Talin has a large number of binding sites for the actin-regulatory protein vinculin. Most sites are hidden inside folded protein domains but are exposed when those domains are unfolded - Vinculin stabilizes talin, actin filaments, and integrins - Vinculin opens up and aids integrin clustering for FA growth, has open and closed conformations

Answer 45

- apical-basal polarity results from the differential distribution of phospholipids, protein complexes, and cytoskeletal components between the various plasma membrane domains, reflecting their specialized functions. - Antagonistic interactions between polarity factors that maintain the identity and control the size of the apical, junctional and lateral domains

Answer 46

1. The Par Complex - cytoplasmic 2. The Crumbs Complex - only transmembrane protein and links aPKC to the membrane 3. The Scribble Complex. - Cytoplasmic - inhibits aPKC Notes: - They are mutually inhibitory - The exact position of the complexes vary slightly between tissues and species.

Answer 47

aPKC - main effector of apical identity because it phosphorylates the junctional and lateral polarity factors Par-3 and Lethal (2) giant larvae (Lgl) to exclude them from the apical domain Phosphorylation of Lgl inhibits its association with the other proteins in the complex - aPKC, a kinase, phosphorylates and inactivates Lgl

Answer 48

- An imaginal disc is a sac-like epithelial structure found inside the larva of insects that undergo metamorphosis. - Without AP polarity the discs overproliferate and form tumors (AP polarity and cancer are linked)

Answer 49

Loss - the loss of apicobasal polarity leads to overproliferation and metastasis EMT - The epithelial–mesenchymal transition (EMT) is a process by which epithelial cells lose their cell polarity and cell–cell adhesion, and gain migratory and invasive properties Note: - tumor suppressors are genes that when mutated lead to uncontrolled proliferation

Answer 50

PCP - coordinated polarization of cells within the plane of a tissue - Ie: Cell orient to the tissue axis, each trichome comes from a single cell controls hair orientation (mutants have disorganized cell structures (eg. trichomes) - regulate trichome positioning via changes in actin cytoskeletal organization Notes: - Z = AB polarity - X+Y is Planar cell polarity

Answer 51

Core proteins - The core proteins self-organize into mutually antagonistic sides - The cytoplasmic proteins mediate the positive and negative interactions Actin-binding proteins - restrict formation of the actin-rich trichome to one side - overall logic is to separate actin processes in the distal and proximal side

Answer 52

1. Core pathway - comprises six distinct proteins that form asymmetrically localized intercellular complexes (ex: Distal and proximal complexes) 2. Ds-Ft pathway - aligns the entire tissue - “global pathway” - Ft and Ds are atypical cadherins - They form heterophilic complexes between adjacent cells, and localize asymmetrically - Asymmetric localization of Ft and Ds is driven by expression gradient of Fj and Ds Notes: - In the Golgi, Fj phosphorylates extracellular cadherin repeats of Ft and Ds. - Modifies Ft-Ds binding affinities

Answer 53

Fluorescence - It is the emission of light by a substance that has absorbed light or other radiation - Fluorescent molecules absorb light at one wavelength and emit it at another, longer wavelength Photons (or e-) - An orbital electron of a fluorochrome molecule can be raised to an excited state after the absorption of a photon. - Fluorescence occurs when the electron returns to its ground state and emits a photon of light at a longer wavelength - Too much exposure to light destroys the fluorochrome molecule in a process called photobleaching

Answer 54

Immunofluorescence is commonly used in molecular and cell biology labs as a robust and simple method to reliably localize molecules on fixed cells or tissues

Answer 55

Origin - original green fluorescent protein comes from a jellyfish (Aequorea victoria) Luminescence - Aequorin is a blue-light-emitting bioluminescent protein. - GFP is a green-light emitting protein (absorbs blue, emits green) - Aequorin and GFP work together to convert Ca2+-induced luminescent signals into the green luminescence More info - GFP does not need a prosthetic group or any other cofactor so it can be entirely genetically encoded. The chromophore is made by amino acids - Fusing GFP to the coding sequence of a gene allows direct visualization of the protein - peptide location signal can be added to the GFP to direct it to a particular cell compartment, such as the endoplasmic reticulum or a mitochondrion - GFP fluorescence responds rapidly and reversibly to pH changes

Answer 56

- Mutations in GFP change the absorption and emission colors - also change other fluorescence properties, like brightness stability, maturation time, etc

Answer 57

Experiment: Track the movement of proteins and cells in-vivo - Visualizing intracellular Ca2+ concentrations by using a fluorescent indicator - calcium imaging is a powerful means for monitoring the activity of distinct neurons in brain tissue in vivo (changes in calcium = neuron activity) Age - Some fluorescent proteins slowly change their color so we can test the “age” of proteins and cells

Answer 58

Photobleaching - photochemical alteration of a fluorophore, that makes it unable to fluoresce. FRAP - Means fluorescence recovery after photobleaching - Indicates dynamics of a protein in a living cell (ie: how fast proteins move)

Answer 59

Technique: - Fluorescence Resonance Energy Transfer (FRET) is a special technique to gauge the distance between two chromophores - FRET can be used to make Genetically encoded fluorescent biosensors many molecular sensors GFP - Split GFP proteins into fragments that spontaneously assemble into a functional protein when in very close proximity Notes: - In proximity = FRET - Not in proximity = No FRET - Test protein to protein binding (1–10 nm)

Answer 60

Hybridization is the process of combining two complementary single-stranded DNA or RNA molecules and allowing them to form a single double-stranded molecule through base pairing.

Answer 61

RNA hybridization - allows detection of mRNA molecules - For example: uses in target hybridization and fluorescence imaging dsRNA - has a strong effect - Once injected and passed onto offspring, the worm twitches in a similar way as if it had defective muscle protein gene dsDNA - inhibits synthesis of specific proteins promotes mRNA degradation - can be introduced experimentally to silence genes of interest

Answer 62

1. Long double-stranded RNA (dsRNA) is cleaved into small interfering RNA (siRNA) by the enzyme Dicer. 2. The siRNA joins the RNA-induced silencing complex (RISC), 3. Argonaute 2 (AGO2) cleaves the sense strand of RNA . 4. The RISC–siRNA complex binds and degrades complementary mRNA 5. The activated RISC–siRNA complex can then be reused

Answer 63

other RNAi info - RNA interference is a defense against viruses and jumping genes especially crucial for plants, worms, and insects allows remove specific proteins in cells and tissues without affecting the genome Applications 1. Therapeutic - Remove malfunctioning proteins that cause problems - Destroy viruses 2. Research - Study the functions of individual proteins Cure Huntington’s Disease (??) - Huntington’s is caused by mutations in huntingtin gene - The mutated huntingtin proteins accumulate in neurons and cause brain damage

Answer 64

- Identify an interesting process → e.g., cell morphology - Get a library of dsRNA that cover all the genes - Incubate cells with specific dsRNA score the changes