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Research Techniques I: Microscopy and Cell Imaging Seeing at the cellular and subcellular level 1  Several Types of Microscopy Light microscope Conventional (e.g. bright field, phase contrast etc.) Fluorescence microscope Confocal micro...

Research Techniques I: Microscopy and Cell Imaging Seeing at the cellular and subcellular level 1  Several Types of Microscopy Light microscope Conventional (e.g. bright field, phase contrast etc.) Fluorescence microscope Confocal microscope Two-photon microscope Electron microscope (next lecture) vesicles mitochondria 2 membrane bilayers  The Light Microscope (LM) * Not for lookinga thick material - Back of path Follow the light ↑ retina T from filters - No blockage ↳ image on Utilizes basic light path. back of retina Used for live or fixed cells and G tissue. Magnification ↑ Loading… Tissues: upright microscope. * Dish with nothing else Isolated cells: inverted lense light + ↑ Light indedent ↳ Inverts microscope. on specimen light goes through/past the An Focus they “upright” specimen on the microscope light specimen before you can focus the image of your 3 on the back eye  Four types of LM 1) Bright Field 1) Bright field illumination transmitted light. 2) Phase contrast 2) Phase contrast · converts phase differences into changes in brightness. · Frits Zernike, 1953 Nobel Prize. 4 * Phase contrast Four types of LM Light moves through cell The Wave properties of light can be exploited. l In unstained cells, a phase shift can will occur as light travels cell through the cell. Loading… cause a Shift in light Phase alignment is related to phase shift L Light increased brightness; if not wave modified aligned, decreased brightness. a Observable with phase Figure 9-7B. Molecular Biology of the Cell, 4th Edition. contrast. can use dye to see more definition 5  ↑ Four types of LM 3) Differential Interference Contrast 3) Differential interference (DIC) contrast similar principles as with phase contrast. 4) Dark polarized light is Field separated and recombined. 3.Prisr more definition. Diffracted light 4) Dark field * used the least lateral light source shows in contrast ↳See changes only scattered light. 6 ↳ source of light modified & angle  Interference Differential contrast Phase and DIC Compared Phase Shift Phase contrast DIC Phase plate brings waves together to E Light rays are produce interference nu recombined num Phase shift Phase shift u Prism separates rays of polarized light Incident light on specimen Incident light on specimen Figure A6, 8. Becker et al. 2006 World of the Cell. 7  Fluorescence Microscopy remit Photons & a specific ↳ Molecules absorb light wavelength Useful for detection of specific molecules or ions. Works on the principle that some molecules absorb and emit photons of light at specific wavelengths. Atomic absorption of a photon is followed by emission at a longer wavelength, and a light signal is detected. specific energy & a ↳ use light + photon excites target molecule wavelength shift back to ↓ then emits a Photon to go Figure A10. Becker et al. 2006 World of the 8 ground energy Cell.  Fluorescence Microscopy A variety of fluorescent molecules are used in fluorescence microscopy (e.g. DAPI, GFP, FITC). ↳ Green Fluorescent Protein Note spectral characteristics of each dye/molecule. GFP L Absorbs blue light ↳ Emits green light 9  Fluorescence Microscopy Fluorescence microscope is optically similar to LM. High energy lamps (Hg) -mercury lamps provide bright source. Filters reduce light of unwanted wavelengths. Chromophores excited at ↳ Allows some light through specific wavelengths. filters Lots of light be there are so many 10  GFP in Research Transgenic mice generated to express GFP and other “FPs”. Permits selective labelling and imaging of cells in live specimens. Loading… Hippocampal Dendritic Spinal cord: double expression (with neurons spines YFP) Neuromuscular junction ACh receptors (GFP+YFP) 11 From Feng et al. 2000. Neuron 28:41- 51. Jackson et al. (2013) Expression of sall4 in taste buds of zebrafish. Dev Neurobiol. 73:543-58. 12  Tissue Preparation To observe cells in tissue, in most cases tissues must be histologically prepared (e.g. not for GFP). Fixation: exposure to chemical -over reagents (aldehydes, acids, time alcohols) to preserve and stabilize. - May produce unwanted effects. Embedding: plastic or polyethylene Sglycol. - Remove Izo + add solvent add plastic Plastic Sectioning: cutting gradually - of thin (1–10 µm) w/ tissue sections on a microtome. Tissue embedded Staining: if applicable, involves exposure to dyes, e.g. hematoxylin, inside eosin, antibodies. 13  Immunofluorescence Protein injected in animal - they produce antibodies Antibodies are produced in host animal and collected.against it Fixed tissue is permeabilized and treated with primary antibodies directed against a specific antigen. ↑ Antibodies bind to antigen on or within cell. Secondary antibodies conjugated with fluorescent marker bind to primary antibodies. Indirect immunohistochemistry labels cell structures. use fluorescence Microscopy to look of the marker method so many * use this one 14 antibodies are in secondary Spot together  The Confocal Microscope ↳ Higher resolution Advantages: Technique that provides clear images with reduced “background” signal. Particularly useful for applications involving thick = sections or whole-mount preparations. Disadvantages: Costly. 15  The Confocal Microscope pinhole “Confocal” refers to d 2 equidistance between light d pinhole 1 source and object, and d1=d 2 object and detector.** Utilizes fluorescence and high energy lasers (He-Ne object and Ar). Pinhole focuses light at a single point in specimen, producing an optical section with low background “noise”. 16  The Confocal Microscope Only light focused at the pinhole will enter the detector.** This allows the confocal to provide clear images a few µm into tissue. In addition to the x and y axes, imaging may occur in the z axis. 3D reconstructions possible. 17  Comparison of Techniques Fluorescenc Confoca e l Glia (red) Figure A15. Becker et al. Neurons (green) 2006 World of the Cell. Actin (green) in Drosophila station embryo - Gives 18  Two-Photon Microscopy (non- linear) ↳ more recent Advantages: resolution ↳ Even higher Non-linear technique that uses higher-order light-matter interactions from multiple photons to generate contrast. Allows deep tissue imaging (up to 1 mm depth possible). - In this process, absorption occurs in the near IR region, and NIR light penetrates deep into tissue. Disadvantage: Very costly. 19 * Non linear  Two-Photon Microscopy Two-photon absorption involves “simultaneous” (~0.5 fs) arrival at excitable molecule. Excitation and emission occurs, as in fluorescence. # Two photons needed (e.g. Confocal) However, signal is dependent upon photon density, so absorption is spatially confined. Speed In confocal, single photon absorption occurs throughout exc. 20 light cone. From Helmchen and Denk. 2005. Nature Meth  2:932 Two-Photon Microscopy Differs from confocal by excitation laser and detection pathway. Rapid high-energy laser pulses (100 fs; 100 MHz) are emitted. Signal collected by detector depending on sample thickness. No pinholes needed, as in confocal. 21  Two-Photon Microscopy Example of in vivo deep-tissue imaging. - Possible configurations. Intact neocortex. 22 From Helmchen and Denk. 2005. Nature Meth  2:932 Things to Consider… 1. What are the primary differences between the different types of microscopy discussed so far? 2. Think about appropriate applications in which you would use regular fluorescence, confocal and two-photon microscopy. 23  Research Techniques II: Microscopy and Cell Imaging Seeing at the cellular and subcellular level (continued...) 1  Not Photons as energy ↑ using Electron Microscopy ↳ Electrons are the Technique with a long history. source of energy We will discuss two major types: 1) Transmission electron microscope (TEM). - 2) Scanning electron microscope (SEM). = Uses bombardment of electrons rather than light. Advantage: Resolution 2,000´ that of LM. 0.1 nm = 1.0 Å (Ångströms). Lower resolution in some biological preparations (2 nm). Disadvantage: Very time-consuming preparation. But for some experiments, this is the only appropriate technique. - 2  In a vacuum Transmission EM ( (m) + ↳ No lens EM LN General configuration analogous to LM. Electrons emitted at filament vacum or cathode. Accelerated by high voltage (105 V) in a vacuum. Magnetic coils focus electron beam like a lens. Sample may be stained, producing “electron dense” detector images. 3  Preparation for TEM Dark-electron dense Lengthy procedure that takes days to weeks. > takes along time - Tissue must be fixed in glutaraldehyde. L preserve tissue Addition of OsO4 increases electron = density. replacing withonal Remove water by Dehydration and infiltration with a plastic resin gives extra support. then put in over Ultrathin = sections (50 – 100 nm) must be cut with a diamond knife. 4 Slices Sections cannot be handled directly. ↳ ! Placed on copper grids. specimen near Objective lense 4 Region of intrest (tissue)  Electron Microscope Transmission TEM Micrograph - Electrons go through # thin sections Only specimen A mitochondrion 100nm A mitochondrion in serial sections - & sections make a 3D image of cell # Multilobed # mitochondria can't see wh a LM ↳ mitochondria single part of 5 ? * midterm-what type of microscope took this image  Immunogold EM particles be its ↳ use gold e-dense - TEM technique C Electron dense - 6 proteins W here they are can identify +  Scanning Electron Microscope (SEM) ↳ detailed Very Produces 3D images of surface structures. Used to study whole cells and z tissues, rather than intracellular structures. Principles of preparation and operation similar to TEM Cells/tissues coated with heavy metal. > coated with gold - Scattered electrons from the specimen surface are collected. ↳ Electrons reflected/scattered bounce of I are The way specimem material determines off the +O 7 produce an the image image  SEM Micrographs ↳ Not looking through tissues Insect head 2.0 mm LD 3D images T4 Bacteriophage 0.1 µm Figure 18.19. Karp. 2008 Cell and Molecular Biology. 8 resolution varying  EM and LM Compared Lu SEM DIC TEM -sections # 3D -sections Put together can be a 3D model ↳ Not so clear but some 3D aspects Stereocilia of hair cell from frog inner ear. 9 * Back to Photons Ion Imaging Changes in intracellular ion concentrations (e.g. Ca2+ and H+) are physiologically important. Ion-selective indicators emit light depending on local ion concentrations. These reveal rapid intracellular dynamics. 10  Ca2+ Imaging Look& in change Voltage Intracellular Ca2+ is low. & probe Bioluminescent aequorin -S C injected into a fish egg reveals Ca2+ wave wave of lights up - when in propagated during depolarization contact fertilization. wi eat Other synthesized indicator molecules produce signal Spreadazt over time (e.g. Fura-2, Fluo-4) Ca2+ wave fert. Ca't binds to fluorescent 11  Ca2+ Imaging in Wavelengths * change correlated with ionic cast is binding and Ca2+ & discrete concentration. one Spot Dyes can be injected, or “AM” analogues can be used to cross Ca2+ sig glia cell membrane. = acetoxymethyl ester S 12 Injecting dye that fluoresence contactwl cast When coming in X-ray Crystallography ‘Seeing’ at the molecular level 13  X-ray Crystallography can understand produce some wave Structure - function interference Macromolecules Atomic resolution X-rays (0.1 nm) Crystallized proteins create Bombardment and diffraction. patterns e.g. Interference patterns ↳ to determine structures Becker. 2006 World of the Cell Problems: Years ago was very time-consuming (first structure...22 yrs!). Large amount of material required. > so much material to see # Insoluble protein crystallization (e.g. membranes)? structure of 14 Protein Hemoglobin would need to - be in O2 free environment  X-ray Crystallography same protein ↑ over over and diffracted beam Synchrotron The structure of one molecule is derived from crystal diffraction pattern. The crystal contains many copies of the same molecule => 15  X-ray Crystallography Protein folding Groups of atoms Individual atoms Electron density Karp. 2008. Cell and Molecular Biology 16  X-ray Crystallography Diffraction pattern Space-filling model of DNA double helix. From Becker. 2006 World of the Cell Fig. 4-5A. Alberts et al. Francis Crick James Watson Maurice Wilkins Rosalind Franklin 17 Cobb M and Comfort N. (2023). Nature. 616:657-660.  X-ray Crystallography Structure of K+ ion channel by X-ray crystallography. Monoclonal antibodies (green) used to “hold” protein. Nobel Prize 2003 3.2 Å resolution Roderick MacKinnon Jiang et al. 2003. Nature 423:33-41. 18  Protein structure from AI Today, protein 3D structure can be predicted by computation and artificial intelligence: https://alphafold.ebi.ac.uk/ e.g. screenshot for Ca2+ channel 19  Things to Consider... 1. Think about applications in Cell Biology in which specific techniques of microscopy are most appropriate. For example, would you use an electron microscope to study intracellular Ca2+ dynamics? 2. What are the major differences between each technique that we’ve discussed? For example, use of light or electrons, linear vs. non-linear, advantages and disadvantages. 20  of cell structure Cytoskeleton I: Filament Proteins 1  3 Types Actin filaments (AFs, or microfilaments) Microtubules (MTs) Intermediate filaments (IFs) 2  be taken a apart can Acytoskeleton Put back together depending on Filament Construction conditions Small subunits form filaments. Actin and tubulin, compact and globular. dismatalled filaments disassembly, diffusion, on reassembly this side Filaments built on this Side 3  Filament Construction * Not how they are ends made just , a visual Protofilaments, e.g. can S fall off MTs easily Weak noncovalent bonds. - All filaments can be built on all sides ↳ Can 2 build a sheet to make it more stable 4 ↳ to break more energy needed ↳ Takes time to form Oligomers (more than Q subunit) ↳ Once oligomer forms reaction set off to Nucleation T Where the build fillament cell likes to L Filament be grows fast ↳ Equilibrium occurs+ the growth of the filament Plateaus - 5  Tubulin ↳two for GTP ↳ binding sites 1 GTp is embeddeda part Structure of + I GTP can part of structure hydrolyzed [Brubuin) so not be & # I different proteins put together - Giptobeneed # Heterodimer = 2 different up(t) proteins, but considered where I # Gip is so it can be hydrolyzed “1 subunit” of tubulin to add to filament embedded each binds GTP; = Gip hydrolyzed at only 1 site 13 protofilaments “plus” and “minus” end (Not a # charge) E NotVeryi down (+) X-tubulin GTP into isintegrated structure so will Equilibrium not be hydrolyzed * Adding subunits toend 6 * Removing subunits from G end  Actin Monomer ATP “plus” and “minus” end 7 mTreadmilling and Dynamic Instability microfilaments * Actin/tubulin SpontaneouslyCome together respectively into their filaments * Spontaneous Polymerization Growth / shrinkage of filament proteins. ATP) (Grp) Actin and tubulin catalyze hydrolysis of ATP or GTP. ↳ can lag behind “T” or “D” form indicates if triphosphate or diphosphate form exists. ATP / GTP caps.# - Amount of subunit Critical concentration (Cc): where subunit addition equals subunit loss. 8  Treadmilling – Actin Filaments ↳ Adding a removing subunits of the same time concentration remains , constant Experiment: 1. Filaments added to ATP-actin. ↳ ATP-actin added to both ends > 2. [ATP-actin] high, addition high occurs at both ends. ATP-actin -solution in - lots of units in cytoplasm 3. [ATP-actin] drops, addition greater at plus end. 4. Steady state. Treadmilling endsa ATP-Actin added going to both keeps L ↳ Once concentration decreases in cytoplasm, there is a greater addition of ATP-Actin & the plus end ATP-actin is removedo added@ same rate , concentration of Atp-Actin ↳@ equilibrium , Nucleation is not a factor here since pre-formed remains constant &Actin - ADD Actin Don't loose - ADP subunits bla L Invites in cytoplasm Falls off - bonding with - filaments were added to actin solution Polymerization of covalent other subunits bic they are Actin-ATP 9  # Is equilibrium Treadmilling – Microtubules = 10  Treadmilling Rate of hydrolysis and Cc (critical Concentration) Linear relationship of [subunit] vs. elongation rate As you a ↑ subunit Conc you add more subunits - · T D ↳ Above zero for F 4 Below Zero for D There is no net change in length 11 of T or D ends at their respective Cc  Treadmilling behaviour – MT Cell injected with rhodamine-labelled tubulin; White arrow, T-end; Red arrow, unlabelled notch 12 Fig. 16-10. Molecular Biology of the Cell  Actin Filaments- MTS * Dynamic Instability – MTs Minus End (-) Plus End (t) Tubulin wI GTP T grow [Tubulin] within continues to W/GDP # Tubulin critical values ↳ Tubulin w/ GTP GTP cap => MT , where GTP hydrolysis ↳ All ↳ End of Polymerizing occurred hydrolyzed Continuous hasn't to GDP transitions ↳ constant transition between Catastrophe ↳ GTP binds again so tubule doesn't fall apart ↓ rescue 13  Dynamic Instability – MTs Hydrolysis affects conformation Stable Unstable 14  Kinetics Dependent on [tubulin] L Tubulin Concentration Blunt tip Tapered tip low concentration high concentration into ↳ Random Polymerize a tapered tip to Surface area At high subunit concentration, the T-end of MTs is modified to promote faster kinetics 15 Gardner et al. (2011). Cell 146:582-292. Dynamic Instability – MTs Epithelial cell, Rhodamine- labelled tubulin Dyn. Inst. Growth cone of neuron 16 Treadmilling and Dynamic Instability What is the purpose? it'll # With time fall apart Constant ATP consumption. Is it worth it? Spatial and temporal flexibility with high turnover. - itsstructure maintain 1 Fastest way to grow filaments without nucleation. Exploration for attachment sites and remodelling. 17 Intermediate Filaments & Better ability to deform themselves withstand much force ↳ can't Epithelial cens 18  Intermediate Filaments (A,B) Dimer formation. (C) Tetramer antiparallel formation. E dimers coiledendtogether I end to (D) Tetramer-tetramer association. ↳ Staggered tetramers 19  Intermediate Filaments Tetramers packed into array of 16 dimers in cross-section. Rope-like appearance. Formation by spontaneous interaction. IDon't need ATp GTp) or Disassembly likely regulated by phosphorylation. 20 “Apocalyptic hagfish spill covers highway in slime” July 2017 21  Storage and deployment of skeins in thread cells of hagfish -storage ↳ Nucleusad down Weingard et al. (2014) Nat. Comm. 5:3534; Weingard and Fudge (2010) J. Exp. Biol. 213:1235-1240 22  IF Types Epithelial (e.g. keratins) Axonal (e.g. neurofilament) Epithelial (keratins) Most diverse family Type I and II keratin chains Strength in hair, nails 23  IF Types Axonal (neurofilaments) Found in central and peripheral axons of vertebrate neurons. Type L, M or H. Growth, increase axon diameter. Axon Glia Axon (cross-section) 24  Things to Consider... 1. Can you differentiate between the processes of treadmilling and dynamic instability? 2. Think about the differences in assembly, growth and shrinkage between each of the 3 filament proteins that we discussed. 3. Where does ATP or GTP fit in? 25 Cytoskeleton II: Regulation 1  Accessory Proteins Filaments (AFs, MTs, IFs) are dynamic and under control of the cell. Form higher-order structures (e.g. mitotic spindle). Accessory proteins modify these cytoskeletal dynamics. ↳ important very L Help speed up or slow down processes w/ filaments ↳ Nucleation 2  Klag Phase)Gamma Nucleation by g-tubulin Protein Complex - ↳ Accessory Protein MTOC (the centrosome) in animal cells. Spindle pole body in yeast. g-tubulin is highly conserved. cells microtubule ↳) find in many gTuRC (ring complex) accelerates MT formation. Nucleation occurs at the (-) end. Brxtubulin - then builds on that template gTuRC 3 ↳ forms complex that is a template  Nucleation by g-tubulin Complex MT initiation also occurs in cytosol = e.g. plants. MTs nucleate daughter MTs. too , (g (parent MT - Daughter 4 MT  Help divide # The Centrioles and Centrosome Centrosome contains 2 cylindrical centrioles. of MT 9 triplets of MTs. ~ + nucleation 9 Triplets Centrioles organize PCM (Pericentriolar of mT L Material). Centriole lumen and PCM contain g-tubulin. PCM initiates MT assembly. s ~ Centries e 2 pairs of centrioles after duplication during cell cycle. Karp. Cell and Molecular Biology 5 The Centrioles and Centrosome cross Section Centrosome near nucleus. - of centriole (-) ends of MTs are anchored. (+) ends of MTs emanate in astral configuration. - l 0.1 µm centrioles inside Centrosome - Astro configura- tion Cooper and Hausman. The Cell: A Molecular Approach 6 Centre of cell/nucleus - See more MT cortex helpsProvide cortex of cell-see more Af Support Nucleation of AFs # just beneath membrane Cell periphery (cortex), where density of AF -Actin filaments proteins is highest. Location related to AF function (cell shape, movement). Facilitated by ABPs (actin binding proteins) including ARPs (actin related proteins). 7  INucleation) Initiation of AFs (unbranched) contraction #Muscle Initiation by formin, an ABP Correct alignment for polymerization e.g. filaments of muscle cells. interact wh more than I actin Bind - - ↑ Strength by &A of covalent bonds Initial actin monomer ↳ Interacts w/ 1 , 3, 4 Formin dimer Cooper and Hausman. The Cell. ↳ Acts as a template to initiate process 8 ↳ Will dissociate actin one enough has binded  Initiation of AF Branches ↳ Locomotion Cell periphery Arp2/3 produces extensive branching. Complex contains 7 proteins. #Daughter Binds at (-) end. 70º favourable angle. - > cell corxex parent 9  subunits are accessible How many # Control of Subunit Pools ↳ Pools of subunits Both actin and tubulin are maintained in the cytosol at high concentrations. Concentration can exceed Cc. Bind temporarily Accessory proteins may sequester unused subunits (sequestering proteins). ↳ Binding Sequestered proteins are not hydrolyzed. Provide control or regulation of filament elongation. 10  Thymosin Sequesters Actin Accelerates growth # Thymosin sequesters, but profilin recruits monomers. Thymosin makes polymerization less favourable. Profilin competes with thymosin and promotes assembly. 11 when a how quickly * Determines actin will grow  Stathmin Sequesters Tubulin Stathmin binding prevents polymerization. Decreases effective [tubulin]. Promotes dynamic instability (catastrophe). 12 &  MT-Associated Proteins (MAPs) ↳ Bind along side MT tau MAP2 Several binding domains. Length of projecting domain determines packing of MTs. MAP2 (neuronal cell bodies); tau (axons). tau and Alzheimer’s Disease (tau unable to bind to MTs). Poorly soluble (hyperphosphorylated) tau may induce neurodegeneration. => Li Help separate microtubules ↳ Helps space ↳ Lengthof MAP determines out MT smaller 13 how far away microtubules are MAP2 overexpression tau overexpression 14  AF Binding Proteins Cofilin destabilizes AFs S end) Accelerates binds to side of proteins.(near negative loss off of binding induces mechanical stress. Gend treadmilling, turnover. ↳ Accelerates loss ↳ Accelerates cell locomotion. overall turnover Tropomyosin stabilizes AFs binds to side of proteins. muscle contraction. 15  Modifications at AF Ends e.g. CapZ “Capping” Recall elongation slower at (-) end. e.g. CapZ (+), elongation occurs only at (-) end. e.g. tropomodulin (-) in muscle ① end - Cap2 binds to contraction. - slows down growth/Shrinkage ↳ slowing down - All thats left is G end + not actin elongation likely to gain 16 Figure 16-43 Molecular Biology of the Cell (© Garland Science 2008)  Modifications at MT Ends Capping in MTs has dramatic effect on dynamic instability. Important during mitosis. (Kinesins) 17  Cross-Linking Proteins and AFs - ↳ formation of Cortex sites Formation of higher-order structures. Binding wi actin Spacing of 2 actin binding domains of cross- linking protein determines type of structure. 2 major groups: bundling and gel-forming. 18  must bind to actin All Cross-Linking Proteins and AFs Cross-linking proteins Form larger Bundling - network Acti Gel-forming · (network) ↳ form dense suber structure I of Af Cactin Red = actin binding domains binding far apart sites 19  Actin Bundling Proteins e.g. Muscle e.g. Filopodia a-actinin fimbrin / ! dinin Fimbrin 3 very Actin Allowsslacement close of myosin 20  Gel-forming Proteins Red blood cell membrane Forms 2-D network. Gives RBC membrane flexibility. & Cooper and Hausman. The Cell. 21  Gel-forming Proteins Filamin e.g. lamellipodia in locomotion 22  Changes in Cell Shape During Embryonic Development Vertebrate embryo. Formation of neural -changes tube during nervous in MT system development. Growing MT Cell height – MTs Folding into tube – AFs. ↳ Becomes cord Spinal 23 Changes in Cell Shape During Embryonic Development 24  Induction by Extracellular Signals Example, crawling neutrophil. Chemical activates WASP... Polymerizing filaments push membrane. Extension of lamellipodium PM (+) (+) ~a ton (-) (-) ~ Network Neutrophil WASP = Wiskott-Aldrich Syndrome Protein At of that ocomotion 25 http://astro.temple.edu/~jbs/courses/204lectures/neutrophil-js.html  Sequence of steps 1. Extracellular signal - Goes across PM 2. Activation of WASP - > Advancing a PM forward ↳ protein 3. Nucleation and branching by Arp2/3 -spontaneous polymerization 4. Promotion of assembly by profilin ↳ Accelerates elongation 5. Elongation reduced by capping proteins 6. Destabilized by cofilin and return of subunits to pool 26  Things to Consider... 1. Think about how each accessory protein affects the stability of AFs and MTs? 2. Many of the dynamic mechanisms that we discussed today do not apply to IFs. Why? 27  Midterm I Format Total of 50 marks, 25% of your final grade. There will be 2 parts to the exam: Part A: Multiple choice (30 x 1 marks) Part B: Long answer (2 x 10 marks) We will discuss further details and go through sample questions together during the review period on Thursday. 1 Cytoskeleton III: Molecular Motors 2  Motor Proteins Use energy derived from hydrolysis of ATP to produce mechanical force. Binds to cytoskeleton (e.g. AFs or MTs). Produces net movement of protein or “cargo”. Divided into 3 families: Bind 1. Myosins: move along AFs M wlactin ~ icrotubule e Bind 2. Kinesins: move along MTs (+) end Fubulin 3. Dyneins: move along MTs (-) 3  Myosins ↳ many types Diverse family of motor proteins. Most eukaryotes (I, II, V). types binds Plants (XI, XIII). in all resume , Protozoa (XIV). O ↑ involved w/ binding to myosins other or other Proteins Head region conserved. O Functions “Conventional” myosins – e.g. type II: muscle, cytokinesis. “Unconventional” myosins L – e.g. type V: organelle transport. some element are the same 4  Myosin II ~ Always -bindin in force Structure Heavy chains (1´ n, n). fails Catalytic parts of – heads, tails. Light chains (2 ´ n, n). Helps articmost ↑ – “essential” and “regulatory” chains. Heads are catalytic, provide force. Tails mediate dimerization with other myosins. Light chains amplify conformational change. 5  Only talking The Myosin Cycle about Q 1 & head Bundle of myosin 1. Attached: no ATP, locked. ↳ Rigor Binds to myosin 2 2. Release: ATP bound, conformational change (away from AF). ↳ caused by binding of ATP - 3 3. Cocked: hydrolysis, conformational change toward (+) end of AF. ↳ movement of myosin forward 4. Force-generating: weak binding, Pi 4 release, power stroke, ADP lost. 5 1 6  Thick Filament Formation Tail-tail interactions. Heads oriented in opposite direction. Formation of bundles in sarcomere of muscle cells. actiprievedNowad is - (-) (-) (-) C No heads interacting w/ ↳ Large bundle - each myosin has two heads -Eventually a head (-) tails other of myosin will bind to actin 7  The Sarcomere Figure 16-74b Molecular Biology of the Cell (2008) Caps stop activ from growing or Shrinking #cappingProtea in decrease growth ⑦ ⑧ O ⑭ ↳ Gives striction to muscles Z disc = α actinin + CapZ 8 -  Ca2+-dependence of Muscle Contraction # If no calcium do contraction & Binds troPOmyosin ↓ actin No Ca2+, tropomyosin blocks > cacium myosin–AF binding - dependent ↳ Interferes wh actin-myosin binding Ca2+ released from SR and binds to troponin C Conformational change in I = “inhibitory” troponin I C = “calcium” T = “tropomyosin” Tropomyosin (bound to Troponin T) moves and exposes binding sites Myosin-AF binding ↳ Given ATD present Becker et al. World of the Cell Muscle contraction 9  Myosin II S1 Fragment Addition of protease -catalytictea enzyme. Myosin cleaved between neck and tail. S1 contains catalytic site. Can be immobilized on glass surface. Slide AFs in vitro. Karp 2008. Cell and Molecular Biology. 10  Experimental Evidence S1 fragments propel AFs on glass slides Myosin labelled with phalloidan Spudich 11  “Contractile Ring” Important during cytokinesis. Myosin II and actin filaments. Redistribution of filaments. (+) (+) Cytokinesis Fig. 17-49B, Alberts 12  Myosin N + actin Myosin V Note: only 1 of 2 heads shown in figures Long neck region. “Hand-over-hand” steps. - Alwaysaheat erate Processivity. contact in Carries cargo (organelles). 13  intrest for Experimental Evidence Optical trap with feedback control. Polystyrene bead bound to myosin V. Steps measured as 30 – 40 nm. Spudich 2000. Nat Rev Mol Cell Biol. 2:387-392 14  Kinesins 1960s: Proteins involved in axonal transport were unidentified. We s Radioactive proteins containing 3H-leucine moved along sciatic nerve Transport in cat. ↳ Track Of Leucine 410 mm/day. - Adapted from Ochs 1972 in Kandel 2000. Principles of Neural Science. 15  Kinesins Discovery MTs adhered to glass coverslips. Axoplasmic extract from squid giant axon + ATP induced organelle movement. ATP but no axoplasm, no movement. With non-hydrolyzable form of ATP (AMP-PNP), vesicles bound to MTs but no movement. Kinesin isolated and identified as motor protein. Vale et al.1985.Cell 42:39-50; Vale et al.1985.Cell 40:559-569. 16  Kinesin Structure Similar basic structure as myosins. Head region conserved. Tail regions diverse. C-terminal domain attaches to cargo. Most travel on MTs toward (+) end. KIFC2 travels toward (-) end. ↳ Dic of its orientation See also Fig. 16-63 in Alberts 17  Kinesin Structure myosin a ATP-binding sites Kinesin (yellow) similar. in MT vs. AF binding sites differ. Linker region interacts with catalytic core, thus swinging arm around. 18  Kinesin Cycle Processive steps along MTs. 1. ATP binding of leading head induces conformational change in its linker region; trailing head advances. 2. ADP induces weak binding of leading head. 3. Hydrolysis of trailing head induces detachment; ADP dissociates from leading head. (Repeat…) 1 2 3 Fig. 16-62 starts at step 2 Longer hydrolysis cycle than with myosin II 19  Myosin and Kinesin Compared Myosin II Kinesins Attach to AFs Attach to MTs Contract muscle Transport organelles Rapid power stroke Longer attachment No head coordination Processive steps 5% of time attached 50% attached ATP binding è release ATP hydrolysis è release 2 - 60 µm/s < 2 µm/s - < 8 pN theoretical force* < 5 pN* Note: binding will occur without ATP 20 *Boal 2006. Mechanics of the Cell  60 in Dyneins opposite (-) end directed. ↳ Another direction MT motor proteins. motor protein 2 major divisions: – Cytoplasmic, e.g. retrograde vesicular transport. – Axonemal, e.g. beating of cilia and flagella. 21  Cytoplasmic Dynein ↳ Don't interact w/ ↳ looks like ants directly cargo Dynein cannot bind directly to cargo. Attachment to MT mediated by dynactin complex So named because of inclusion of actin (red). ↓related Other accessory proteins. dunein Actin (Arp) 22  Axonal Transport E Organelles – Kin/Dyn 23  Axonal Transport SEM micrograph of vesicle being transported along axon MTs by kinesin or dynein. ↳ 3D image 24 Role of AF vs. MT-based Motors 25  Things to Consider... 1. Structure is related to function. Think about the differences between types of motors at the molecular level that lead to differences in function. 2. Why isn’t there a single motor protein that performs all of the roles discussed? 26 Cell Cycle I: Intracellular Control 1 Cell Cycle I: Intracellular Control 1 Cell Cycle I: Intracellular Control - Non disjunction 1  * Refresh The Cell Cycle We will not discuss at length the cell cycle itself. Focus on mechanisms of control that occur at specific stages. small only ~ of time “growth” “mitosis” amount t Frog Egg - Most cells “growth” “synthesis” 2 The main checkpoints that monitor and control the cell cycle to prevent catastrophic events are: 1. **DNA Replication Checkpoint** - Detects unreplicated DNA - Blocks M-Cdk activation and prevents cell from progressing into mitosis 2. **Spindle Attachment Checkpoint** - Checks if all chromosomes are attached to the spindle - Blocks chromatid separation if unattached kinetochores are detected 3. **DNA Damage Checkpoint (G1)** - Activated by DNA damage (e.g. from X-rays) - Involves p53 protein, which prevents cell from entering S-phase 4. **G2/M Checkpoint** - Checks if all DNA is replicated and if environment is favorable - Controls entry into mitosis 5. **Metaphase-to-Anaphase Transition Checkpoint** - Ensures all chromosomes are attached to the spindle - Triggers anaphase and cytokinesis when conditions are met These checkpoints act as "molecular switches" to regulate important events in the cell cycle, including DNA replication, mitosis, chromosome segregation, and cell proliferation.  Why Control the Cell Cycle? So cell will turn out correctly The cell cycle is a complex system of coordinated processes that must occur in a specific sequence. If performed incorrectly or out of sequence, results may be catastrophic. something onloff biochemically Turn ↑ Regulatory proteins and biochemical switches control progression through the cell cycle. This system monitors intracellular and extracellular environments. -checkpoints * want same end point 3 each time  Cdks Control the Cell Cycle Menzyme-protein kinases that Phosphorylate Cdk = “cyclin-dependent kinase”. proteins) - target Activity of cyclin (and thus Cdks) rises and falls with cell cycle. Molecular switches that regulate important events: or Activate – DNA replication inactivate – mitosis proteins in – chromosome segregation cell cycle progression – cell proliferation activates cyclin 4 CdK  Classification of Cyclins and Cdks Cyclins in all eukaryotes (4 major classes): S 4 G1/S cyclins: bind Cdk near end of G1 and lead cell into DNA replication. levels fall is s Phase = major S-cyclins: bind Cdk during S phase and are required for cyclins DNA replication, control early mitotic events. M-cyclins: promote the events of mitosis. G1-cyclins: (in most cells) promote passage through of the restriction point in late G1. -Helps govern the activity G /s cyclings. 5  dependent Cyclin kinases Cdks are Protein Kinases Protein - Phosphorylates protein that gets - Takes away phosphate phosphorylated - Phosphorylating activates target protein - inactivates target activation Protein depends on 6 where the phosphate is added ? Cdk Activity is Regulated 7  Activation of Cdk-cyclin kinase ↑ Phosphorylates ( ( Protein ↳ can phosphorylate Proteins in cell = cycle bound, active wo cyclin blocked by site is cyrclin T-100P Out o 8 the Pulls Site to be phosphorylated  Inhibition of Cdk - Weel Phosphorylates the site above e.g. M-Cdk the active site ↓ inhibits CdK - Cdc25 dephosphorylates activity this site and reactivates Prevents by blocking active the Cdk Sites ↳temporary p27 is a CKI block · kI-Cdk inhibitor Proteins 9 The activation of Cdk-cyclin complexes involves several mechanisms: **1. Cyclin Binding** - Cdks are initially inactive and require binding to a cyclin to become partially active - The cyclin binds to the Cdk, exposing its active site and T-loop **2. CAK Phosphorylation** - After cyclin binding, a Cdk-activating kinase (CAK) phosphorylates the Cdk - This phosphorylation fully activates the Cdk-cyclin complex **3. Removal of Inhibitory Phosphates** ↳ Phosphorylate - Some Cdks (like M-Cdk) can be inhibited by phosphorylation from kinases like Wee1 ↓ deactivate the Cdk - Phosphatases like Cdc25 can remove these inhibitory phosphates, activating the Cdk 3 **4. Degradation of CKI** - Cyclin-dependent kinase inhibitors (CKIs) like p27 can bind and inactivate Cdk-cyclin complexes - Phosphorylation of CKIs can lead to their ubiquitination by SCF and subsequent degradation, allowing Cdk activation These mechanisms work together to ensure precise control of Cdk activity throughout the cell cycle. Ubiquitin and Protein Degradation Ubiquitin * Figure 6-90 Molecular Biology of the Cell, 5th ed. (2008) The proteasome A ubiquitin ligase is required to catalyze addition of a polyubiquitin chain to a protein 10  SCF and APC are Ubiquitin Ligases SCF can lead to destruction of G1/S cyclins (see slide 5). SCF can lead to destruction of CKI (e.g. below and slide 5, 9). a as adapr & tats Spot - Forosphate S-Cdk not deactivated; CKI must first be phosphorylated e.g. Cell proceeds to S phase before it is recognized by SCF/F-box Positive effect on cell cycle - 11 Skp/Cullin/F box family of proteins; Anaphase Promoting Complex  SCF and APC are Ubiquitin Ligases APC can lead to destruction of securin, leading to chromatid separation (see slide 17). APC can lead to destruction of M-cyclin (e.g. below and slide 5). ↳ Recognize M-Cal Cell permitted to leave M-phase Positive effect on cell cycle Need M-cyclin to go downin e can leave 12 Skp/Cullin/F box family of proteins; Anaphase Promoting Complex Mphase Yes, cyclin concentration needs to decrease during M-phase of the cell cycle. This is evident from the information provided in the document: 1. **M-cyclin levels**: The graph in shows that M-cyclin concentration rises during G2 and peaks at the beginning of M-phase, then sharply decreases towards the end of M-phase. 2. **APC activation**: The APC (Anaphase-Promoting Complex) becomes active during M-phase. One of its functions is to target M- cyclins for degradation, which leads to the decrease in cyclin concentration. 3. **Cell cycle progression**: The decrease in M-cyclin levels is crucial for the cell to exit M-phase and enter G1 of the next cell cycle. 4. **Regulation mechanism**: The APC-Cdc20 complex becomes active during metaphase-anaphase transition, contributing to the degradation of M-cyclins. This cyclin concentration decrease is an essential part of the cell cycle control mechanism, ensuring proper progression through mitosis and preparation for the next cell cycle. SCF and APC are Active During Different Stages = => SCF APC -M-cyclin Conc keeps M-cdk M sell to stop down to M-phase from going (-) Keeps M-cyclin êM-cyclin êM-Cdk éAPC-Cdh1 APC-Cdc20 (slide 5 and 12) (above) levels low (slide 5) activation and cell leaves M-phase Separase activation 13 and anaphase (slide 17) Activation of M-Cdk Triggers Mitosis Here, we are in the latter stages of G2 on the brink of mitosis… (Phosphorylation by Wee1 blocks activity) innib Adds Enhances Feedback S-Cdk phosphorylates (activates) Cdc25 during M-phase (see slide 5) 14 The activation of M-Cdk triggers mitosis through the following process : **1. Initial state:** M-cyclin binds to inactive Cdk1 The complex is initially inactive **2. Activation steps:** Cdk-activating kinase (CAK) phosphorylates the complex This creates an activating phosphate, but the complex remains inactive due to inhibitory phosphate **3. Final activation:** Cdc25 phosphatase removes the inhibitory phosphate This fully activates the M-Cdk complex **4. Positive feedback loop:** Active M-Cdk enhances its own activation by: - Further activating Cdc25 - Inhibiting Wee1 (which normally adds inhibitory phosphates) **5. Mitosis initiation:** The rapid activation of M-Cdk through this positive feedback loop triggers the onset of mitosis This process ensures a swift and decisive transition into mitosis once the necessary conditions are met.  “Checkpoints” 2 1 1. DNA replication checkpoint 2. Spindle attachment checkpoint 3. DNA damage checkpoints (several) 3 Fig. 17-14. Alberts, 5th ed. 15  DNA Replication Checkpoint Detection of unreplicated DNA activated - Doesn't get Cdc 25 not activated; M-Cdk activation is blocked / (See slide 14) Protein Phosphatase Cell does not progress into mitosis 3 16 Chromatid Separation 17 Slide 17 explains the process of chromatid separation during cell division. The key points are: **Activation of APC (Anaphase-Promoting Complex)**: M-Cdk activates APC by phosphorylating it Active APC then binds to Cdc20 **Degradation of Securin**: Active APC targets securin for ubiquitylation and degradation Securin degradation releases and activates separase **Cohesin Cleavage**: Activated separase cleaves the cohesin complexes holding sister chromatids together This allows the chromatids to separate and move to opposite poles during anaphase **Progression from Metaphase to Anaphase**: The image shows the transition from metaphase (aligned chromosomes) to anaphase (separated chromatids) This process is crucial for proper chromosome segregation during cell division  Spindle Attachment Checkpoint Unattached kinetochore Mad2 binds to attached Kinetochore Cdc20-APC activation blocked No chromatid separation Nondisjunction 18 “Mad” = mitotic arrest deficient DNA Damage Checkpoint (G1) p53 is a gene regulatory protein, GRP Negative effect on cell cycle Cell does not enter S-phase 19 The DNA damage checkpoint is a crucial control mechanism in the cell cycle that helps prevent cells with damaged DNA from progressing through the cycle. Here's an explanation of the DNA damage checkpoint: **G1 DNA Damage Checkpoint:** - Activated when DNA damage is detected in the G1 phase of the cell cycle - Key player: **p53**, a gene regulatory protein (GRP) - Function: - Has a negative effect on the cell cycle - Prevents the cell from entering S-phase (DNA synthesis phase) The DNA damage checkpoint ensures that cells with compromised genetic material do not replicate or divide, which could lead to the propagation of mutations or chromosomal abnormalities. By halting the cell cycle progression, this checkpoint allows time for DNA repair mechanisms to fix the damage before the cell continues through the cycle. If the damage is too severe to be repaired, the checkpoint can trigger programmed cell death (apoptosis) to eliminate potentially harmful cells.  Pg 1080 General Name functions and comments Z Cdk-activating Kinase (CAK) Phosphorylates an active site in Cdks Wee' kinase phosphorylates inhibitory sites in Laks ; primarily involved in Surpressing Cdk) activity before mitosis Cdc25 phosphatase Removes inhibitory phosphates from Cdks ; Control CdkI activation of the onset of mitosis piL (CDI) Surpresses G , /S-cdk and s-cdk activities in 61 ; helps cell Withdraw from cell cycle Ubiquitin 3 Sef Employs ADC CdC 20 D53 p2l mamz securine separase Cell Cycle II: Extracellular Control Fig. 22-103. Alberts et al. 1  Total Cell Mass Total cell mass (size of organ / organism) Total cell number Cell Growth Cell division – Cell death 2  Total Cell Mass Dependent on Extracellular Factors: Cells produce proteins stimulate cell Grow , growth 1. Growth Factors: ­ synthesis and ¯ degradation Sof proteins of cell division ↑ cell increase probability by r mass promoting of 2. Mitogens: ­ cell division ↳Stimulate cell division See & 3. Survival Factors: ¯ apoptosis (next lecture) ↳Promote cell survival Note: It’s confusing, but some “growth factors” also have mitogenic and - survival effects. Therefore, please remember processes and not - specific factors. 3  Growth Factors